Bifunctional molecules that selectively induce degradation of extracellular targets in lysosomes

Abstract

The present invention relates to bifunctional molecules containing a protein binding moiety of interest linked by a linker group to a cellular receptor binding moiety, preferably a moiety that binds to a receptor sort protein encoded by the gene SORT1.

Description

Technical Field

[0001] The present invention relates to bifunctional molecules containing a protein-of-interest binding portion connected by a linker group to a cellular receptor binding portion, preferably a portion that binds to the receptor sortilin encoded by the gene SORT1. Pharmaceutical compositions based on these bifunctional molecules represent additional aspects of the invention. These compounds and / or compositions can be used to treat disease states and conditions by removing secreted proteins and / or transmembrane proteins by degradation inside the cells of a patient or subject in need of therapy. Also described herein are methods of treating disease states and / or conditions in which circulating proteins are associated with disease states and / or conditions. Background Art

[0002] Protein degradation is an essential part of biomolecule turnover and turnover and is a naturally occurring process in all cells. Cytoplasmic proteins are typically degraded in the proteasome following ubiquitination, whereas extracellular biomolecules are degraded in the lysosomal compartment. Specific ubiquitination of cytoplasmic disease-associated proteins (DAPs) is facilitated by bifunctional molecules called proteolysis targeting chimeras (PROTACs) [1]. PROTACs are bifunctional molecules that include a DAP-binding warhead at one end and are linked to an E3 ubiquitin ligase-binding small molecule at the other end. Thus, PROTACs link the E3 ligase to the DAP, leading to proteasomal ubiquitination and subsequent degradation, effectively acting as a chemical DAP knockout. However, since PROTACs rely on the proteasome to function, they are only applicable to intracellular proteins. Data suggest that non-cytoplasmic proteins can be directed for degradation in the lysosomal compartment by using bifunctional molecules to link the protein-sorting mannose-6-phosphate receptor (M6P-R) [2]. Banik et al. demonstrated that antibodies tagged with M6P-R binding carbohydrate moieties promoted lysosomal degradation of extracellular and membrane-bound targets, and these types of molecules are called lysosomal targeting chimeras (LYTACs), which provided the first preclinical proof-of-concept (PoC) that enhanced lysosomal delivery and degradation of DAPs have therapeutic potential. The M6P-R binding motif is a phosphorylated sugar polymer, so a lot of effort is needed to develop an orally available form. Both PROTACs and LYTACs are designed to exploit natural cellular mechanisms, so the warhead does not need to provide function itself. This is particularly advantageous relative to traditional drug development, because warhead development only requires optimization of binding affinity and linker coupling strategies. In addition, due to current limitations, these requirements are not required to further connect targets that are considered undruggable.

[0003] The function of LYTACs depends on the successful recruitment of lysosomal transport receptors, exemplified by M6P-R [2]. Another lysosomal receptor protein is sortilin. Sortilin shares many features with M6P-R, including rapid internalization from the cell surface and transport of cargo to lysosomes [3]. Sortilin is expressed in most tissues and promotes lysosomal degradation of several known ligands [4][5][6]. Sortilin's internalization capacity is illustrated by the plasma accumulation of its natural ligand and frontotemporal dementia (FTD)-associated protein progranulin, which is increased 3.5-fold in the plasma of mice deficient in sortilin [6,7]. Increasing extracellular progranulin levels by inhibiting sortilin-mediated progranulin degradation is considered a therapeutic approach for the treatment / prevention of FTD, and several independent attempts to inhibit this interaction have been explored. The result is a number of small molecule high-affinity sortilin binders with diverse pharmacological properties [8-10], including orally bioavailable compounds and compounds with CNS exposure [8]. Summary of the invention

[0004] In one main aspect, the present invention relates to bifunctional compounds having a structure according to formula (I):

[0005] T L –L I –S L (I)

[0006] in,

[0007] S L is the part that binds to sortilin;

[0008] L I is a joint or a bond; and

[0009] T L The part that binds to the extracellular target molecule

[0010] or a pharmaceutically acceptable salt thereof.

[0011] In another aspect, the invention relates to bifunctional compounds for use in treating a disorder or condition in a subject.In one aspect, the disorder or condition is mediated by an extracellular protein.

[0012] In yet another aspect, the present invention provides a method for targeting lysosomal degradation of extracellular proteins, the method comprising administering an effective amount of a bifunctional compound of formula (I) as described herein.

[0013] In another aspect, the invention provides a method of removing extracellular proteins from the plasma of a subject in need thereof, the method comprising administering to the subject an effective amount of a bifunctional compound of formula (I) as described herein.

[0014] Thus, in one aspect, the present disclosure provides a method for targeting lysosomal degradation of TNFα, comprising administering an effective amount of a bifunctional compound as described herein.

[0015] In another aspect, the present disclosure provides a method of removing TNFα from the plasma of a patient or subject in need thereof, comprising administering a bifunctional compound as described herein.

[0016] In one aspect, the present disclosure relates to a pharmaceutical composition comprising a bifunctional compound as described herein.

[0017] In one aspect, the invention relates to novel small molecule Sortilin binders as described herein.

[0018] In another aspect, the invention relates to novel Sortilin-binding peptides as described herein.

[0019] In another aspect, the present invention relates to an isolated polynucleotide encoding a peptide or bifunctional compound described herein. In another aspect, the present invention relates to a vector comprising a polynucleotide encoding a peptide or bifunctional compound described herein. In another aspect, the present invention relates to a host cell comprising a polynucleotide or vector encoding a peptide or bifunctional compound described herein.

[0020] In another aspect, the invention relates to novel TNFα binding agents as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : MST binding assay using fluorescent Sortilin-6his (100 nM) and compounds according to SEQ ID NO.: 147 (A) and SEQ ID NO.: 157 (B).

[0022] Figure 2 : Change of proximity-induced HTRF ratio as a function of compound concentration for compounds according to SEQ ID NO:147 and SEQ ID NO.:157.

[0023] Figure 3: A) shows the cell-associated fluorescence intensity (FI) signal after HEK293 / sortilin was incubated with a series of concentrations of SEQ ID NO.: 147, SEQ ID NO.: 157 or SEQ ID NO.: 155 and a fluorescent derivative of Neutravidin (NA650) for 3 hours; B) shows the cell-associated fluorescence intensity (FI) signal after incubation of HEK293 / sortilin with a series of concentrations of SEQ ID NO.: 147, SEQ ID NO.: 157 or SEQ ID NO.: 155 and a fluorescent derivative of Neutravidin (NA650) for 3 hours; Cell-associated FI signals after 3 hours of incubation of HEK293 / sortilin with SEQ ID NO.: 147 (300 nM) and NA650 (Invitrogen) (100 nM) in the presence of SEQ ID NO.: 147 (300 nM) and NA650 (Invitrogen) (100 nM) in the presence of SEQ ID NO.: 172 (a peptide without a biotin warhead) (0.1 nM-100 μM) or biotin alone (0.1 nM-100 μM) (n=2). Data points are shown as mean + / - SEM.

[0024] Figure 4: A) Fluorescence microscopy imaging of lysosomal markers in HEK293 / sortilin cells fixed and immunostained with anti-LAMP1 (Alexa-488) after incubation for 2 hours with the bifunctional molecule SEQ ID NO.: 147 (300 nM) and NA650 (100 nM). Nuclei were stained with Hoechst. Scale bar is 20 μm. B) SDS-PAGE gel showing NA650 in cell lysates of HEK293 / sortilin after incubation for 24 hours with the peptide bifunctional compound (300 nM) and leupeptin (80 μM) (Sigma Aldrich) before harvesting the cell lysates and separating the proteins on SDS-PAGE gels. The bar graph shows the quantification of the upper band of NA650 (mean with std).

[0025] Figure 5: NA650 fluorescence signal in HEK293 / sortilin cell culture supernatants after incubation with SEQ ID NO.: 147 (20 nM to 10 μM) and NA650 (Invitrogen) (100 nM) up to 24 hours, 48 ​​hours and 72 hours.

[0026] Figure 6 : NA650 fluorescence signal in HEK293 / sortilin cell culture supernatant after incubation with NA650 (Invitrogen) (100 nM) and SEQ ID NO.: 147 (20 nM-5 μM) or SEQ ID NO.: 155 (20 nM to 5 μM).

[0027] Figure 7: A) SDS-PAGE analysis of the coupling of NHS-linker (SEQ ID NO.: 169) or NHS-linker-RQLL (SEQ ID NO.: 168) to alirocumab under non-reducing or reducing (2 mM DTT + heating) conditions. B) SDS-PAGE analysis of the coupling of NHS-linker or NHS-linker-RQLL to adalimumab under non-reducing or reducing (2 mM DTT + heating) conditions.

[0028] Figure 8 : Shown are the responses of MST binding to Sortilin as a function of the concentrations of amorobumab-linked-RQLL and amorobumab-linked antibody.

[0029] FIG. 9 shows the affinity of PCSK9-6HIS with Amorobizumab-Linker-RQLL ( Fig. 9A ) or Amorobismub-Linker ( Fig. 9B ) complex formation between. SDS-PAGE analysis of protein complex peak fractions ( Fig. 9C ).

[0030] Figure 10: Baseline corrected HTRF (665 / 620 nm) ratios: A) Ratios normalized to Amorobumab-linkage plotted for a concentration gradient (290 nm-3.5 fM) of Amorobumab conjugated to a linker with or without RQLL after 2.5 hours of incubation. (Error bars = SD; n = 2); B) Ratios corrected to the lowest global signal plotted for a concentration gradient (400 nM-4.7 fM) of Adalimumab conjugated to a linker with or without RQLL after 2.5 hours of incubation (Error bars = SD; n = 3). C) Ratios corrected to the lowest global signal plotted for a concentration gradient (400 nM-4.7 fM) of Adalimumab conjugated to Sortilin binding peptides SEQ ID NO.: 184 to SEQ ID NO.: 187 after 2.5 hours of incubation. (Error bars = SD; n = 3).

[0031] Fig.11: Cell-associated fluorescence signal after 3 hours of incubation of HEK293 / sortilin with: A) a range of concentrations of amorobumab-linked-RQLL (SEQ ID NO.: 159) (1 nM to 0.5 μM) and Cy5-PCSK9 (200 nM). In a control experiment, cells were incubated with amorobumab with a peptide lacking the C-terminal sortilin binding sequence, i.e., amorobumab-linker (SEQ ID NO.: 160); B) a range of concentrations of adalimumab-linked-RQLL (SEQ ID NO.: 161) (250 pM to 250 nM) and TNFα (100 nM). In control experiments, cells were incubated with adalimumab with a peptide lacking the C-terminal sortilin binding sequence, i.e., adalimumab-linker (SEQ ID NO.: 162); and C) a range of concentrations of adalimumab (250 pM to 250 nM) and TNFα (100 nM) conjugated to sortilin binding peptides of SEQ ID NO.: 184 to SEQ ID NO.: 187.

[0032] Figure 12: Shows the size exclusion chromatography elution profiles and final samples of A) NB-Linker-RQLL (SEQ ID NO.: 158) and B) NB-Linker (SEQ ID NO.: 71). Pooled and concentrated size exclusion fractions are indicated by grey boxes.

[0033] Figure 13: shows the formation of IgG target and ( Fig.13A )NB-Linker-RQLL (SEQ ID NO.: 158) or ( Fig. 13B )NB-linker (SEQ ID NO.: 71) complex is formed and can be purified by gel-filtration. SDS-PAGE analysis of the main peak is shown on the right, and the pooled and concentrated size exclusion fractions are represented by gray boxes.

[0034] Figure 14: A) shows the cell-associated FI signal after HEK293 / sortilin was incubated with a range of concentrations of NB-linker-RQLL (SEQ ID NO.: 158) (8 nM to 8 μM) and Cy5-conjugated IgG (5 nM, 50 nM or 500 nM) for 3 hours; B) Cy5 FI in cell culture supernatants (upper blots) and lysates (middle blots) of HEK293 / sortilin cells after incubation with Cy5-conjugated IgG (kappa light chain) (50 nM) and NB-linker-RQLL (SEQ ID NO.: 158) as indicated for 72 hours. The lower blot confirms the expression of sortilin in the cells by Western blotting. C) Western blot of Cy5 FI (upper blot) and IgG signals in lysates of HEK293 / sortilin cells after incubation for 6:30 h with Cy5-conjugated IgG (κ light chain) (50 nM), NB-linker-RQLL (SEQ ID NO.: 158) or control Nanobody (NB-linker, SEQ ID NO.: 71) (250 nM) and the lysosomal protease inhibitor leupeptin (80 μM) as indicated (n=2). The two lower blots confirm the loading of the gel (anti-β-actin) and the expression of sortilin in the cells by Western blotting. The bar graph shows the quantification of Ig HC expressed as mean + / - SEM.

[0035] Fig.15 . Detection of binding of BF025, BF023 and BF020 (highest concentration 12.5 μM) titrated with constant NTA-labeled Sortilin-6His (100 nM) in MST analysis. (Error bars = SD; n = 2)

[0036] Fig.16 HTRF (665 / 620 nm) signal baseline corrected to the minimum local signal plotted against bifunctional compound concentrations (5 μM-0.6 pM) after 2.5 h of incubation.

[0037] Fig.17 . Shown are cell-associated FI signals after 3 h incubation of HEK293 / sortilin with a range of concentrations (2 nM to 2 μM) of compounds B025, B023 or B020 and NA650 (100 nM).

[0038] Fig.18 NA650 FI signal in HEK293 / sortilin cell culture supernatants after 72 h incubation with NA650 (100 nM) and the bifunctional compounds BF025, BF023 or BF20 (20 nM-5 μM) as indicated.

[0039] Fig.19 FI signals in lysates of HEK293 / sortilin harvested after 24 h incubation with NA650 (100 nM), B025 (300 nM), AF38469 (10 μM), and leupeptin (80 μM) as indicated.

[0040] Fig. 20 Cell-associated FI signals after HEK293 / sortilin incubated with a range of concentrations (1.9 nM to 2 μM) of compounds BF011, BF006 or BF005 and AlexaFluor488-anti-DNP antibody (100 nM) for 3 h (n=2).

[0041] Figure 21. A) FI signal in lysates of HEK293 / sortilin obtained by incubation with AlexaFluor488-anti-DNP antibody (Thermo Fisher) (100 nM) and BF005 (30 nM) for 3 hours, followed by removal of target and degrader by replacing the medium with assay medium, and incubation for 0 to 24 hours, followed by lysis of cells as previously described. Anti-β-actin and anti-sortilin western blots are shown as controls. The bar graph shows quantification of the FI signal of the HC band normalized to the β-actin signal (mean + / - SEM) (n=2). B) FI in lysates of HEK293 / sortilin incubated for 6:30 hours with AlexaFluor488-anti-DNP antibody (100 nM), BF005 (30 nM) and leupeptin (80 μM) as indicated. SB001 (30 nM) and AF38469 (10 μM) were included as controls. The bar graph shows quantification of the FI signal of the HC band normalized to the β-actin signal (mean + / - SEM) (n=2).

[0042] Fig. 22 . Cell-associated FI signals after 3 hours of incubation of HEK293 or HEK293 / sortilin with a range of concentrations (2 nM to 2 μM) of three different bifunctional compounds: SEQ ID NO.: 155, NB-linked-RQLL (SEQ ID NO.: 158) or a small molecule bifunctional compound (BF005) and the corresponding targets as shown.

[0043] Fig.23 is a scheme representing a non-limiting example of the preparation of bifunctional compounds.

[0044] Fig.24.A) Detection of binding of BF080, BF081 and BF082 (highest concentration 12.5 μM) titrated with constant NTA labeled TNFα-6His (100 nM) in an MST assay is shown. (Error bars = SD; n = 4-6). B) Mouse anti-TNFα (Invitrogen, MA5-23720) Western blot of cell lysates after incubation of HEK393 / sortilin with a range of concentrations (100 nM-10 μM) of compounds BF040 and BF043 and TNFα (100 nM) for 24 hours. Control cells were incubated without addition of compound and TNFα or with TNFα alone. Anti-β-actin (Sigma, A5441) Western blot is shown as a control. C) Cell-associated FI signals after 24 h incubation of HEK293 / sortilin with a range of concentrations (0.5 nM to 20 μM) of BF040, BF043 and BF042 and Cy5-TNFα (100 nM) in the presence of 80 μM leupeptin. D) Cell-associated FI signals after 24 h incubation of HEK293 / sortilin cells with BF042 (0.5 nM-20 μM) and Cy5-TNFα with or without the addition of leupeptin (80 μM). E) TNFα levels in the conditioned medium of HEK293 / sortilin cells 72 h after addition of 20 nM TNFα and the bifunctional molecules BF040, (BF043) or BF042 are shown. TNFα is shown as % normalized to the level measured in the conditioned medium of cells without the addition of the bifunctional molecule. F) Anti-TNFα Western blot of HEK293 / sortilin cell lysates harvested at the indicated time points (0 to 24 hours) after preincubation with BF042 (3 μM) and TNFα (100 nM) for 24 hours. The preincubation medium was replaced at the 0 hour time point. Western blot of anti-β-actin is shown as a control. G) Cell-associated FI signals after HEK293 / sortilin was incubated with a range of concentrations (0.5 to 20 μM) of BF042 and Cy5-TNFα (100 nM) for 24 hours with the addition of the sortilin binder SB013 (1.25 μM, 5.0 μM, and 10 μM) or DMSO. H) shows the cell-associated FI signal after 24 h incubation of HEK293 / sortilin with BF077 (1 μM) and Cy5-TNFα (100 nM) in the presence of increasing concentrations of TF018 or TF005 as competitors for TNFα binding.

[0045] Fig.25 The efficacy of TNFα degraders in an in vivo model of LPS-induced acute systemic inflammation is shown.

[0046] definition

[0047] As used herein, the term "alkyl" refers to a straight or branched chain hydrocarbon moiety.

[0048] As used herein, the term "alkoxy" refers to a group of the formula -O-alkyl, wherein alkyl is as defined above. In particular, C 1 -C 3 -Alkoxy is intended to mean such hydrocarbons having 1, 2 or 3 carbon atoms. Examples of alkoxy include methoxy, ethoxy, n-propoxy and isopropoxy.

[0049] As used herein, the term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms have been replaced by a halogen atom, for example, one or more hydrogen atoms are replaced by any of F, Cl, Br or I.

[0050] As used herein, the term "cycloalkyl" or "carbocycle" refers to a monocyclic or polycyclic ring system. Also as used herein, the term "cycloalkyl" may optionally contain one or more unsaturations or substituents.

[0051] As used herein, the term "heterocyclic" or "heterocycle", alone or in combination, refers to a saturated or unsaturated aromatic or non-aromatic ring containing 3 to 7 ring atoms, wherein at least one ring atom is a heteroatom. As used herein, the term "heteroaromatic" or "heteroaryl", alone or in combination, refers to an aromatic ring containing 5 to 6 ring atoms, wherein at least one ring atom is a heteroatom. "Heteroatom" means sulfur, oxygen or nitrogen.

[0052] The term "aromatic" or "aryl" refers to a cyclic or polycyclic moiety having a conjugated unsaturated (4η+2)π electron system (where n is a positive integer), sometimes also referred to as a delocalized π electron system.

[0053] The term "alkenyl" includes groups having at least one carbon-carbon double bond.

[0054] As used herein, the term "substituent" or "substituted" alone or in combination refers to a group that can be used to replace hydrogen. In some embodiments of the present invention, the substituted molecule itself can be further substituted. As used herein, "a substituent derived from..." refers to a group of atoms derived from a particular molecule or formula at any position of the molecule or formula. In some embodiments, a substituent derived from a molecule is a corresponding molecule in which a hydrogen atom has been removed. For example, a substituent derived from CH 4 The substituent may be -CH 3 .

[0055] Dissociation constant (K D) or binding affinity is a measure of the degree of reversible association between two molecular species. The smaller the dissociation constant, the stronger the binding affinity.

[0056] As described herein, a ternary complex is a complex containing three different molecules bound together. As described herein, a bifunctional compound is capable of forming a ternary complex between Sortilin and a target molecule. This refers to a three-member complex in which Sortilin and a target protein are simultaneously bound to the bifunctional compound.

[0057] As used herein, an extracellular molecule or protein refers to a molecule or protein that is not completely enclosed within a cell. This refers, for example, to proteins that are completely outside of a cell, but also includes membrane-bound or membrane-associated proteins that have an extracellular domain.

[0058] As used herein, TNF-α may be referred to as TNFa, TNF-a, TNF-αTNFα, or TNFα. DETAILED DESCRIPTION OF THE INVENTION

[0060] In one main aspect, the present invention relates to bifunctional compounds having a structure according to formula (I):

[0061] T L –L I –SL(I)

[0062] in,

[0063] S L is the part that binds to sortilin;

[0064] L I is a joint or a bond; and

[0065] T L The part that binds to the extracellular target molecule

[0066] or a pharmaceutically acceptable salt thereof.

[0067] The compounds of formula (I) act to bind the desired extracellular target molecule or protein to Sortilin in a ternary complex. This recruits the target molecule or protein to the lysosomal pathway of the cell, resulting in degradation of the target molecule or protein. Thus, the present invention relates to a chemical entity platform that is capable of targeted degradation of extracellular disease targets by engaging the Sortilin receptor.

[0068] Advantages of the present invention are, for example:

[0069] Ability to induce targeted degradation of extracellular disease proteins.

[0070] • The ability to target a broad disease space due to the wide range of body compartments where sortilin is expressed: blood stream, CNS, PNS, CSF, immune system cells and tumor subtypes.

[0071] The inventors have demonstrated the ability to build such a platform by producing bifunctional compounds according to formula (I) in a variety of formats: from small molecules through medium-sized peptides to large proteins.

[0072] Binding of Sortilin:

[0073] Sortilin (SEQ ID NO.: 1) is a membrane protein expressed in most tissues and promotes lysosomal degradation of several known ligands [4][5][6]. Structural studies performed by several independent investigators have demonstrated that sortilin interacts with ligands within the lumen of large 10-bladed β-propellers [10,11] and that maturation and acidification of endosomal vesicles release cargo, leading to lysosomal degradation of the cargo and recycling of sortilin back to the plasma membrane

[12] . Overall, sortilin is a well-described lysosomal trafficking receptor with a variety of different high-affinity small-molecule binding partners.

[0074] The present disclosure relates to a novel bifunctional compound platform capable of targeting sortilin to recruit target molecules or proteins and internalize them into the lysosomal compartment, wherein their degradation occurs under lysosomal conditions. Thus, a variety of different sortilin binders can be used.

[0075] Due to the potential of targeted Sortilin inhibition as a therapeutic approach for disease conditions, there are many disclosures in the art regarding Sortilin binding agents. These are based on small molecule scaffolds as well as peptides and proteins.

[0076] Small molecules

[0077] For example, several small molecule scaffolds have been used to develop compounds with high affinity for sortilin. The first identified small molecule sortilin binder was compound AF40431 (Andersen et al., 2013

[14] ). Since then, several promising scaffolds for developing sortilin binders have been proposed, such as Formula IV in WO 2014 / 114779; Formulas II and VI in Stachel et al., 2020

[10] ; or Formula VII in Andersent et al., 2017[9]. Other scaffolds capable of binding to sortilin are disclosed in US2016 / 0331646, such as scaffolds based on norbornene anhydride amino acid adducts (Formulas VIII and IX), benzyl-substituted glutaric acid phenyl-amide-acids (Formula X), and 2-substituted 3-oxo-1,2,3,4-tetrahydro-2-quinoxaline (Formula XI). Other examples of reported sortilin binding compounds are SB013 and SB014 disclosed in Sparks et al., 2020. The contents of the above-mentioned references on protein scaffolds are incorporated herein by reference.

[0078]

[0079]

[0080] Thus, in one embodiment, the bifunctional compounds according to the present invention are capable of binding to Sortilin via substituents derived from the above structures or derivatives thereof.

[0081] In one embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L ) has a structure according to formula (II):

[0082]

[0083] in

[0084] R 1 is selected from heteroaryl, aryl, heterocyclic, C 3 -C 10 Cycloalkyl, -(C 1 -C 5 Alkyl)-aryl and C 1 -C 10 alkyl, -BO-aryl, each of which is optionally substituted by one or more identical or different substituents R IIa substituted; wherein B is optionally substituted C 1 -C 5 alkyl;

[0085] R 2 Selected from C 1 -C 10Alkyl and C 2 -C 10 alkenyl, each of which is optionally substituted by one or more identical or different substituents R IIb replace;

[0086] R IIa Selected from -O-aryl, -CH 2 -Aryl, Cl, Br, F, C 1 -C 3 Alkoxy and C 1 -C 3 Alkyl, among which -O-aryl, -CH 2 -Aryl, C 1 -C 3 Alkoxy and C 1 -C 3 Each of the alkyl groups is optionally substituted with one or more substituents which may be the same or different and which are selected from the group consisting of H, -O-CH 2 -C(=O)O-, -O-CH 2 -C(=O)NH-, halogen, alkoxy, -CF 3 and optionally substituted C 1 -C 4 alkyl;

[0087] R IIb Selected from C 1 -C 10 Alkoxy, C 3 -C 10 Cycloalkyl, C 1 -C 10 Alkyl and -CH 2 - aryl;

[0088] Where S L By R 1 or R IIa With L I Coupling.

[0089] In one embodiment, R 1 With two identical R IIa In one embodiment, R 1 With two different R as above IIa In one embodiment, R 1 With only one R as above IIa Substituent.

[0090] In one embodiment, R 1 is an optionally substituted phenyl. 1is phenyl having one or two substituents selected from halogen or alkoxy. 1 is phenyl substituted with two chlorine atoms. 1 is a phenyl group substituted with two chlorine atoms at the meta position of the attachment point of formula II. 1 is a phenyl group having one alkoxy substituent. In another embodiment, the alkoxy substituent is -OCH 3 In one embodiment, R 1 According to formula B:

[0091]

[0092] Where R L Indicates that L I connection, and * Represents the connection with formula II.

[0093] In one embodiment, R 2 C 1 -C 6 In one embodiment, R 2 To replace C 1 Alkyl, where C 1 The alkyl group is optionally substituted with C 3 -C 6 In one embodiment, R 2 To replace C 1 Alkyl, where C 1 Alkyl is C 6 In one embodiment, R 2 In one embodiment, R 2 -CH 2 -C-(CH 3 ) 3 .

[0094] In one embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L ) has a structure according to formula (III):

[0095]

[0096] Where Q 1 is a bond or -CH 2 -;and

[0097] R 3 Having formula (IIIa), formula (IIIb) or formula B;

[0098]

[0099] in

[0100] R 3a Selected from H, halogen, alkoxy, -CF 3 and optionally substituted C 1 -C 4 alkyl;

[0101] in * represents a connection with formula (III), and R L Indicates that L I connection.

[0102] As described in this article, R L Indicates L I and S L The connection point between L and L I The connection points between.

[0103] In some embodiments, S L According to any one selected from Formula IIIc, IIId, IIIe, IIIf, IIIg and IIIh:

[0104]

[0105]

[0106] Where R 3a Selected from H, halogen, alkoxy, -CF 3 and optionally substituted C 1-4 alkyl; and

[0107] R L Indicates that L I connection.

[0108] In one embodiment, the bifunctional compounds according to the present disclosure have S according to Formula IIIc L In one embodiment, the bifunctional compound according to the present disclosure has S according to Formula IIId L In one embodiment, the bifunctional compounds according to the present disclosure have S according to Formula IIIe L In one embodiment, the bifunctional compounds according to the present disclosure have S according to Formula IIIf L In one embodiment, the bifunctional compounds according to the present disclosure have S according to Formula IIIg L In one embodiment, the bifunctional compounds according to the present disclosure have S according to Formula IIIh LIn each of Formulae IIIc, IIId, IIIe, IIIf, IIIg and IIIh, R 3a Selected from H, halogen, alkoxy, -CF 3 and optionally substituted C 1-4 alkyl; and R L represents the same as L in formula (I) I connection.

[0109] In one embodiment, R 3a is H. In another embodiment, R 3a Halogen or -CF 3 In one embodiment, R 3a In yet another embodiment, R 3a is an optionally substituted C 1-4 alkyl.

[0110] In some embodiments, S L Having a structure according to any one of Formulas IIIi, IIIj, IIIk, IIIm, IIIn and IIIo:

[0111]

[0112]

[0113] R L Indicates that L I connection.

[0114] In one embodiment, the bifunctional compounds according to the present disclosure have S according to Formula IIIi L In one embodiment, the bifunctional compound according to the present disclosure has S according to Formula IIIj L In one embodiment, the bifunctional compounds according to the present disclosure have S according to Formula IIIk L In one embodiment, the bifunctional compounds according to the present disclosure have S according to Formula IIIm L In one embodiment, the bifunctional compounds according to the present disclosure have S according to Formula IIIn L In one embodiment, the bifunctional compounds according to the present disclosure have S according to Formula IIIo L In formulae IIIi, IIIj, IIIk, IIIm, IIIn and IIIo, R L represents the same as L in formula (I) I connection.

[0115] In another embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I)L ) has a structure according to formula (DI):

[0116]

[0117] Where R L Indicates that L I connection.

[0118] In another embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L ) has a structure according to formula (D-II):

[0119]

[0120] Where R L Indicates that L I connection.

[0121] As described herein, structures according to Formula III and IIIa through IIIo have a stereocenter at the alpha position to the carboxylic acid group (as marked with "α" in Formula III below):

[0122]

[0123] The stereogenic center can be present in one of two configurations, R or S. These formulae III and IIIa to IIIo exist in the S-stereoisomer configuration or the R-stereoisomer configuration.

[0124] Therefore, in one embodiment, S L is the S-stereoisomer according to any one of formula III or IIIa to IIIo as described herein.

[0125] In another embodiment, S L is the R-stereoisomer according to any one of formula III or IIIa to IIIo as described herein.

[0126] In another embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L ) has a structure according to formula (IV):

[0127]

[0128] R 4 is H or F;

[0129] R 4’ is H;

[0130] R 5 For halogen, H, C 1 -C 6 Alkyl, C2-6 Alkenyl or C 1 -C 6 Haloalkyl;

[0131] R 6 For halogen, H, C 1 -C 6 Alkyl or C 1 -C 6 Haloalkyl;

[0132] Q 2 is a bond or CH 2 ;

[0133] R 7 is a 5-6 membered heteroaromatic monocyclic ring having one or two heteroatoms, wherein the heteroaromatic ring is optionally substituted by one or two independently selected from -CN; C 1 -C 3 Alkyl; HaloC 1 -C 3 Alkyl; C 1 -C 3 Alkoxy, halogen; -C(O)NH-C 1 -C 3 Alkyl; optionally substituted by -C(O)NH-C 1 -C 3 alkyl substituted aryl; and optionally substituted heteroaryl substituents,

[0134] or a pharmaceutically acceptable salt thereof, wherein S L By R 7 With L I Coupling.

[0135] In one embodiment, R 5 is halogen, H, or C 1 -C 6 haloalkyl; and R 6 For halogen, H, C 1 -C 6 Alkyl or C 1 -C 6 Halogenated alkyl.

[0136] In another embodiment, R 4 For H, R 5 For-CF 3 or H; R 6 For-CF 3 or H; and R 4 'For H.

[0137] In one embodiment, Q 2 In another embodiment, Q 2 For –CH2 -.

[0138] In one embodiment, R 7 To be C 1 an alkyl-substituted 6-membered heteroaromatic monocyclic ring having one heteroatom, wherein the heteroaromatic ring is optionally further substituted with an optionally substituted aryl group, and S L By R 7 With L I Coupling.

[0139] In one embodiment, Q 2 is the key, R 4 For H, R 5 For-CF 3 or H; R 6 For-CF 3 or H; R 4 'For H,

[0140] Q 2 is the key, R 7 For C 1 an alkyl-substituted 6-membered heteroaromatic monocyclic ring having one heteroatom, wherein the heteroaromatic ring is optionally further substituted with an optionally substituted aryl group, and wherein S L By R 7 With L I Coupling.

[0141] In one embodiment, R 5 or R 6 At least one of them is -CF 3 In one embodiment, R 5 or R 6 At least one of them is -CF 3 , and the other is hydrogen.

[0142] In one embodiment, R 7 Having a structure according to Formula IVa or Formula IVb:

[0143]

[0144] in * represents a connection with formula (IV), and wherein R L Indicates that L I connection.

[0145] In another embodiment, S L Selected from any one of Formula IVc, IVd, IVe and IVf:

[0146]

[0147] Where RL Indicates that L I connection.

[0148] In one embodiment, the bifunctional compounds according to the present disclosure have S according to Formula IVc L In one embodiment, the bifunctional compound according to the present disclosure has S according to Formula IVd L In one embodiment, the bifunctional compound according to the present disclosure has S according to Formula IVe L In one embodiment, the bifunctional compound according to the present disclosure has S according to Formula IVf L In Formulae IVc, IVd, IVe and IVf, R L Indicates that L I connection.

[0149] In another embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L ) is a substituent derived from a compound of formula VI:

[0150]

[0151] in

[0152] R 8 C 1 -C 10 alkyl; and

[0153] R 9 Selected from C 1 -C 10 Alkyl, aryl and heteroaryl, each of which is optionally substituted by one or more identical or different substituents R VIa replace;

[0154] R VIa is selected from the group consisting of aryl, heteroaryl, -O-aryl, -O-heteroaryl and halogen, wherein each of aryl, heteroaryl, -O-aryl and -O-heteroaryl is optionally substituted with one or more halogens.

[0155] In one embodiment, R 8 C 1 -C 5 In one embodiment, R 8 In another embodiment, R 8 For –CH 2 -C-(CH 3 ) 3 .

[0156] In one embodiment, R 9 is an optionally substituted aryl group.9 is an optionally substituted benzyl group. 9 For -C 1 -C 3 -Aryl.

[0157] In one embodiment, R 9 One of the following:

[0158]

[0159] in * Indicates connection with formula VI.

[0160] In one embodiment, R 9 By one or two substituents R VIa In a further embodiment, one or two substituents R VIa It is a halogen.

[0161] In one embodiment, Formula VI is 9 Connected to the linker part (L) in formula (I) I ).

[0162] In another embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L ) is a substituent derived from a compound of formula VI:

[0163]

[0164] in

[0165] R 10 for and

[0166] R 11 is an optionally substituted heteroaryl group or an optionally substituted aryl group.

[0167] In one embodiment, R 11 In one embodiment, Formula VII is represented by R 11 Connected to the linker part (L) in formula (I) I ).

[0168] In one embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L ) is a substituent derived from the following compound:

[0169]

[0170] In one embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I)L ) is a substituent derived from the following compound:

[0171]

[0172] In one embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L ) is a substituent derived from the following compound:

[0173]

[0174] In another embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L ) is a substituent derived from a compound of Formula VIII or Formula IX:

[0175]

[0176] in,

[0177] R VIIIa is a carboxylic acid or an ester thereof, wherein any of the carbon atoms of the norbornene ring may be replaced by C 1-6 Alkyl substitution, or carboxylic acid can be condensed to C 1-5 ester.

[0178] Where R IXa is an optionally substituted divalent C 1 -C 5 alkyl, wherein one or more methylene groups are optionally replaced by -CO(NH)-,

[0179] R IXb is a group selected from hydroxy, alkoxy, amino or aminoalkyl.

[0180] In one embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L ) is a substituent derived from the compound 2-methyl-3,5-dioxo-4-azatricyclo[5.2.1.0(2,6)]dec-8-en-4-yl)acetic acid.

[0181] In one embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L ) is a substituent derived from the compound methyl 2-(1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-methanoisoindol-2-yl)propanoate.

[0182] In one embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L) is a substituent derived from the compound 2-(1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-methanoisoindol-2-yl)-4-(methylthio)butanoic acid.

[0183] In one embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L ) is a substituent derived from the compound 2-(3,5-dioxo-4-azatricyclo[5.2.1.0(2,6)]dec-8-en-4-yl)-4-methylpentanoic acid.

[0184] In another embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L ) is a substituent derived from a compound of formula X:

[0185]

[0186] in

[0187] R Xa and R Xb Independently for C 1- C 5 alkyl, acyl, amino, sulfonyl (sulfono), chloro, bromo, iodo or fluoro; and

[0188] R Xc C 1- C 5 The alkyl group, or the acid is replaced by a tetrazole.

[0189] In one embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L ) is a substituent derived from the compound 4-[(3,4-dichlorophenyl)amino]-3-(3-methylbenzyl)-4-oxobutanoic acid or 3-benzyl-4-[(3-chloro-2-methylphenyl)amino]-4-oxobutanoic acid.

[0190] In another embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L ) is a substituent derived from a compound of formula XI:

[0191]

[0192] in

[0193] R XIa C 1-5 Alkyl, acyl, amino, sulfonyl, chlorine, bromine, iodine or fluorine;

[0194] R XIb is a bond or optionally C1 -C 5 Alkyl substituted C 1- C 3 Alkyl, and

[0195] R XIc C of carboxylic acid or carboxylic acid 1 -C 5 ester or tetrazole.

[0196] In one embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L ) is a substituent derived from the compound 3-oxo-1,2,3,4-tetrahydro-2-quinoxalinyl)acetic acid.

[0197] Other sortilin binding agents are described in WO 2023 / 031440 (which application is incorporated herein by reference), such as those described in formula (A):

[0198] in

[0199] A 1 , A 2 and A 3 are independently selected from halogen, H, C 1 -C 4 Alkyl, C 1 -C 4 Haloalkyl, C 2 -C 5 Alkenyl and C 2 -C 5 haloalkenyl;

[0200] A 4 Selected from H, C 1 -C 3 Alkyl, C 1 -C 3 Haloalkyl, C 3 -C 8 Aryl, C having one or more halogen substituents 3 -C 8 Aryl, C 3 -C 8 Heteroaryl and C having one or more halogen substituents 3 -C 8 heteroaryl;

[0201] A 5 Selected from C 3 -C 20 Aryl, C 3 -C 20heteroaryl and 3 to 12 membered heterocyclic rings; wherein the aryl, heteroaryl or heterocyclic ring is optionally substituted by one or more substituents independently selected from halogen, -OH, cyano, carbonyl, C 1 -C 4 Alkyl, C 1 -C 4 Hydroxyalkyl, C 1 -C 4 Haloalkyl, acetyl, C 1 -C 4 Alkoxy, C 1 -C 4 Haloalkoxy, C 3 -C 8 Aryl and C 3 -C 8 heteroaryl; or

[0202] A 4 and A 5 together to form a 6- to 20-membered heterocyclic ring, wherein

[0203] wherein the heterocyclic ring is monocyclic, bicyclic or tricyclic and is optionally substituted by one or more substituents independently selected from halo, -OH, cyano, carbonyl, C 1 -C 4 Alkyl, C 1 -C 4 Haloalkyl, acetyl, C 1 -C 4 Haloalkoxy, C 1 -C 4 Halogenated alkoxy.

[0204] For example, for the following compound:

[0205]

[0206] Thus, in one embodiment, the bifunctional compounds according to the present invention are capable of binding to Sortilin via substituents derived from the above structures or derivatives thereof.

[0207] In one embodiment, the Sortilin binding portion (S L ) is a substituent derived from the above-mentioned compound according to formula (A).

[0208] In one embodiment, the sortilin binding moiety (S) in the bifunctional compound according to formula (I) L ) is any one of formulas AI to A-III:

[0209]

[0210] or its derivatives, wherein RL Indicates that L I connection.

[0211] In one embodiment, the Sortilin binding moiety (S L ) is according to Formula AI. In one embodiment, the sortilin binding portion (S L ) is according to formula A-II. In one embodiment, the sortilin binding portion (S L ) is according to formula A-III.

[0212] Other sortilin binding agents are described in WO 2023 / 101595 (the contents of which are incorporated herein by reference), for example as described by formula (E):

[0213]

[0214] in

[0215] Ring E A is a 5- or 6-membered aromatic, heteroaromatic or heterocyclic ring having 0 to 2 heteroatoms selected from N, O and S, or an 8- to 10-membered bicyclic heterocyclic or heteroaromatic ring having 1 or 2 heteroatoms selected from N, O and S, the aromatic, heteroaromatic, heterocyclic or bicyclic ring A being optionally replaced by one or more independently selected from C 1 -C 4 Alkyl, -N(E 1 )(E 2 )、-C(=O)C(E Z ) 3 、-OE 4 , halogen and =O substituents;

[0216] E Y Does not exist, -O-, -OCH 2 -、-CH 2 -、-NE 3 -, or –CH(NH 2 )-;

[0217] Ring E B is a 5- or 6-membered aromatic or heteroaromatic ring having no or 0 to 4 heteroatoms selected from N, O and S, the aromatic or heteroaromatic ring E B Optionally one or more independently selected from C 1 -C 4 Alkyl, -N(E 1 )(E 2 )、-C(=O)C(E Z ) 3 、-OE 4, halogen and =O substituents;

[0218] E 1 、E 2 、E 3 and E 4 are each independently selected from hydrogen or C 1 -C 4 alkyl;

[0219] E Z is a halogen; and

[0220] E 5 Hydroxyl or C 1 -C 4 Alkoxy.

[0221] In one embodiment, the Sortilin binding portion (S L ) is a substituent derived from a compound according to the above formula (E).

[0222] In one aspect, the present invention provides a novel sortilin ligand having high affinity to sortilin. Accordingly, one embodiment of the present disclosure provides a compound according to formula III or a pharmaceutically acceptable salt thereof:

[0223]

[0224] Where Q 1 is a bond or -CH 2 -;and

[0225] R 3 Having formula (IIIp) or formula (IIIq) or formula B-II;

[0226]

[0227] Where R 3b Selected from H, halogen, alkoxy, -CF 3 and optionally substituted C 1-5 Alkyl, where C 1-5 The one or more methylene groups of the alkyl group are optionally replaced individually by -O-, -NH-, -C(O)-, ester, amide, carbamate, thiourea, sulfonamide, urea, optionally substituted carbocyclic ring; optionally substituted heterocyclic ring and One or more groups in the group consisting of: wherein X is NH or O; and

[0228] in * It indicates the connection with formula (III).

[0229] In one embodiment, when R 3When having formula (IIIq), R 3b Not H.

[0230] In one embodiment, Q 1 -CH 2 -. In one embodiment, Q 1 is the key.

[0231] In one embodiment, R 3 Having the formula IIIr:

[0232]

[0233] Where R 3b Selected from H, halogen, alkoxy, -CF 3 and optionally substituted C 1-5 Alkyl, where C 1-5 The one or more methylene groups of the alkyl group are optionally replaced by -O-, -NH-, -C(O)-, ester, amide, carbamate, thiourea, or One or more groups in the group consisting of are replaced; and

[0234] in * It indicates the connection with formula (III).

[0235] In one embodiment of the present disclosure, the compound is selected from Formula IIIs, Formula IIIt or Formula B-III:

[0236]

[0237] Where R 3b Selected from H, halogen, alkoxy, -CF 3 and optionally substituted C 1-5 Alkyl, where C 1-5 The one or more methylene groups of the alkyl group are optionally replaced individually by -O-, -NH-, -C(O)-, ester, amide, carbamate, thiourea, sulfonamide, urea, optionally substituted carbocyclic ring; optionally substituted heterocyclic ring and One or more groups in the group consisting of: wherein X is NH or O.

[0238] In one embodiment, R 3b For H.

[0239] In one embodiment, R 3b Selected from halogen or -CF 3 .

[0240] In one embodiment, R 3b is an optionally substituted C 1-C 5 Alkyl, where C 1-5 The one or more methylene groups of the alkyl group are optionally replaced by -O-, -NH-, -C(O)-, ester, amide, carbamate, thiourea and One or more groups in the group are replaced.

[0241] In one embodiment, at least one methylene group is replaced by -O-, -NH-, -C(O)-, ester, amide, carbamate, thiourea, and One or more groups in the group are replaced.

[0242] In one embodiment, R 3b C 1- C 5 Alkyl, where C 1-5 The one or more methylene groups of the alkyl group are optionally replaced by -O-, amide and One or more groups in the group are replaced.

[0243] In one embodiment, R 3b C 1- C 5 Alkyl, where C 1-5 One or more methylene groups of the alkyl group are optionally replaced individually by -O-.

[0244] In one embodiment, R 3b C 1- C 5 Alkyl, where C 1-5 One or more methylene groups of the alkyl group are optionally replaced individually by an amide.

[0245] In one embodiment, R 3b C 1- C 5 Alkyl, where C 1-5 One or more methylene groups of the alkyl group are optionally replace.

[0246] In one embodiment, R 3b C 1- C 5 Alkyl, where C 1-5 One or more methylene groups of the alkyl group are optionally replaced individually by an optionally substituted carbocyclic ring, for example according to wherein n is an integer selected from 0, 1, 2 or 3.

[0247] In one embodiment, R 3b C 1- C 5 Alkyl, where C1-5 One or more methylene groups of the alkyl group are optionally replaced individually by an optionally substituted heterocycle, for example according to wherein n is an integer selected from 0, 1, 2 or 3; or of heterocyclic ring.

[0248] In one embodiment, R 3b -O-CH 2 -C(=O)N(H)R IIIa , where R IIIa Selected from H and C 1 -C 6 alkyl.

[0249] In one embodiment, R 3b -O-CH 2 -CO 2 R IIIa , where R IIIa Selected from H and C 1 -C 6 alkyl.

[0250] In one embodiment, R 3b C 1- C 5 Alkyl, where C 1-5 One or more methylene groups of the alkyl group are optionally replaced individually with one of the groups shown in Table Z in the "Linker" portion. In one embodiment, the compound is an S-stereoisomer based on the configuration of the carbon alpha to the carboxylic acid.

[0251] In one embodiment, the compound is the R-stereoisomer based on the configuration of the carbon alpha to the carboxylic acid.

[0252] In one embodiment, the present disclosure provides compounds:

[0253]

[0254] In another embodiment, the present disclosure provides the following compounds:

[0255]

[0256]

[0257] or a pharmaceutically acceptable salt thereof.

[0258] In one embodiment, the present disclosure provides compounds:

[0259]

[0260] or a pharmaceutically acceptable salt thereof.

[0261] In one aspect, the present disclosure provides a compound according to any one of the following:

[0262] or a pharmaceutically acceptable salt thereof.

[0263] The present invention demonstrates several bifunctional compounds capable of binding to sortilin. As shown in the Examples, these compounds have affinity for sortilin. Those skilled in the art will appreciate that the compounds comprising S L and L I A fragment of a bifunctional compound containing part or all of the compound will also be used as a sortilin binder. Therefore, in one embodiment, the present disclosure provides a S as described below L -L I or any combination of fragments thereof.

[0264] In one embodiment, the present invention provides a compound according to any one of formula IIIt or B-III, wherein R 3b is a fragment of any of the structures shown in Table Z-III of the "Linker" section.

[0265] In addition, the present disclosure provides a compound capable of binding to sortilin, wherein the compound is an intermediate for preparing a bifunctional compound as described herein in the "Synthetic Schemes" section.

[0266] Peptides

[0267] Peptides have also been shown to bind to sortilin. For example, the neuropeptide neurotensin (SEQ ID NO.: 4) and fragments thereof, such as the 4 amino acid C-terminal neurotensin fragment (SEQ ID NO.: 5) or the amidated 6 amino acid N-terminal neurotensin fragment (SEQ ID NO.: 6). Smaller peptide fragments are also able to bind to sortilin, such as the C-terminal sequence of progranulin (SEQ ID NO.: 3), a natural sortilin binder, which has been shown to play a crucial role in the binding of progranulin to sortilin.

[0268] Other examples of synthetic peptides have been used to target sortilin, such as the peptides described in WO 2020 / 037434, for example:

[0269] IKLSGGVQAKAGVINMDKSESM(SEQ ID NO.:7)

[0270] IKLSGGVQAKAGVINMFKSESY(SEQ ID NO.:8)

[0271] IKLSGGVQAKAGVINMFKSESYK(SEQ ID NO.:9)

[0272] GVQAKAGVINMFKSESY(SEQ ID NO.:10)

[0273] GVRAKAGVRNMFKSESY(SEQ ID NO.:11)

[0274] GVRAKAGVRN(Nle)FKSESY(SEQ ID NO.:12)

[0275] YKSLRRKAPRWDAPLRDPALRQLL(SEQ ID NO.:13)

[0276] YKSLRRKAPRWDAYLRDPALRQLL(SEQ ID NO.:14)

[0277] YKSLRRKAPRWDAYLRDPALRPLL(SEQ ID NO.:15)

[0278] According to the bifunctional compound of the present invention, the sortilin binding group (S L ) may also be a peptide.

[0279] There are several proteins that have affinity for sortilin. Therefore, in one embodiment of the present disclosure, the sortilin binding group (S L ) is a peptide fragment comprising the amino acid sequence of this protein.

[0280] In one embodiment, the sortilin binding group comprises a peptide fragment of the amino acid sequence of a protein selected from the group consisting of:

[0281] a) progranulin (SEQ ID NO.: 3),

[0282] b) neurotensin (SEQ ID NO.: 4),

[0283] c) Brain-derived neurotrophic factor (BDNF) (SEQ ID NO.: 177)

[0284] d) Apolipoprotein B (ApoB) (SEQ ID NO.: 176)

[0285] e) Nerve Growth Factor (NGF) (SEQ ID NO.: 178)

[0286] or a variant of said fragment, which has at least 60% sequence identity, such as at least 80%, such as at least 90%, such as at least 95% sequence identity with the corresponding original protein fragment of any one of a) to e).

[0287] In one embodiment, S L A peptide fragment comprising the amino acid sequence of progranulin (SEQ ID NO.: 3) or a variant of said fragment or consisting thereof, wherein the variant of said fragment has at least 60% sequence identity, such as at least 80% sequence identity, such as at least 90%, such as at least 95%, such as at least 99% sequence identity with the corresponding original fragment of SEQ ID NO.: 3, or consists thereof.

[0288] In one embodiment, S L A peptide fragment comprising the amino acid sequence of progranulin (SEQ ID NO.: 3) or a variant of said fragment or consisting thereof, wherein the variant of said fragment has up to 5 amino acid substitutions, such as up to 4 amino acid substitutions, such as up to 3 amino acid substitutions, such as up to 2 amino acid substitutions, such as up to 1 amino acid substitution compared to the corresponding original fragment of SEQ ID NO.: 3.

[0289] In one embodiment, S L A peptide fragment comprising the amino acid sequence of neurotensin (SEQ ID NO.: 4) or a variant of said fragment or consisting thereof, wherein the variant of said fragment has at least 60% sequence identity, such as at least 80% sequence identity, such as at least 90%, such as at least 95%, such as at least 99% sequence identity with the corresponding original fragment of SEQ ID NO.: 4.

[0290] In one embodiment, S L A peptide fragment comprising the amino acid sequence of neurotensin (SEQ ID NO.: 4) or a variant of said fragment or consisting thereof, wherein the variant of said fragment has up to 5 amino acid substitutions, such as up to 4 amino acid substitutions, such as up to 3 amino acid substitutions, such as up to 2 amino acid substitutions, such as up to 1 amino acid substitution compared to the corresponding original fragment of SEQ ID NO.: 4.

[0291] In one embodiment, S L A peptide fragment comprising the amino acid sequence of ApoB (SEQ ID NO.: 176) or a variant of said fragment or consisting thereof, wherein the variant of said fragment has at least 60% sequence identity with the corresponding original fragment of SEQ ID NO.: 176, such as at least 80% sequence identity, such as at least 90%, such as at least 95%, such as at least 99% sequence identity.

[0292] In one embodiment, S L A peptide fragment comprising the amino acid sequence of ApoB (SEQ ID NO.: 176) or a variant of said fragment or consisting thereof, wherein the variant of said fragment has up to 5 amino acid substitutions, such as up to 4 amino acid substitutions, such as up to 3 amino acid substitutions, such as up to 2 amino acid substitutions, such as up to 1 amino acid substitution compared to the corresponding original fragment of SEQ ID NO.: 176.

[0293] In one embodiment, S L A peptide fragment comprising the amino acid sequence of BDNF (SEQ ID NO.: 177) or a variant of said fragment or consisting thereof, wherein the variant of said fragment has at least 60% sequence identity with the corresponding original fragment of SEQ ID NO.: 177, such as at least 80% sequence identity, such as at least 90%, such as at least 95%, such as at least 99% sequence identity.

[0294] In one embodiment, S L A peptide fragment comprising the amino acid sequence of BDNF (SEQ ID NO.: 177) or a variant of said fragment or consisting thereof, wherein the variant of said fragment has up to 5 amino acid substitutions, such as up to 4 amino acid substitutions, such as up to 3 amino acid substitutions, such as up to 2 amino acid substitutions, such as up to 1 amino acid substitution compared to the corresponding original fragment of SEQ ID NO.: 177.

[0295] In one embodiment, S L A peptide fragment comprising the amino acid sequence of NGF (SEQ ID NO.: 178) or a variant of the fragment or consisting thereof, wherein the variant of the fragment has at least 60% sequence identity with the corresponding original fragment of SEQ ID NO.: 178, for example, at least 80% sequence identity, for example, at least 90%, for example, at least 95%, for example, at least 99% sequence identity.

[0296] In one embodiment, S L A peptide fragment comprising the amino acid sequence of NGF (SEQ ID NO.: 178) or a variant of the fragment or consisting thereof, wherein the variant of the fragment has up to 5 amino acid substitutions compared with the corresponding original fragment of SEQ ID NO.: 178, for example, up to 4 amino acid substitutions, for example, up to 3 amino acid substitutions, for example, up to 2 amino acid substitutions, for example, up to 1 amino acid substitution.

[0297] In one embodiment, SL is a peptide comprising a sequence selected from:

[0298] H-PYMKLAPGELTIIL-OH (SEQ ID NO.:44),

[0299] H-NEKLSQLQTYMI-OH(SEQ ID NO.:45),

[0300] H-KDADLYTSRVMLSSQVP-OH (SEQ ID NO.:46),

[0301] H-RLFKRRLRRSPRVLF-NH2 (SEQ ID NO.:47),

[0302] H-ITVDPRLFKRRLRRSPRVLF-NH2 (SEQ ID NO.: 48),

[0303] H-ITVDPRLFKRRLRRSPRVLFSTQPPR-OH (SEQ ID NO.:49),

[0304] H-WSGPIGVSWGLRAAAAGGAFP-OH (SEQ ID NO.:50),

[0305] H-WSGPIGVSWGLRAAAAGGAFPRGGRWRR-OH (SEQ ID NO.:51), H-GVSWGLR-OH (SEQ ID NO.:52),

[0306] WSGPIGVSWGLRAAAAGFQLL-OH (SEQ ID NO.:179).

[0307] When S L When comprising or consisting of a peptide, L I Can be connected to any amino acid residue in this peptide. In one embodiment, when S L When peptide is included, L I Attach to any free amino group of the peptide.

[0308] In a preferred embodiment, when S L When comprising or consisting of a peptide, L I Attached to the N-terminus of the peptide.

[0309] The present inventors have demonstrated that the C-terminal tetrapeptide RQLL (SEQ ID NO.: ) of progranulin is sufficient to bind sortilin and promote lysosomal-mediated degradation of progranulin. In addition, the present inventors have demonstrated that several peptide analogs with selected substitutions on the RQLL sequence are good sortilin binders.

[0310] Therefore, in one embodiment, SL Comprising or consisting of a peptide, the peptide comprising the following sequence SEQ ID NO.: 16

[0311] X 5 -X 1 X 2 X 3 X 4 (SEQ ID NO.: 16)

[0312] in,

[0313] X 5 is an optional amino acid residue or a peptide comprising 2 to 30 amino acid residues or a bond,

[0314] X 1 is R, P, F, Y, L, K, G or H;

[0315] X 2 is Q, Y, L, E or G;

[0316] X 3 is Y, F, L, I, Q, E or N; and

[0317] X 4 is M, K, L or a conservative substitution of L.

[0318] In one embodiment, X 5 is an optional amino acid residue or a peptide comprising at least 2 amino acid residues, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 11, such as at least 12, such as at least 13, such as at least 14, such as at least 15, such as at least 20, such as at least 25 amino acid residues, such as at least 28 amino acid residues, such as at least 30 amino acid residues.

[0319] In one embodiment, X 5 A peptide comprising no more than 32 amino acid residues, such as no more than 29, such as no more than 28, such as no more than 27, such as no more than 26, such as no more than 25, such as no more than 24, such as no more than 23, such as no more than 22, such as no more than 21, such as no more than 20, such as no more than 15, such as no more than 10, such as no more than 5 amino acid residues.

[0320] In one embodiment, X 5 A peptide fragment of the amino acid sequence of a protein selected from the group consisting of:

[0321] a) progranulin (SEQ ID NO.: 3),

[0322] b) neurotensin (SEQ ID NO.: 4),

[0323] c) Brain-derived neurotrophic factor (BDNF) (SEQ ID NO.: 177)

[0324] d) Apolipoprotein B (ApoB) (SEQ ID NO.: 176)

[0325] e) Nerve Growth Factor (SEQ ID NO.: 178)

[0326] or a variant of said fragment, which has at least 60% sequence identity, such as at least 80%, such as at least 90%, such as at least 95% sequence identity with the corresponding original protein fragment of any one of a) to e).

[0327] In one embodiment, X 5 A peptide fragment comprising the amino acid sequence of progranulin (SEQ ID NO.: 3) or a variant of said fragment or consisting thereof, wherein the variant of said fragment has at least 60% sequence identity, such as at least 80% sequence identity, such as at least 90%, such as at least 95%, such as at least 99% sequence identity with the corresponding original fragment of SEQ ID NO.: 3.

[0328] In one embodiment, X 5 A peptide fragment comprising the amino acid sequence of progranulin (SEQ ID NO.: 3) or a variant of said fragment or consisting thereof, wherein the variant of said fragment has up to 5 amino acid substitutions, such as up to 4 amino acid substitutions, such as up to 3 amino acid substitutions, such as up to 2 amino acid substitutions, such as up to 1 amino acid substitution compared to the corresponding original fragment of SEQ ID NO.: 3.

[0329] In one embodiment, X 5 A peptide fragment comprising the amino acid sequence of neurotensin (SEQ ID NO.: 4) or a variant of said fragment or consisting thereof, wherein the variant of said fragment has at least 60% sequence identity, such as at least 80% sequence identity, such as at least 90%, such as at least 95%, such as at least 99% sequence identity with the corresponding original fragment of SEQ ID NO.: 4.

[0330] In one embodiment, X 5A peptide fragment comprising the amino acid sequence of neurotensin (SEQ ID NO.: 4) or a variant of said fragment or consisting thereof, wherein the variant of said fragment has up to 5 amino acid substitutions, such as up to 4 amino acid substitutions, such as up to 3 amino acid substitutions, such as up to 2 amino acid substitutions, such as up to 1 amino acid substitution compared to the corresponding original fragment of SEQ ID NO.: 4.

[0331] In one embodiment, X5 is selected from:

[0332] an amino acid residue selected from L, T or Y;

[0333] REAPRWDAPLRDPAL(SEQ ID NO.:17);

[0334] REALRWDAPLRDPAP(SEQ ID NO.:18);

[0335] PYILKRQLYENKPRR(SEQ ID NO.:19);

[0336] LYENKPR (SEQ ID NO.: 20); and

[0337] APLRDPAP (SEQ ID NO.: 21);

[0338] WSGPIGVSWGLRAAAAG(SEQ ID NO.:180);

[0339] CREAPRWDAPLRDPAL(SEQ ID NO.:181),

[0340] or a variant thereof having 1 to 5 amino acid substitutions.

[0341] In one embodiment, X 5 is the key.

[0342] In one embodiment, X 5 is APLRDPAP (SEQ ID NO.: 21). In one embodiment, X 5 is REAPRWDAPLRDPAL (SEQ ID NO.: 17). In one embodiment, X 5 is REALRWDAPLRDPAP (SEQ ID NO.:18).

[0343] In one embodiment, X 5 X is an amino acid residue selected from L, T or Y. In one embodiment, X 5is PYILKRQLYENKPRR (SEQ ID NO.: 19). In one embodiment, X 5 It is LYENKPR (SEQ ID NO.: 20).

[0344] In one embodiment, X 1 is F or R. In one embodiment, X 2 is Q. In another embodiment, X 3 is L. In one embodiment, X 4 For L.

[0345] In one embodiment,

[0346] X 1 is selected from Y, F, R, P, L or H,

[0347] X 2 Selected from Q, Y or L,

[0348] X 3 is selected from Y, F, L, I or Q,

[0349] X 4 For L.

[0350] In one embodiment,

[0351] X 1 is selected from Y, F, R or P,

[0352] X 2 Select from Q or Y,

[0353] X 3 is selected from L, Y, F or I,

[0354] X 4 For L.

[0355] In one embodiment,

[0356] X 1 is selected from Y, F or R,

[0357] X 2 Select from Q or Y,

[0358] X 3 For L,

[0359] X 4 For L.

[0360] In one embodiment,

[0361] X 1 Selected from F or R,

[0362] X2 For Q,

[0363] X 3 For L,

[0364] X 4 For L.

[0365] In one embodiment, S L comprising or consisting of a sequence selected from any one of SEQ ID NOs: 22 to 65, 182 to 187, 196 and 197, wherein L I Connected to the N-terminus.

[0366] In one embodiment, S L A peptide comprising any of the sortilin binding sequences described herein at the C-terminus.

[0367] In one embodiment, S L is a peptide consisting of RQLL-OH (SEQ ID NO.: 22). In one embodiment, S L is a peptide comprising RQLL-OH (SEQ ID NO.: 22) at the C-terminus.

[0368] In one embodiment, S L is a peptide consisting of FQLL-OH (SEQ ID NO.: 23). In one embodiment, S L is a peptide comprising FQLL-OH (SEQ ID NO.: 23) at the C-terminus.

[0369] In one embodiment, S L is a peptide consisting of RYLL-OH (SEQ ID NO.: 27). In one embodiment, S L is a peptide comprising RYLL-OH (SEQ ID NO.: 27) at the C-terminus.

[0370] In one embodiment, S L is a peptide consisting of FYLL-OH (SEQ ID NO.: 28). In one embodiment, S L is a peptide comprising FYYL-OH (SEQ ID NO.: 28) at the C-terminus.

[0371] In one embodiment, S L is a peptide consisting of YQLL-OH (SEQ ID NO.: 29). In one embodiment, S L It is a peptide comprising YQLL-OH (SEQ ID NO.: 29) at the C-terminus.

[0372] In one embodiment, S L is a peptide consisting of REAPRWDAPLRDPALRQLL-OH (SEQ ID NO.: 53). In one embodiment, S L It is a peptide comprising REAPRWDAPLRDPALRQLL-OH (SEQ ID NO.: 53) at the C-terminus.

[0373] In one embodiment, S L is a peptide consisting of REALRWDAPLRDPAPRQLL-OH (SEQ ID NO.: 54). In one embodiment, S L It is a peptide comprising REALRWDAPLRDPAPRQLL-OH (SEQ ID NO.: 54) at the C-terminus.

[0374] In one embodiment, S L is a peptide consisting of REAPRWDAPLRDPALFQLL-OH (SEQ ID NO.: 58). In one embodiment, S L It is a peptide comprising REAPRWDAPLRDPALFQLL-OH (SEQ ID NO.: 58) at the C-terminus.

[0375] In one embodiment, S L is a peptide consisting of REAPRWDAPLRDPALRYLL-OH (SEQ ID NO.: 59). In one embodiment, S L It is a peptide comprising REAPRWDAPLRDPALRYLL-OH (SEQ ID NO.: 59) at the C-terminus.

[0376] In one embodiment, S L is a peptide consisting of REAPRWDAPLRDPALRQYL-OH (SEQ ID NO.: 60). In one embodiment, S L is a peptide comprising REAPRWDAPLRDPALRQYL-OH (SEQ ID NO.: 60) at the C-terminus.

[0377] In one embodiment, S L is a peptide consisting of REAPRWDAPLRDPALRYYL-OH (SEQ ID NO.: 61). In one embodiment, S L It is a peptide comprising REAPRWDAPLRDPALRYYL-OH (SEQ ID NO.: 61) at the C-terminus.

[0378] In one embodiment, S L is a peptide consisting of APLRDPAPRQLL-OH (SEQ ID NO.: 57). In one embodiment, S L It is a peptide comprising APLRDPAPRQLL-OH (SEQ ID NO.: 57) at the C-terminus.

[0379] In one embodiment, S L is a peptide consisting of PYILKRQLYENKPRRPYIL-OH (SEQ ID NO.: 55). In one embodiment, S L It is a peptide comprising PYILKRQLYENKPRRPYIL-OH (SEQ ID NO.: 55) at the C-terminus.

[0380] In one embodiment, S L is a peptide consisting of LYENKPRRPYIL-OH (SEQ ID NO.: 56). In one embodiment, S L It is a peptide comprising LYENKPRRPYIL-OH (SEQ ID NO.: 56) at the C-terminus.

[0381] In one embodiment, S L is a peptide consisting of AARL-OH (SEQ ID NO.: 196). In one embodiment, S L is a peptide comprising AARL-OH (SEQ ID NO.: 196) at the C-terminus.

[0382] In one embodiment, S L is a peptide consisting of PIPLV-OH (SEQ ID NO.: 197). In one embodiment, S L It is a peptide comprising PIPLV-OH (SEQ ID NO.: 197) at the C-terminus.

[0383] In a preferred embodiment, L I At the N-terminus, it is linked to S L .

[0384] In one embodiment, S L 1 to 4 amino acid residues are substituted by other amino acids, for example, one amino acid is substituted, for example, 2, for example, 3, for example, 4 amino acids.

[0385] In one embodiment, S L In another embodiment, S L Two amino acid residues were substituted.

[0386] In one embodiment, the substitution is a conservative amino acid substitution. In another embodiment, the substitution is made with a non-naturally occurring amino acid. Thus, in one embodiment, S L Can have 1 substitution with 1 non-naturally occurring amino acid. In another embodiment, S L There can be two substitutions with non-naturally occurring amino acids.

[0387] In one embodiment, S L 1 to 4 amino acid residues are chemically modified, for example 1 amino acid residue is modified, for example 2, for example 3, for example 4 amino acids.

[0388] In one embodiment, the chemical modification can be any chemical modification. In one embodiment, the chemical modification is selected from acylation, amidation, acetylation, esterification and / or alkylation. L Can have 1 chemically modified amino acid. In another embodiment, S L Can have 2 chemically modified amino acids.

[0389] In one embodiment, S L A peptide comprising no more than 50 amino acid residues, such as no more than 45, such as no more than 40, such as no more than 35, such as no more than 32, such as no more than 30, such as no more than 28, such as no more than 26, such as no more than 24, such as no more than 22, such as no more than 20, such as no more than 19, such as no more than 18, such as no more than 17, such as no more than 16, such as no more than 15, such as no more than 14, such as no more than 13, such as no more than 12, such as no more than 11, such as no more than 10, such as no more than 9, such as no more than 8, such as no more than 7, such as no more than 6, such as no more than 5, such as no more than 4 amino acid residues.

[0390] In one embodiment, S L is a peptide comprising no more than 28 amino acid residues. In another embodiment, S L A peptide comprising no more than 4 amino acid residues.

[0391] In one embodiment, S LThe invention relates to a polypeptide comprising or consisting of a peptide of at least 4 amino acid residues, for example at least 5, for example at least 6, for example at least 7, for example at least 8, for example at least 9, for example at least 10, for example at least 12, for example at least 14, for example at least 16, for example at least 18, for example at least 20, for example at least 22, for example at least 24, for example at least 26, for example at least 28 amino acid residues.

[0392] In one embodiment, S L The invention comprises or consists of a peptide having a length of 4 to 50 amino acids, for example 4 to 32 amino acids, for example 4 to 28 amino acids, for example 4 to 24 amino acids, for example 4 to 20 amino acids, for example 4 to 19 amino acids, for example 4 to 18, for example 4 to 17 amino acids, for example 4 to 16 amino acids, for example 4 to 15 amino acids, for example 4 to 14 amino acids, for example 4 to 13 amino acids, for example 4 to 12 amino acids, for example 4 to 11 amino acids, for example 4 to 10 amino acids, for example 4 to 9 amino acids, for example 4 to 8 amino acids, for example 4 to 7 amino acids, for example 4 to 6 amino acids, for example 4 to 5 amino acids.

[0393] In one embodiment, S L In one embodiment, S L In one embodiment, S L In one embodiment, S L In one embodiment, S L In one embodiment, S L In one embodiment, S L In one embodiment, S L In one embodiment, S L It comprises or consists of a peptide, wherein the peptide has a length of 12 amino acid residues.

[0394] In one embodiment, S LThe invention relates to a peptide which can bind to sortilin with a dissociation constant of less than 50 μM, such as less than 40 μM, such as less than 30 μM, such as less than 20 μM, such as less than 10 μM, such as less than 5 μM, such as less than 4 μM, such as less than 3 μM, such as less than 2 μM, such as less than 1 μM, such as less than 0.8 μM, such as less than 0.6 μM, such as less than 0.5 μM, such as less than 0.4 μM, such as less than 0.3 μM, such as less than 0.2 μM, such as less than 0.1 μM, such as less than 0.05 μM, such as less than 0.04 μM, such as less than 0.03 μM, such as less than 0.02 μM, such as less than 0.01 μM.

[0395] One aspect of the present invention is to provide a sortilin binding peptide. In one embodiment, the peptide is a peptide according to the S disclosed herein. L .

[0396] One aspect of the present invention is to provide a sortilin binding peptide. In one embodiment, the sortilin binding peptide comprises the following sequence:

[0397] X 5 -X 1 X 2 X 3 X 4 (SEQ ID NO.: 16)

[0398] Where X 1 , X 2 , X 3 , X 4 and X 5 The definition of is as described herein.

[0399] In one embodiment, the peptide is a peptide of 4 to 50 amino acids in length, for example 4 to 32 amino acids, for example 4 to 28 amino acids, for example 4 to 24 amino acids, for example 4 to 20 amino acids, for example 4 to 18 amino acids, for example 4 to 14 amino acids, for example 4 to 12 amino acids, for example 4 to 11 amino acids, for example 4 to 10 amino acids, for example 4 to 9 amino acids, for example 4 to 8 amino acids, for example 4 to 7 amino acids, for example 4 to 6 amino acids, for example 4 to 5 amino acids.

[0400] In one embodiment, the peptide is a peptide consisting of RQLL-OH (SEQ ID NO.: 22). In one embodiment, the peptide is a peptide comprising RQLL-OH (SEQ ID NO.: 22) at the C-terminus.

[0401] In one embodiment, the peptide is a peptide consisting of FQLL-OH (SEQ ID NO.: 23). In one embodiment, the peptide is a peptide comprising FQLL-OH (SEQ ID NO.: 23) at the C-terminus.

[0402] In one embodiment, the peptide is a peptide consisting of RYLL-OH (SEQ ID NO.: 27). In one embodiment, the peptide is a peptide comprising RYLL-OH (SEQ ID NO.: 27) at the C-terminus.

[0403] In one embodiment, the peptide is a peptide consisting of FYLL-OH (SEQ ID NO.: 28). In one embodiment, the peptide is a peptide comprising FYYL-OH (SEQ ID NO.: 28) at the C-terminus.

[0404] In one embodiment, the peptide is a peptide consisting of YQLL-OH (SEQ ID NO.: 29). In one embodiment, the peptide is a peptide comprising YQLL-OH (SEQ ID NO.: 29) at the C-terminus.

[0405] In one embodiment, the peptide is a peptide consisting of REAPRWDAPLRDPALRQLL-OH (SEQ ID NO.: 53). In one embodiment, the peptide is a peptide comprising REAPRWDAPLRDPALRQLL-OH (SEQ ID NO.: 53) at the C-terminus.

[0406] In one embodiment, the peptide is a peptide consisting of REALRWDAPLRDPAPRQLL-OH (SEQ ID NO.: 54). In one embodiment, the peptide is a peptide comprising REALRWDAPLRDPAPRQLL-OH (SEQ ID NO.: 54) at the C-terminus.

[0407] In one embodiment, the peptide is a peptide consisting of REAPRWDAPLRDPALFQLL-OH (SEQ ID NO.: 58). In one embodiment, the peptide is a peptide comprising REAPRWDAPLRDPALFQLL-OH (SEQ ID NO.: 58) at the C-terminus.

[0408] In one embodiment, the peptide is a peptide consisting of REAPRWDAPLRDPALRYLL-OH (SEQ ID NO.: 59). In one embodiment, the peptide is a peptide comprising REAPRWDAPLRDPALRYLL-OH (SEQ ID NO.: 59) at the C-terminus.

[0409] In one embodiment, the peptide is a peptide consisting of REAPRWDAPLRDPALRQYL-OH (SEQ ID NO.: 60). In one embodiment, the peptide is a peptide comprising REAPRWDAPLRDPALRQYL-OH (SEQ ID NO.: 60) at the C-terminus.

[0410] In one embodiment, the peptide is a peptide consisting of REAPRWDAPLRDPALRYYL-OH (SEQ ID NO.: 61). In one embodiment, the peptide is a peptide comprising REAPRWDAPLRDPALRYYL-OH (SEQ ID NO.: 61) at the C-terminus.

[0411] In one embodiment, the peptide is a peptide consisting of APLRDPAPRQLL-OH (SEQ ID NO.: 57). In one embodiment, the peptide is a peptide comprising APLRDPAPRQLL-OH (SEQ ID NO.: 57) at the C-terminus.

[0412] In one embodiment, the peptide is a peptide consisting of PYILKRQLYENKPRRPYIL-OH (SEQ ID NO.: 55). In one embodiment, the peptide is a peptide comprising PYILKRQLYENKPRRPYIL-OH (SEQ ID NO.: 55) at the C-terminus.

[0413] In one embodiment, the peptide is a peptide consisting of LYENKPRRPYIL-OH (SEQ ID NO.: 56). In one embodiment, the peptide is a peptide comprising LYENKPRRPYIL-OH (SEQ ID NO.: 56) at the C-terminus.

[0414] In one embodiment, the peptides according to the present disclosure can be expressed as peptides with a dissociation constant (K) of less than 50 μM, such as less than 40 μM, such as less than 30 μM, such as less than 20 μM, such as less than 10 μM, such as less than 5 μM, such as less than 4 μM, such as less than 3 μM, such as less than 2 μM, such as less than 1 μM, such as less than 0.8 μM, such as less than 0.6 μM, such as less than 0.5 μM, such as less than 0.4 μM, such as less than 0.3 μM, such as less than 0.2 μM, such as less than 0.1 μM, such as less than 0.05 μM, such as less than 0.04 μM, such as less than 0.03 μM, such as less than 0.02 μM, such as less than 0.01 μM. D ) binds to sortilin.

[0415] In one aspect, the present disclosure provides an isolated polynucleotide encoding a peptide or protein described herein.

[0416] In one aspect, the disclosure provides a vector comprising a polynucleotide described herein. In one embodiment, the vector is thus an expression vector, such as a bacterial vector or a viral vector.

[0417] In one aspect, the present disclosure provides a host cell comprising a polynucleotide and / or a vector described herein.

[0418] protein

[0419] The bifunctional compounds according to the present invention can be prepared by using any antibody or antibody fragment capable of binding to sortilin. L It can be selected from antibodies or antibody fragments that are able to bind to Sortilin.

[0420] Antibodies or antibody fragments having the ability to bind to sortilin have been disclosed, as described in WO 2017 / 009327, WO 2019 / 016247, WO 2021 / 263279, WO 2021 / 116290, WO 2020 / 252066, WO 2020 / 014617, US2017 / 0218058 or WO 2016 / 164637, the contents of which are incorporated herein by reference.

[0421] Therefore, in one embodiment, S L is selected from one of the antibodies or antibody fragments described in the above disclosure that are capable of binding to sortilin. L is selected from the antibodies or antibody fragments described in the above disclosure, and L I and / or T L The selection is as described herein.

[0422] In one embodiment, S L It is an antibody, antibody fragment or nanobody that is able to bind to sortilin.

[0423] In one embodiment, S L It is an antibody, antibody fragment or Nanobody capable of binding to Sortilin with a dissociation constant of at least 50 μM, such as at least 2 μM, such as at least 0.1 μM.

[0424] As indicated above, there are many examples of Sortilin binding agents suitable for preparing bifunctional compounds according to the present disclosure. Thus, in one embodiment, S in the bifunctional compound L The group is selected from any one of the above-mentioned structures, peptides and proteins.

[0425] In one embodiment, the bifunctional compounds according to the present disclosure can be synthesized with a dissociation constant (K) of less than 50 μM, such as less than 40 μM, such as less than 30 μM, such as less than 20 μM, such as less than 10 μM, such as less than 5 μM, such as less than 4 μM, such as less than 3 μM, such as less than 2 μM, such as less than 1 μM, such as less than 0.8 μM, such as less than 0.6 μM, such as less than 0.5 μM, such as less than 0.4 μM, such as less than 0.3 μM, such as less than 0.2 μM, such as less than 0.1 μM, such as less than 0.05 μM, such as less than 0.04 μM, such as less than 0.03 μM, such as less than 0.02 μM, such as less than 0.01 μM. D ) binds to sortilin.

[0426] In one embodiment, the bifunctional compounds according to the present disclosure can be present in an amount of 50 μM to 0.001 μM, such as 50 μM to 40 μM, such as 40 μM to 30 μM, such as 30 μM to 20 μM, such as 20 μM to 10 μM, such as 10 μM to 5 μM, such as 5 μM to 4 μM, such as 4 μM to 3 μM, such as 3 μM to 2 μM, such as 2 μM to 1 μM, such as 1 μM to 0.9 μM, such as The dissociation constant (K) of 0.9 μM to 0.8 μM, for example 0.8 μM to 0.7 μM, for example 0.7 μM to 0.6 μM, for example 0.5 μM to 0.4 μM, for example 0.4 μM to 0.3 μM, for example 0.3 μM to 0.2 μM, for example 0.2 μM to 0.1 μM, for example 0.1 μM to 0.05 μM, for example 0.05 μM to 0.01 μM, for example 0.01 μM to 0.001 μM D ) binds to sortilin.

[0427] Targeted Warhead:

[0428] The bifunctional compounds according to the present disclosure comprise a portion capable of binding to an extracellular target molecule or protein, such as growth factors, cytokines, hormones, lipoproteins, neurotransmitters, capsids, extracellular secretory proteins, antibodies.

[0429] In one embodiment, the extracellular target molecule is a protein. As described herein, an extracellular protein refers to a protein that is not completely enclosed within a cell. This refers to, for example, a protein that is completely outside the cell, but also includes membrane-bound or membrane-associated proteins that have an extracellular domain.

[0430] The bifunctional compound according to formula (I) is obtained by binding the target ligand (T L ) binds to an extracellular target molecule. In one embodiment, T LIt is a substituent of a small organic molecule (i.e., a non-biological molecule) that sufficiently binds to a target molecule, or a substituent derived from a pharmaceutically active compound that binds to a target extracellular protein, or a peptide, protein, or biologic or binding fragment thereof that sufficiently binds to an extracellular target molecule.

[0431] T L The moiety may be, for example, but not limited to, a substituent derived from an approved or clinical stage drug, or a substituent derived from a compound to be reviewed as a drug by a regulatory agency (eg, the FDA or EMA).

[0432] The extracellular target protein may be a protein comprising T L Any amino acid sequence to which the bifunctional compound can bind, producing a beneficial therapeutic effect through its degradation. In one embodiment, the target protein is a non-endogenous peptide, such as a non-endogenous peptide from a pathogen or toxin. In another embodiment, the target protein can be an endogenous protein that mediates a disease. The endogenous protein can be a normal form of the protein or an abnormal form of the protein. For example, the target protein can be an extracellular mutant protein, or a protein in which a partial or complete gain of function or loss of function is encoded by a nucleotide polymorphism, for example. In some embodiments, the bifunctional compound targets an abnormal form of the protein rather than the normal form of the protein.

[0433] According to the targeting ligand (T L ) moiety is a ligand that covalently or non-covalently binds to a target protein that has been selected for lysosomal degradation. A targeting ligand is a small molecule or moiety (e.g., a peptide, nucleotide, antibody fragment, aptamer, biomolecule, or other chemical structure) that binds to a target protein that is a mediator of disease in a host, as described in detail below.

[0434] Many of the exemplary extracellular proteins targeted for medical therapy described below have characteristic structural information in the well-known Protein Data Bank ("PDB"), which is a database of three-dimensional structural information of large biological molecules such as proteins and nucleic acids. The PDB includes x-ray crystallographic and other information submitted by scientists around the world and can be accessed free of charge. For example, see www.rcsb.org: wwwrwwpdb.org and www.uniprot.org for the codes provided below.

[0435] For example, one skilled in the art can use available visualization tools, including those available on the PDB website, to determine T L The docking position of the extracellular protein. Those skilled in the art can also compare the crystal structure and the selected target T LThe bifunctional compound T is then imported into modeling software (including, for example, PyMOL, Glide, Maestro, RasMol, Visual Molecular Dynamics, Jrnol, and AutoDock) to determine which part of the extracellular protein targeting ligand binds to the extracellular protein. L The linker (L) is attached at a point that does not unduly adversely affect binding to extracellular proteins. I ) or sortilin binding group (S L )combined.

[0436] Non-limiting examples of extracellular proteins:

[0437] In one embodiment, the extracellular target protein is one selected from the group consisting of PCSK9, TNF-α, ANGPTL-3, antibody light chain, IgG, IgE, IgA IL-1, IL-2, IL-6, IFN-γ, VEGF, TFG-β1, IL-21, IL-22, IL-5, IL-10, IL-8, cholinesterase, human CCL2, carboxypeptidase B-2, neutrophil elastase, factor Xa, factor XI, factor XIa, factor XII, factor XIII, prothrombin, coagulation factor VII, coagulation factor IX, fibroblast growth factor 1, FGF-2, fibronectin 1 (fibronectin 1), kallikrein-1, lipoprotein lipase, human matrix metallopeptidase 1, macrophage migration inhibitory factor, transforming growth factor-p (TGF-p), thrombospondin-1 (TSP-T), CD40 ligand, urokinase-type plasminogen activator, tissue-type plasminogen activator (TPA), plasminogen (PLG), plasminogen activator inhibitor-1, placental growth factor, phospholipase A2 group IB, phospholipase A2 group IIA, complement factor B, complement factor D, complement factor H, complement component 5, and complement C1s.

[0438] Immunoglobulin G (IgG)

[0439] In some embodiments, the target protein is human immunoglobulin G (IgG). IgG accounts for about 75% of human serum antibodies. IgG is the most common type of antibody in the blood circulation. IgG antibodies are large globular proteins with a molecular weight of about 150kDa composed of four peptide chains. It contains two identical g (γ) heavy chains of about 50kDa and two identical light chains of about 25kDa, so it is a tetrameric quaternary structure. The two heavy chains are connected to each other and each to the light chain by a disulfide bond. The resulting tetramer has two identical halves, which together form a Y-like shape. Each end of the fork contains the same antigen binding site. The various regions and domains of a typical IgG are shown on the left in the figure. The Fc region of IgG has a highly conserved N-glycosylation site at asparagine 297 in the heavy chain constant region. The N-glycans attached to this site are mainly complex types of core fucosyl biantennary structures. In addition, a small amount of these N-glycans also have bisecting GlcNAc and a-2,6-linked sialic acid residues. The N-glycan composition of IgG has been implicated in several autoimmune, infectious, and metabolic diseases. In addition, overexpression of IgG4 is associated with IG4-related diseases (which often involve multiple organs) and conditions, including type 1 autoimmune pancreatitis, interstitial nephritis, Riedel's thyroiditis, Mikulicz's disease, Kuttner's tumor, inflammatory'pseudotumors (in various parts of the body), mediastinal fibrosis and some cases of retroperitoneal fibrosis, aortitis, retroperitoneal fibrosis, proximal biliary strictures, tubulointerstitial nephritis, pachymeningitis, pancreatic enlargement, and pericarditis.

[0440] The protein database website provides IgG crystal structures that can be searched through 1H3X (Krapp, S., et al., J. Mol. Biol., 2003, 325:979); and 5V43 (Lee, CH, et al., Nat. Immunol., 2017, 18:889-898); and 5YC5 (Kiyoshi M., et al., Sei. Rep., 2018, 8:3955-3955); 5XJE (Sakae Y., et al., Sci. Rep., 2017, 7:13780-13780); 5GSQ (Chen, CL, et al., ACS Chem Biol, 2017, 12:1335-1345); and 1HZH (Saphire E. ( ., et al., Science, 2001, 293: 1155-1159) searched for crystal structures of IgG bound to various compounds. In addition, Kiyoshi, M. et al. studied in depth the structural basis of human IgG1 binding to its high affinity human receptor FcγRI. (Kiyosi M., et al., Nat Commun., 2015, 6, 6866).

[0441] TNF-α

[0442] In some embodiments, the target protein is human TNF-α (UniProtKB-PC) 1375 (TNFA_HUMAN). TNF-α is a proinflammatory cytokine active in the body's immune response and severe inflammatory diseases. TNF-α is associated with a variety of conditions, including but not limited to rheumatoid arthritis, inflammatory bowel disease, graft-vs-host disease, ankylosingspondylitis, psoriasis, hidradenilis suppurativa, refractory asthma, systemic lupus erythematosus, diabetes, and induction of cachexia. As used herein, TNF-α may be referred to as TNFa, TNF-a, TNF-α, or TNFα or TNFα.

[0443] The protein database website provides the crystal structure of TNF-α that can be searched through 6RMJ (Va!entinis, B., et al., Int. J. Mol. Sci., 2019, 20), 5UUI (Carrington et al., Biophys J., 2017, 113371-380); 600Y, 600Z and 60PO (O'Connell, J. et al., Nat. Commun., 2019, 105795-5795) and 5TSVV (Cha, SS, J Biol Che., 1998, 2732153-2160); and 5YOY (Ono et al., Protein The crystal structures of TNF-α bound to various compounds were searched by 2AZ5 (He., M.VI. et al., Science, 2005, 310: 1022-1025); 5WUX (Lee, JU, Int JMol Sci., 2017, 18); 5MU8 (Blevitt et al., J Med Chem., 2017, 603511-3517); 4Y60 (Feldman JI, et al., Biochemistry, 2015, 543037-3050); 3WD5 (Hu, S. et al., J Biol Chem, 2013, 28827059-27067); and 4G3Y (Liang, SY, J Biol Chem., 2013, 28813799-13807).

[0444] Proprotein convertase subtilisin / Kexm type 9 (PCSK-9)

[0445] In some embodiments, the target protein is human proprotein convertase subtilisin / kexin type 9 (PCSK-9) (UniProtKB-Q8NBP7 (PCSK9_HUMAN)). PCSK-9 plays a vital role in regulating plasma cholesterol homeostasis. PCSK-9 binds to members of the low-density lipid receptor family, i.e., low-density lipoprotein receptor (LDLR), very low-density lipoprotein receptor (VLDLR), apolipoprotein E receptor (LRP1 / APOER) and apolipoprotein receptor 2 (LRP8 / APOER2), and promotes their degradation in intracellular acidic compartments. It acts through a non-proteolytic mechanism to enhance the degradation of liver LDLR through a clathrin LDLRAP1 / ARH-mediated pathway, and may prevent LDLR from recycling from endosomes to the cell surface or direct it to lysosomes for degradation. PCSK-9 is associated with the development of hypercholesterolemia and cardiovascular disease.

[0446] The protein database website provides crystal structures of PCSK-9 that can be searched by 2P4E (Cunningham, D. et al., Nat Struct Mol Biol., 2007, 14413-419); and by 3BPS (Kwon, HJ et al., Proc Natl Acad Sei USA, 2008, 1051820-1825); 6U26, 6U2N, 6U2P, 6U36, 6U38, and 6U3X (Petrilli, WL et al., Ceil Chem Biol., 2019, 2732-40.e3); 50CA (Gustafsen, C. et al., Nat Commun., 2017, 8503-503); 4NE9 (Schroeder, CL, et al., Chem The crystal structures of PCSK-9 bound to various compounds were searched by 40V6 (Mitchell, T. et al., J Pharmacol Exp Ther., 2014, 350412-424); and 4NMX (Zhang, Y. et al., J Biol Chem., 2014, 289942-955). In addition, Piper et al. studied the crystal structure of PCSK9 in depth (Piper, DE, et al., Structure, 2007, 15 (5), 545-52).

[0447] TNFα

[0448] In one embodiment, the present disclosure provides a bifunctional compound as described herein capable of targeting TNFα. Thus, in one embodiment, the target molecule is TNFα.

[0449] In one embodiment, the targeting moiety (TL) is according to Formula XVIIa:

[0450]

[0451] Among them, R XVIIa With formula Where R XVIId and R XVIIe Independently selected from Formula C 1 -C 5 Alkyl, C 1 -C 5 Alkoxy, cyano, halogen and C 1 haloalkyl, each of which is optionally independently substituted; X is an atom selected from N or CH, and * represents a connection with formula (XVIIa);

[0452] R XVIIb and R XVIIb’ Each independently selected from H and C 1 -C 3 alkyl;

[0453] R XVIIc For R L or selected from Formula XVIIa-1 and Formula XVIIa-2:

[0454]

[0455] Where R XVIIf Selected from optionally substituted C 1 -C 5 an alkyl group, wherein one or more methylene groups are substituted by one selected from a carbonyl group, an ester, an amide, -NH- or -O-, and * represents a connection to Formula XVIIa; and

[0456] R L Indicates that L I connection.

[0457] In one embodiment, R XVIIb and / or R XVIIb ' is -CH 3 In one embodiment, R XVIIb and R XVIIb ' is -CH 3 .

[0458] In one embodiment, R XVIIaSelected from:

[0459]

[0460] in * Indicates linkage to Formula XVIIa.

[0461] In one embodiment, T L According to Formula XVIIa-3:

[0462]

[0463] Where R L Indicates that L I connection.

[0464] In one embodiment, T L According to Formula XVIIa-4:

[0465]

[0466] Where R L Indicates that L I connection.

[0467] In one embodiment, T L According to Formula XVIIa-5:

[0468]

[0469] Where R L Indicates that L I connection.

[0470] In one embodiment, T L According to Formula XVIIb

[0471]

[0472] Where R XVIIi is selected from a bond and an optionally substituted piperazine group;

[0473] R XVIIj Selected from in * represents a connection to Formula XVIIb; and,

[0474] Where R L Indicates that L I connection.

[0475] In one embodiment, T L According to Formula XVIIb-1:

[0476]

[0477] Formula (XVIIb-1)

[0478] Where R L Indicates that L I connection.

[0479] In one embodiment, T L According to Formula XVIIb-2

[0480]

[0481] Where R L Indicates that L I connection.

[0482] In one embodiment, T L According to Formula XVIIc

[0483]

[0484] Where R XVIIg C 1 -C 4 Alkyl, and R XVIIh is optionally substituted by one or more selected from halogen, haloalkyl, cyano, hydroxy, amino, hydroxy, alkoxy, C 3 -C 6 Cycloalkyl and C 3 -C 6 an aromatic or heteroaromatic ring substituted with a heterocycloalkyl group; and

[0485] Where R L Indicates that L I connection.

[0486] In one embodiment, R XVIIg -CH 3 .

[0487] In one embodiment, T L According to Formula XVIIc-1

[0488]

[0489] Formula (XVIIc-1), wherein R L Indicates that L I connection.

[0490] In one embodiment, T L According to Formula XVIIc-2

[0491]

[0492] Formula (XVIIc-2), wherein RL Indicates that L I connection.

[0493] In one embodiment, T L According to Formula XVIId

[0494]

[0495] In one aspect, the present disclosure provides a compound selected from any one of compounds TF001 to TF022 shown in Table C in the "List of Compounds" section.

[0496] TL Biotin and DNP

[0497] In one embodiment, T L In another embodiment, T L The section has the following structure:

[0498]

[0499] Where R L Indicates that L I connection.

[0500] Many proteins bind strongly to biotin. Therefore, in one embodiment, the extracellular target protein is a biotin-binding protein. For example, the biotin-binding protein can be selected from avidin, streptavidin or neutravidin or a derivative thereof. In some cases, the biotin-binding protein is labeled with a tag moiety, such as a fluorophore, a chromophore, or a radiolabeled compound.

[0501] In one embodiment, T L In another embodiment, T L The section has the following structure:

[0502]

[0503] Where R L Indicates that L I connection.

[0504] In one embodiment, the extracellular target protein is a DNP binding protein. For example, the extracellular target protein is an anti-DNP antibody, an anti-DNP antibody fragment capable of binding DNP, or a derivative thereof. In some cases, the anti-DNP antibody or its fragment is labeled with a tag moiety, such as a fluorophore, a chromophore, or a radiolabeled compound.

[0505] T L For protein

[0506] In some embodiments, T L is an antibody, an antigen-binding fragment or a nanobody capable of binding to an extracellular target molecule. L It comprises an antibody, an antigen-binding fragment of an antibody, or a nanobody that is capable of binding to an extracellular target protein.

[0507] According to the present disclosure, T L Can comprise or consist of a full-length antibody or an antibody fragment. In one embodiment, T L The antibody fragments include or consist of antibody fragments, such as antibody light chain (LC), antibody heavy chain (HC), antigen binding region (Fab), light chain variable region (V L ) or heavy chain variable region (V H ).

[0508] In one embodiment, T L It comprises or consists of an antibody heavy chain and / or an antibody light chain.

[0509] In one embodiment, T L The variable region of the antibody light chain (V L ) or consisting thereof. In another embodiment, T L The variable region of the heavy chain of the antibody (V H ) or consists of.

[0510] In one embodiment, T L In one embodiment, T L Comprising or consisting of a monoclonal antibody.

[0511] In one embodiment, T L A fusion protein comprising or consisting of two or more antibody fragments, such as two antibody fragments, such as three antibody fragments, such as four antibody fragments, such as five antibody fragments, such as six antibody fragments, such as seven antibody fragments, such as eight antibody fragments.

[0512] For example, T L The antibody comprises or consists of a single-chain antigen binding region (scAb) or a single-chain variable fragment (scFv). L Contains a single-chain variable fragment (scFv). scFv is the heavy chain variable region (V H ) and light chain variable region (V L ) fusion protein.

[0513] In one embodiment, TL The antibody comprises or consists of a single-chain antigen binding region (scAb). L ) and the heavy chain variable region (V H ) and heavy chain constant region (C H ) or light chain constant region (C L ) together with one or more regions.

[0514] In one embodiment, T L Nanobodies (also called single-domain antibodies) are composed of a single monomeric variable heavy chain (V H H) domain-composed antibody fragments.

[0515] In one embodiment, T L Consists of an antibody or antibody fragment or a fusion protein of an antibody fragment as described herein.

[0516] Alirocumab is a monoclonal antibody that binds to PCSK-9 and has been approved for the treatment of hypercholesterolemia. The heavy chain sequence of the amorobumab antibody is SEQ ID NO.: 66, and the light chain sequence is SEQ ID NO.: 67.

[0517] In one embodiment, T L An antibody or antigen-binding fragment thereof having binding specificity to PCSK-9, comprising:

[0518] a light chain region comprising SEQ ID NO.:67 or an amino acid sequence having at least 70% sequence identity, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO.:67;

[0519] and / or

[0520] A heavy chain region comprising SEQ ID NO.:66 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO.:66, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity.

[0521] In one embodiment, T L Comprising or consisting of an antibody or antigen-binding fragment thereof having binding specificity for PCSK-9, comprising or consisting of:

[0522] A light chain region comprising SEQ ID NO.: 67

[0523] and / or

[0524] A heavy chain region comprising SEQ ID NO.:66.

[0525] In one embodiment, T L Comprising or consisting of the full length monoclonal antibody amorobumab (an antibody having a light chain according to SEQ ID NO.: 67 and a heavy chain according to SEQ ID NO.: 66).

[0526] Adalimumab is a monoclonal antibody that binds to TNF-α. The heavy chain sequence of Adalimumab is SEQ ID NO.: 68, and the light chain sequence is SEQ ID NO.: 69.

[0527] In one embodiment, T L An antibody or antigen-binding fragment thereof having binding specificity to TNF-α comprises:

[0528] a light chain region comprising SEQ ID NO.:69 or an amino acid sequence having at least 70% sequence identity, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO.:69;

[0529] and / or

[0530] A heavy chain region comprising SEQ ID NO.:68 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO.:68, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity.

[0531] In one embodiment, T L It comprises or consists of an antibody or antigen-binding fragment thereof having binding specificity for TNF-α, which comprises or consists of:

[0532] A light chain region comprising SEQ ID NO.: 69

[0533] and / or

[0534] A heavy chain region comprising SEQ ID NO.:68.

[0535] In one embodiment, T L It comprises or consists of the full-length monoclonal antibody adalimumab.

[0536] In one embodiment, T L The method comprises a Nanobody with binding specificity for the kappa light chain of a second antibody, the Nanobody comprising the following sequence:

[0537] MGGTHHHHHHENLYFQGQVQLQESGGGLVQPGGSLRLSCAASGRT ISRYAMSWFRQAPGKEREFVAVARRSGDGAFYADSVQGRFTVSRDDAKN TVYLQMNSLKPEDTAVYYCAIDSDTFYSGSYDYWGQGTQVTVSSE(SEQID NO.:70)

[0538] Or an amino acid sequence having at least 70% sequence identity to SEQ ID NO.: 70, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity.

[0539] In one embodiment, T L Contains or consists of the following sequence:

[0540] MGGTHHHHHHENLYFQGQVQLQESGGGLVQPGGSLRLSCAASGRTISRYAMSWFRQAPGKEREFVAVARRSGDGAFYADSVQGRFTVSRDDAKNTVYLQMNSLKPEDTAVYYCAIDSDTFYSGSYDYWGQGTQVTVSSE

[0541] SEQ ID NO.:70

[0542] In one embodiment, the bifunctional compounds according to the present disclosure can be synthesized with a dissociation constant (K) of less than 50 μM, such as less than 40 μM, such as less than 30 μM, such as less than 20 μM, such as less than 10 μM, such as less than 5 μM, such as less than 4 μM, such as less than 3 μM, such as less than 2 μM, such as less than 1 μM, such as less than 0.8 μM, such as less than 0.6 μM, such as less than 0.5 μM, such as less than 0.4 μM, such as less than 0.3 μM, such as less than 0.2 μM, such as less than 0.1 μM. D ) binds to the target molecule.

[0543] As known to those skilled in the art, binding to a target protein can be measured by different methods, for example, microscale thermophoresis (MST).

[0544] In one embodiment, the bifunctional compound is according to any one of Formula XVIIIa, XVIIIb, XVIIIb-1, XVIIIc, XVIIId, XVIIId-1, XVIIIe, XVIIIf, XVIIIf-1, XVIIIg, XVIIIh, and XVIIIh-1:

[0545]

[0546]

[0547]

[0548] in

[0549] Q 3 is a bond or –CH 2 -;

[0550] R 3a Selected from H, halogen, alkoxy, -CF 3 , and optionally substituted C 1-4 alkyl; and

[0551] L I Indicates a connector.

[0552] In one embodiment, R 3a For H.

[0553] In one embodiment, R 3a Halogen or –CF 3 .

[0554] In one embodiment, R 3a It is an alkoxy group.

[0555] In one embodiment, R 3a is an optionally substituted C 1-4 alkyl.

[0556] In one embodiment, Q 3 is the key.

[0557] In one embodiment, Q 3 -CH 2 -.

[0558] In one embodiment, the bifunctional compound is according to any one of formula D-III, D-IV, DV and D-VI:

[0559]

[0560] In one embodiment, the bifunctional compound is according to any one of formula XVIIIm, XVIIIn, XVIIII-1, XVIIIo, XVIIIp:

[0561]

[0562]

[0563] Where LI Indicates a connector.

[0564] In one embodiment, the bifunctional compound is according to any one of Formula XIVa, XIVb, XIVc and XIVd:

[0565]

[0566]

[0567] in

[0568] Q 3 is a bond or -CH 2 -;

[0569] R 3a Selected from H, halogen, alkoxy, -CF 3 and optionally substituted C 1-4 alkyl.

[0570] In one embodiment, in any of Formulas XIVa, XIVb, XIVc or XIVd, R 3a For H.

[0571] In one embodiment, in any of Formulas XIVa, XIVb, XIVc or XIVd, R 3a Halogen or -CF 3 .

[0572] In one embodiment, in any of Formulas XIVa, XIVb, XIVc or XIVd, R 3a It is an alkoxy group.

[0573] In one embodiment, in any of Formulas XIVa, XIVb, XIVc or XIVd, R 3a is an optionally substituted C 1-4 alkyl.

[0574] In one embodiment, in any of Formulas XIVa, XIVb, XIVc or XIVd, Q 3 is the key.

[0575] In one embodiment, in any of Formulas XIVa, XIVb, XIVc or XIVd, Q 3 -CH 2 -.

[0576] In one embodiment, the bifunctional compound is according to any one of Formulas XVa, XVb, XVc and XVd:

[0577]

[0578] in,

[0579] R F1 and R F2 Independently selected from CF 3 or H, provided that R F1 or R F2 At least one of them is CF 3 .

[0580] In one embodiment, in any of Formulas XIVa, XIVb, XIVc or XIVd, R F1 For-CF 3 And R F2 For H.

[0581] In one embodiment, the bifunctional compound is selected from the group consisting of:

[0582] X T -X L -RQLL-OH (SEQ ID NO.: 72),

[0583] X T -X L -FQLL-OH (SEQ ID NO.: 73),

[0584] X T -X L -KQLL-OH (SEQ ID NO.: 74),

[0585] X T -X L -PQLL-OH (SEQ ID NO.: 75),

[0586] X T -X L -PYIL-OH (SEQ ID NO.: 76),

[0587] X T -X L -RYLL-OH (SEQ ID NO.: 77),

[0588] X T -X L -FYLL-OH (SEQ ID NO.: 78),

[0589] X T -X L -YQLL-OH (SEQ ID NO.: 79),

[0590] X T -X L-LLQL-OH(SEQ ID NO.:80),

[0591] X T -X L -FYIL-OH(SEQ ID NO.:81),

[0592] X T -X L -RYIL-OH(SEQ ID NO.:82),

[0593] X T -X L -PYLL-OH(SEQ ID NO.:83),

[0594] X T -X L -PYYL-OH(SEQ ID NO.:84),

[0595] X T -X L -PYFL-OH(SEQ ID NO.:85),

[0596] X T -X L -RYYL-OH(SEQ ID NO.:86),

[0597] X T -X L -RYFL-OH(SEQ ID NO.:87),

[0598] X T -X L -RQFL-OH(SEQ ID NO.:88),

[0599] X T -X L -RQYL-OH(SEQ ID NO.:89),

[0600] X T -X L -LQLL-OH(SEQ ID NO.:90),

[0601] X T -X L -HQLL-OH(SEQ ID NO.:91),

[0602] X T -X L -IQLL-OH(SEQ ID NO.:92),

[0603] XT -X L -FYYL-OH(SEQ ID NO.:93),

[0604] X T -X L -PYMKLAPGELTIIL-OH(SEQ ID NO.:94),

[0605] X T -X L -NEKLSQLQTYMI-OH(SEQ ID NO.:95),

[0606] X T -X L -KDADLYTSRVMLSSQVP-OH(SEQ ID NO.:96),

[0607] X T -X L -ITVDPRLFKKRRLRSPRVLFSTQPPR-OH(SEQ ID NO:99),

[0608] X T -X L -WSGPIGVSWGLRAAAAGGAFP-OH(SEQ ID NO:100),

[0609] X T -X L -WSGPIGVSWGLRAAAAGGAFPRGGRWRR-OH(SEQ ID NO.:101),

[0610] X T -X L -GVSWGLR-OH(SEQ ID NO.:102),

[0611] X T -X L -REAPRWDAPLRDPALRQLL-OH(SEQ ID NO.:103),

[0612] X T -X L -REALRWDAPLRDPAPRQLL-OH(SEQ ID NO.:104),

[0613] X T -X L -PYILKRQLYENKPRRPYIL-OH(SEQ ID NO.:105),

[0614] X T -X L -LYENKPRRPYIL-OH(SEQ ID NO.:106),

[0615] X T -X L -APLRDPAPRQLL-OH(SEQ ID NO.:107),

[0616] X T -X L -REAPRWDAPLRDPALFQLL-OH(SEQ ID NO.:108),

[0617] X T -X L -REAPRWDAPLRDPALRYLL-OH(SEQ ID NO.:109),

[0618] X T -X L -REAPRWDAPLRDPALRQYL-OH(SEQ ID NO.:110),

[0619] X T -X L -REAPRWDAPLRDPALRYYL-OH(SEQ ID NO.:111),

[0620] X T -X L -TGGFM-OH (SEQ ID NO.: 112), and

[0621] X T -X L -YGGFL-OH (SEQ ID NO.: 113);

[0622] Where X L For L I And X T Selected from:

[0623] a) a group comprising biotin;

[0624] b) a group comprising dinitrophenol;

[0625] c) an antibody with binding specificity for PCSK-9 comprising or consisting of:

[0626] A light chain region comprising SEQ ID NO.: 67

[0627] and / or

[0628] A heavy chain region comprising SEQ ID NO.: 66

[0629] or an antigen-binding fragment thereof.

[0630] d) an antibody with binding specificity for TNF-α, comprising or consisting of:

[0631] A light chain region comprising SEQ ID NO.: 69

[0632] and / or

[0633] A heavy chain region comprising SEQ ID NO.: 68

[0634] or an antigen-binding fragment thereof.

[0635] In one embodiment, the bifunctional compound is according to:

[0636] Biotin-X L -X 5 -X 1 X 2 X 3 X 4 (SEQ ID NO.: 116)

[0637] in,

[0638] X 5 is an optional amino acid residue or a peptide comprising 2 to 30 amino acid residues or a bond,

[0639] X 1 is R, P, F, Y, L, K, G or H;

[0640] X 2 is Q, Y, L, E or G;

[0641] X 3 is Y, F, L, I, Q, E or N;

[0642] X 4 is M, K, L or a conservative substitution of L;

[0643] Where X L For L I .

[0644] In one embodiment, X 5 is the key.

[0645] In one embodiment, X 5 For X 5 It is APLRDPAP (SEQ ID NO.: 21).

[0646] In one embodiment, X5 is REAPRWDAPLRDPAL (SEQ ID NO.: 17). In one embodiment, X 5 is REALRWDAPLRDPAP (SEQ ID NO.: 18). In one embodiment,

[0647] X 1 is selected from Y, F or R,

[0648] X 2 Select from Q or Y,

[0649] X 3 For L,

[0650] X 4 For L.

[0651] In one embodiment, X 1 Is F or R.

[0652] In one embodiment, X 2 For Q.

[0653] In one embodiment, X 3 For Q.

[0654] In one embodiment, X 4 For L.

[0655] In one embodiment, the bifunctional compound is any one of the group consisting of: biotin-X L -RQLL-OH (SEQ ID NO.: 117)

[0656] Biotin-X L -FQLL-OH (SEQ ID NO.: 118)

[0657] Biotin-X L -RYLL-OH (SEQ ID NO.: 119)

[0658] Biotin-X L -FYLL-OH (SEQ ID NO.: 120)

[0659] Biotin-X L -YQLL-OH (SEQ ID NO.: 121)

[0660] Biotin-X L -REAPRWDAPLRDPALRQLL-OH (SEQ ID NO.: 122) Biotin-X L-REALRWDAPLRDPAPRQLL-OH (SEQ ID NO.: 123) Biotin-X L -REAPRWDAPLRDPALFQLL-OH(SEQ IDNO.:124)

[0661] Biotin-X L -REAPRWDAPLRDPALRYLL-OH (SEQ ID NO.: 125) Biotin-X L -REAPRWDAPLRDPALRQYL-OH (SEQ ID NO.: 126) Biotin-X L -REAPRWDAPLRDPALRYYL-OH(SEQ IDNO.:127)Biotin-X L -APLRDPAPRQLL-OH(SEQ ID NO.:128)

[0662] Biotin-X L -PYILKRQLYENKPRRPYIL-OH(SEQ ID NO.:129)

[0663] Biotin-X L -LYENKPRRPYIL-OH(SEQ ID NO.:130)

[0664] Where X L For L I .

[0665] Antibody conjugates

[0666] In one embodiment, by combining a monoclonal antibody and a group-L I -S L The reactive precursors of the group –L I -S L The free amino groups in the antibody sequence can be coupled, such as the side chain of a lysine amino acid or the N-terminal group. A variety of methods and conditions are known in the art for coupling groups to free amino groups in antibodies. For example, L I -S L N-hydroxysuccinimide (NHS) derivatives of the peptide and react with antibodies.

[0667] Thus, in one embodiment, the bifunctional compound is provided by -L I -S L The NHS derivatives were prepared by reacting with monoclonal antibodies.

[0668] Those skilled in the art will understand that –LI -S L The reaction of NHS derivatives with monoclonal antibodies will produce a product in which –L I -S L More than one unit of may be coupled to an antibody.

[0669] Thus, in one embodiment, the bifunctional compound has T L Group, the T L The group consists of at least 1 -L I -S L Unit, for example with 2-L I -S L Unit, for example with 3-L I -S L Unit, for example with 4-L I -S L Unit, for example with 5-L I -S L Unit, for example with 6-L I -S L Unit, for example with 7-L I -S L Unit, for example with 8-L I -S L Unit, for example with 9-L I -S L The monoclonal antibody is composed of a unit-coupled monoclonal antibody.

[0670] In one embodiment, the monoclonal antibody is amorobizumab (an antibody having a heavy chain of SEQ ID NO.: 66 and a light chain of SEQ ID NO.: 67), and S L and / or L I As described herein.

[0671] In one embodiment, the monoclonal antibody is amorobizumab (an antibody having a heavy chain of SEQ ID NO.: 66 and a light chain of SEQ ID NO.: 67) and S L A peptide comprising at its C-terminus a sequence selected from the group consisting of:

[0672] RQLL-OH (SEQ ID NO.: 22)

[0673] FQLL-OH (SEQ ID NO.: 23)

[0674] RYLL-OH (SEQ ID NO.: 27)

[0675] FYLL-OH (SEQ ID NO.: 28)

[0676] YQLL-OH (SEQ ID NO.: 29) and

[0677] PYILKRQLYENKPRRPYIL-OH (SEQ ID NO.:55);

[0678] Where L I Attached at the N-terminus.

[0679] In one embodiment, the monoclonal antibody is amorobizumab (an antibody having a heavy chain of SEQ ID NO.: 66 and a light chain of SEQ ID NO.: 67), and S L is a peptide comprising the following sequence at the C-terminus: RQLL-OH (SEQ ID NO.: 22).

[0680] In one embodiment, the monoclonal antibody is amorobizumab (an antibody having a heavy chain of SEQ ID NO.: 66 and a light chain of SEQ ID NO.: 67), and S L is a peptide comprising the following sequence at the C-terminus: FQLL-OH (SEQ ID NO.: 23).

[0681] In one embodiment, the monoclonal antibody is adalimumab (an antibody having a heavy chain of SEQ ID NO.: 68 and a light chain of SEQ ID NO.: 69) and S L As described herein.

[0682] In one embodiment, the monoclonal antibody is adalimumab (an antibody having a heavy chain of SEQ ID NO.: 68 and a light chain of SEQ ID NO.: 69) and S L A peptide comprising at its C-terminus a sequence selected from the group consisting of:

[0683] RQLL-OH (SEQ ID NO.:22); FQLL-OH (SEQ ID NO.:23); RYLL-OH (SEQ ID NO.:27); FYYL-OH (SEQ ID NO.:28); YQLL-OH (SEQ ID NO.:29) and

[0684] PYILKRQLYENKPRRPYIL-OH (SEQ ID NO.:55);

[0685] Wherein LI is linked to the N-terminus.

[0686] In one embodiment, the monoclonal antibody is adalimumab (an antibody having a heavy chain of SEQ ID NO.: 68 and a light chain of SEQ ID NO.: 69), and S Lis a peptide comprising the following sequence at the C-terminus: RQLL-OH (SEQ ID NO.: 22).

[0687] In one embodiment, the monoclonal antibody is adalimumab (an antibody having a heavy chain of SEQ ID NO.: 68 and a light chain of SEQ ID NO.: 69), and S L is a peptide comprising the following sequence at the C-terminus: FQLL-OH (SEQ ID NO.: 23).

[0688] Connectors:

[0689] The bifunctional compounds according to the present disclosure comprise a covalently linked sortilin binding moiety (S L ) and targeting moiety (T L ) connector. L and T L Covalent coupling of groups through linkers to form bifunctional compounds can be achieved by a variety of strategic chemical strategies known in the art.

[0690] In one embodiment, the bifunctional compounds according to the present disclosure have L according to the following structure: I :

[0691]

[0692] in

[0693] * Indicates that L or S L The connection point;

[0694] L 1 and L 2 are each independently selected from a bond, -C(H 2 )-, -O-, -N(H)-; a functional group selected from carbonyl, ester, amide, carbamate, thiourea, urea, sulfonamide and triazole; or C 1 -C 3 hydrocarbon chains in which one or more methylene groups are individually and optionally replaced by carbonyl, ester, amide, carbamate, thiourea, urea, sulfonamide and triazole;

[0695] Z is selected from: divalent saturated or unsaturated straight or branched chain C 1- C 30 A hydrocarbon chain in which one or more methylene groups are individually and optionally replaced by one or more groups selected from: -O-, -N(H)-, -N(R L1 )-, -OC(=O)-, -C(=O)O-, -C(=O)-, -N(H)C(=O)-, -N(R L1)C(=O)-, -C(=O)N(H)-, -NHC(=O)NH-, -NHC(=O)O--C(=O)N(R L1 )-, -S-, -S(=O)-, -S(=O) 2 -、-N(R L1 )S(=O) 2 -、-S(=O) 2 N(R L1 )-; an optionally substituted divalent aromatic group; an optionally substituted carbocyclic ring; an optionally substituted heterocyclic ring; an optionally substituted divalent aromatic heterocyclic ring;

[0696] Indicates that L 1 or L 2 connection;

[0697] R L1 Selected from C 1-5 alkyl;

[0698] n and w are each independently an integer of 1 to 8.

[0699] In one embodiment, the bifunctional compounds according to the present disclosure have L according to formula (XVI-2): I ,in:

[0700]

[0701] in

[0702] * Indicates that L or S L The connection point,

[0703] L 1 and L 2 Each independently selected from -C(H 2 )-, -O-, -N(H)-, and amides;

[0704] Z is a divalent saturated or unsaturated straight or branched chain C 1- C 30 A hydrocarbon chain in which one or more methylene groups are individually and optionally replaced by one or more groups selected from: -O-, -N(H), -N(R L1 )-, -N(H)C(=O)-, -N(R)C(=O)-, -C(=O)N(H)-, -C(=O)N(R L1 )-、

[0705]

[0706] Indicates that L 1 or L 2 connection;

[0707] R L1 Selected from C 1-5 alkyl;

[0708] n and w are each independently an integer of 1 to 8.

[0709] In one embodiment, Z is a divalent C 1 -C 30 Hydrocarbon chains, such as C 5 -C 30 Hydrocarbon chains, such as C 8 -C 30 Hydrocarbon chains, such as C 10 -C 30 Hydrocarbon chains, such as C 12 -C 30 A hydrocarbon chain in which one or more methylene groups are replaced as described in Formula XVI or Formula XVI-2.

[0710] In one embodiment, Z is a divalent C 10 -C 25 Hydrocarbon chains, such as C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 or C 25 A hydrocarbon chain wherein one or more methylene groups are replaced as described in Formula XVI or Formula XVI-2.

[0711] In one embodiment, Z is a divalent C 14 -C 20 A hydrocarbon chain wherein one or more methylene groups are replaced as described in Formula XVI or Formula XVI-2. In one embodiment, Z is a divalent C 7 -C 13 A hydrocarbon chain in which one or more methylene groups are replaced as described in Formula XVI or Formula XVI-2.

[0712] In one embodiment, L 1 In one embodiment, L 2 In one embodiment, L1 and L 2 is the key.

[0713] In one embodiment, Z is C 1- C 30 A hydrocarbon chain in which one or more methylene groups are individually and optionally replaced by one or more groups selected from: -O-, -N(H), -N(H)-C(=O)-, -C(=O)-N(H)-, urea, triazole, optionally substituted carbocycle; optionally substituted heterocycle and -CH 2 -CH 2 -O-.

[0714] In one embodiment, Z comprises C 1 -C 30 Hydrocarbon chains, such as C 1 -C 20 , for example C 1 -C 15 , for example C 1 -C 10 wherein one or more methylene groups are individually and optionally replaced by one or more groups selected from the group consisting of: -O-, -N(H)-, -N(R L1 )-, -OC(=O)-, -C(=O)O-, -C(=O)-, -N(H)C(=O)-, -NHC(=O)NH-, -NHC(=O)O-, -N(R L1 )C(=O)-, -C(=O)N(H)-, -C(=O)N(R L1 )-, -S-, -S(=O)-, -S(=O) 2 -、-N(R L1 )S(=O) 2 -、-S(=O) 2 N(R L1 )-、-CH 2 -CH 2 -O-, optionally substituted carbocycle; optionally substituted heterocycle, and triazole; R L1 Selected from C 1-5 alkyl.

[0715] In one embodiment, one or more methylene groups, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 methylene groups, of the hydrocarbon chain in Z are individually and optionally replaced by one or more groups selected from: -O-, -N(H)-, -N(R L1 )-, -OC(=O)-, -C(=O)O-, -C(=O)-, -N(H)C(=O)-, -N(R L1)C(=O)-, -C(=O)N(H)-, -C(=O)N(R L1 )-, -S-, -S(=O)-, -S(=O) 2 -、-N(R L1 )S(=O) 2 -、-S(=O) 2 N(R L1 )-、-CH 2 -CH 2 -O-, optionally substituted carbocycle; optionally substituted heterocycle, and triazole; R L1 Selected from C 1-5 alkyl.

[0716] In one embodiment, Z comprises one or more groups -NH-SO 2 -group.

[0717] In one embodiment, Z comprises one or more triazole groups. In one embodiment, triazole is

[0718] In one embodiment, Z comprises one or more groups selected from the following: optionally substituted carbocyclic groups and optionally substituted heterocyclic groups. In one embodiment, Z comprises two groups, each of which is individually selected from: triazole, optionally substituted carbocyclic groups and optionally substituted heterocyclic groups. In one embodiment, Z comprises three groups, each of which is individually selected from: triazole, optionally substituted carbocyclic groups and optionally substituted heterocyclic groups.

[0719] Non-limiting examples of carbocyclic and heterocyclic rings are described below. In one embodiment, one or more carbocyclic or heterocyclic rings are as described below.

[0720] In one embodiment, Z comprises wherein n is an integer selected from 0, 1, 2 or 3.

[0721] In one embodiment, Z comprises one or more heterocyclic groups. For example, a heterocyclic group may be an optionally substituted 3 to 6 membered ring in which one or two carbon atoms of the ring have been replaced by N. In one embodiment, Z comprises wherein n is an integer selected from 0, 1, 2 or 3.

[0722] In one embodiment, Z comprises one or more groups, each of which is individually selected from the groups shown in Table Z:

[0723] Table Z

[0724]

[0725] In one embodiment, Z comprises two groups, each group being individually selected from the groups shown in Table Z. In one embodiment, Z comprises three groups, each group being individually selected from the groups shown in Table Z.

[0726] In one embodiment, Z comprises one or more groups, each group being individually selected from the groups shown in Table ZI:

[0727] Table ZI

[0728]

[0729] wherein n and / or n′ are each independently an integer from 1 to 10, and t, t′ and / or w are each independently an integer from 1 to 20.

[0730] In one embodiment, Z comprises wherein n is an integer from 1 to 10.

[0731] In one embodiment, Z comprises wherein n is an integer from 1 to 10, and t or w is an integer from 1 to 20.

[0732] In one embodiment, Z comprises wherein n is an integer from 1 to 10, and each of t and t′ is independently an integer from 1 to 20.

[0733] In one embodiment, Z comprises wherein each of n and n′ is independently an integer from 1 to 10, and t is an integer from 1 to 20.

[0734] In one embodiment, L 1 or L 2 In one embodiment, L 1 and L 2 It is an amide group.

[0735] In one embodiment, L 1 or L 2 In one embodiment, L 1 and L 2 A carbonyl group.

[0736] In one embodiment, L 1 or L 2 is an ester group. 1 and L 2 It is an ester group.

[0737] In one embodiment, L 1 or L 2is a carbamate group. In one embodiment, L 1 and L 2 A carbamate group.

[0738] In one embodiment, L 1 or L 2 is a urea group. 1 and L 2 It is a urea group.

[0739] In one embodiment, L 1 or L 2 is –NH-S(=O) 2 -. In one embodiment, L 1 and L 2 It is –NH-S(=O)2-.

[0740] In one embodiment, L 1 or L 2 is a triazole group. In one embodiment, L 1 and L 2 The triazole group can be prepared by reacting an azide group with an alkyne group, optionally in the presence of a catalyst, for example using copper ions as a catalyst.

[0741] In one embodiment, L 1 or L 2 In one embodiment, L 1 and L 2 It is –O-.

[0742] In one embodiment, L 1 or L 2 In one embodiment, L 1 and L 2 It is –NH-.

[0743] In one embodiment, L 1 or L 2 =S(=O) 2 -. In one embodiment, L 1 and L 2 -S(=O) 2 -.

[0744] In one embodiment, L 1 or L 2 for In one embodiment, L 1 and L 2 for

[0745] In one embodiment, L 1 or L 2 for In one embodiment, L 1 and L 2 for

[0746] In one embodiment, L 1 or L 2 for wherein X is NH or O. In one embodiment, L 1 and L 2 for wherein X is NH or O. In one embodiment, L 1 or L 2 for In one embodiment, L 1 and L 2 for

[0747] In one embodiment, L 1 or L 2 for wherein X is NH or O. In one embodiment, L 1 and L 2 for wherein X is NH or O. In one embodiment, L 1 or L 2 for In one embodiment, L 1 and L 2 for

[0748]

[0749] In one embodiment, L 1 and L 2 In one embodiment, L 1 and L 2 for the same.

[0750] In one embodiment, the bifunctional compounds according to the present disclosure have L according to one selected from the following I :

[0751] n and w are each independently an integer from 1 to 9 and

[0752] * Indicates that T L or S L connection.

[0753] In one embodiment, L I Based on

[0754]

[0755] Wherein w is an integer from 1 to 9, * Indicates that T L or S L connection.

[0756] In one embodiment, L I Based on

[0757]

[0758] Where n is an integer from 1 to 8, * Indicates that T L or S L connection.

[0759] In one embodiment, L I Based on

[0760]

[0761] Where n and w are integers from 1 to 8, * Indicates that T L or S L connection.

[0762] In one embodiment, L I Based on

[0763]

[0764] Where n is an integer from 1 to 9, * Indicates that T L or S L connection.

[0765] In one embodiment, L I One selected from Formula (XVIa) to Formula (XVIae) in Table Z-II: Table Z-II

[0766]

[0767]

[0768]

[0769] Where R L Indicates that S L The connection point, and R T Indicates that T L connection point.

[0770] In one embodiment, L I One of the formulas XVIaf to XVIbw selected from Table Z-III: Table Z-III

[0771]

[0772]

[0773]

[0774]

[0775]

[0776] in * Indicates that S L or T L connection.

[0777] In one embodiment, the linker L I According to any one of formulas XVIaf to XVIbw in Table Z-III. The linkers shown in Table Z-III, such as formulas XVIaf to XVIbw, can be in the form of * Connect any position to S L and T L For example, in one embodiment, the linkers shown in Table Z-III are represented by * The position is connected to T L and the right side of the formula shown in Table Z-III is marked with * The location is connected to S L .

[0778] In one embodiment, the linker is a peptide. Thus, the linker can be produced by methods known in the art for preparing peptides, such as by solid phase synthesis using protected amino acids or by expressing a suitable vector in a chosen organism to produce a peptide linker.

[0779] In one embodiment, L I It is a peptide with a length of between 1 and 30 amino acids, such as 3 and 20 amino acids.

[0780] In one embodiment, L I A peptide between 3 and 20 amino acids in length, consisting of any combination of glycine, serine, and cysteine.

[0781] In one embodiment, L I It is a peptide between 3 and 20 amino acids in length, consisting of any combination of glycine and serine.

[0782] In one embodiment, L I is a peptide selected from the group consisting of:

[0783] GGSGGGGSGGGGSGG(SEQ ID NO.:131)

[0784] GGSGGGG (SEQ ID NO.: 132)

[0785] GGGGSGGGGSGGGGSGG(SEQ ID NO.:133)

[0786] GGSGGGGSGGGGS (SEQ ID NO.: 134)

[0787] GGSGGGGSGGG (SEQ ID NO.: 135)

[0788] GGSGGGGSG (SEQ ID NO.: 136)

[0789] GGSGGGG (SEQ ID NO.: 137)

[0790] GGSGG (SEQ ID NO.: 138)

[0791] GGS (SEQ ID NO.: 139)

[0792] CGGSGGGGSGGGGSGG(SEQ ID NO.:140)

[0793] In one embodiment, L I is GGSGGGSGGGGSGG (SEQ ID NO.: 131).

[0794] In one embodiment, L I It is GGSGGGG (SEQ ID NO.: 132).

[0795] In one embodiment, L I is GGGGSGGGSGGGGSGG (SEQ ID NO.: 133).

[0796] In one embodiment, L I is GGSGGGSGGGGS (SEQ ID NO.: 134).

[0797] In one embodiment, L I is GGSGGGGSGGG (SEQ ID NO.:135).

[0798] In one embodiment, L I It is GGSGGGGSG (SEQ ID NO.: 136).

[0799] In one embodiment, L I It is GGSGGGG (SEQ ID NO.: 137).

[0800] In one embodiment, L I is GGSGG (SEQ ID NO.: 138).

[0801] In one embodiment, L I It is GGS (SEQ ID NO.: 139).

[0802] In one embodiment, L I is CGGSGGGGSGGGGSGG (SEQ ID NO.:140)

[0803] In one embodiment, L I By L I The C-terminus is connected to S L , and L I By L I The N-terminus is connected to T L .

[0804] Bifunctional compounds:

[0805] Compound

[0806] In one embodiment, the bifunctional compound is selected from any one of compounds BF030 to BF149 shown in Table A in the "Compound Listing" section, or a pharmaceutically acceptable salt thereof.

[0807] In one embodiment, the bifunctional compounds according to the present disclosure have:

[0808] a) S L , which is selected from any one of Formulae IIIi, IIIj, IIIk, IIIm, IIIn and IIIo; or selected from Formula DI described above in the "Binding of Sortilin" section; and

[0809] b) capable of dissociating with a dissociation constant (K) of less than 50 μM, for example, less than 40 μM, for example, less than 30 μM, for example, less than 20 μM, for example, less than 10 μM, for example, less than 5 μM, for example, less than 4 μM, for example, less than 3 μM, for example, less than 2 μM, for example, less than 1 μM, for example, less than 0.8 μM, for example, less than 0.6 μM, for example, less than 0.5 μM, for example, less than 0.4 μM, for example, less than 0.3 μM, for example, less than 0.2 μM, for example, less than 0.1 μMD ) binds to the target molecule.

[0810] In one embodiment, the bifunctional compounds according to the present disclosure have:

[0811] a) S L , which is selected from any one of Formulae IIIi, IIIj, IIIk, IIIm, IIIn and IIIo; or selected from Formula DI described above in the "Binding of Sortilin" section; and

[0812] b)L I , which is selected from any one of Formulas XVIaf to XVIbw in Table Z-III in the "Linkers" section above; and

[0813] c)T L , which is according to any of Formulas XVIIa-3, XVIIa-4, XVIIc-1 and XVIIc-2 described above in the "Targeted Warhead" section.

[0814] In one embodiment, the bifunctional compound according to the present disclosure is selected from any one of compounds BF001 to BF028 shown in Table A in the "Compound Listing" section.

[0815] Peptides

[0816] In one embodiment, the bifunctional compound is selected from any one of the following:

[0817] Biotin-GGSGGGGSGRQLL-OH (SEQ ID NO: 141)

[0818] Biotin-GGSGGGGSGGGGSRQLL-OH (SEQ ID NO.: 142)

[0819] Biotin-GGSGGGGFQLL-OH (SEQ ID NO.: 143)

[0820] Biotin-GGSGGFQLL-OH (SEQ ID NO.: 144)

[0821] Biotin-GGSGGGGSGFQLL-OH (SEQ ID NO.: 145)

[0822] Biotin-GGSGGGGSGGGGSFQLL-OH (SEQ ID NO.:146)

[0823] Biotin-GGSGGGGSGGGGSGGRQLL-OH (SEQ ID NO.:147)

[0824] Biotin-GGSGGGGSGGGFQLL-OH (SEQ ID NO.:148)

[0825] Biotin-GGSGGGGSGGGRQLL-OH (SEQ ID NO.: 149)

[0826] Biotin-GGSGGGGRQLL-OH (SEQ ID NO.: 150)

[0827] Biotin-GGSGGRQLL-OH (SEQ ID NO.: 151)

[0828] Biotin-GGSRQLL-OH (SEQ ID NO.: 152)

[0829] Biotin-GGRQLL-OH (SEQ ID NO.: 153)

[0830] Biotin-RQLL-OH (SEQ ID NO.: 154)

[0831] Biotin-GGSGGGGSGGGGSGGFQLL-OH((SEQ ID NO.:155)

[0832] Biotin-REAPRWDAPLRDPALFQLL-NH2 (SEQ ID NO.: 188)

[0833] Biotin-REAPRWDAPLRDPALFQLL-OH (SEQ ID NO.: 189)

[0834] Biotin-REAPRWDAPLRDPALRQLL-NH2 (SEQ ID NO.: 190), and

[0835] Biotin-REAPRWDAPLRDPALRQLL-OH (SEQ ID NO.: 191).

[0836] protein

[0837] In one embodiment, the bifunctional compound is:

[0838] MGGTHHHHHHENLYFQGQVQLQESGGGLVQPGGSLRLSCAASGRTISRYAMSWFQAPGKEREFVAVARRSGDGAFYADSVQGRFTVSRDDAKNTVYLQMNSLKPEDTAVYCAIDSDTFYSGSYDYWGQGTQVTVSSEGGGGSGGGGSGGGGSGGRQLL(SEQ ID NO.:158)

[0839] In one embodiment, the bifunctional compound has T L , the T L Composed of the monoclonal antibody amorobumab with at least 1 unit–L I -S L The coupling composition, the –L I -S L Consists of CGGSGGGGSGGGGSGGRQLL (SEQ ID NO.: 175); wherein SEQ ID NO.: 175 is coupled via its N-terminus. The conjugate of amorobizumab is described herein as

[0840] Amorobizumab-CGGSGGGGSGGGGSGGRQLL (SEQ ID NO.: 159) or Amorobizumab-Linker-RQLL or Amorobizumab-Linker-RQLL.

[0841] A variant conjugate without the sequence RQLL is also disclosed and is referred to herein as:

[0842] Amorobumab-CGGSGGGGSGGGGSGG (SEQ ID NO.: 160) or Amorobumab-linkage or Amorobumab-linker.

[0843] The product described in SEQ ID NO.: 159 may have more than one unit of (SEQ ID NO.: 175) coupled to an antibody, such as 2 or more units, such as 3 or more units, such as 4 or more units.

[0844] In one embodiment, the bifunctional compound has T L , the T L Composed of the monoclonal antibody adalimumab and at least 1 unit – L I -S L The coupling composition, the –L I -S LIt is composed of CGGSGGGGSGGGGSGGRQLL (SEQ ID NO.: 175), wherein SEQ ID NO.: 175 is coupled via its N-terminus. The conjugate of adalimumab is referred to herein as:

[0845] Adalimumab-CGGSGGGGSGGGGSGGRQLL (SEQ ID NO.: 161) or Adalimumab-Linker-RQLL or Adalimumab-Linker-RQLL.

[0846] In one embodiment, the bifunctional compound has T L , the T L Composed of the monoclonal antibody adalimumab and at least 1 unit – L I -S L The coupling composition, the –L I -S L Depend on

[0847] CGGSGGGGSGGGGSGGRQLL (SEQ ID NO.:175),

[0848] CREAPRWDAPLRDPALRQLL(SEQ ID NO.:184)

[0849] CREAPRWDAPLRDPALRQLL-NH2(SEQ ID NO.:185)

[0850] CREAPRWDAPLRDPALFQLL-OH (SEQ ID NO: 186) and

[0851] CREAPRWDAPLRDPALFQLL-NH2 (SEQ ID NO.: 187),

[0852] Wherein SEQ ID NO.: 175 or SEQ ID NO.: 184 to 187 are coupled via their N-terminus. These adalimumab conjugates are referred to herein as:

[0853] Adalimumab-CGGSGGGGSGGGGSGGRQLL (SEQ ID NO.: 161)

[0854] Adalimumab-CREAPRWDAPLRDPALRQLL (SEQ ID NO.: 192)

[0855] Adalimumab-CREAPRWDAPLRDPALRQLL-NH2 (SEQ ID NO.: 193)

[0856] Adalimumab-CREAPRWDAPLRDPALFQLL-OH (SEQ ID NO: 194)

[0857] Adalimumab-CREAPRWDAPLRDPALFQLL-NH2 (SEQ ID NO.: 195).

[0858] A variant conjugate without the sequence RQLL is also disclosed and is referred to herein as:

[0859] Adalimumab-CGGSGGGGSGGGGSGG (SEQ ID NO.: 162) or Adalimumab-linkage or Adalimumab-linker.

[0860] The product described in SEQ ID NO.: 161 may have more than one unit of (SEQ ID NO.: 175) coupled to an antibody, such as 2 or more units, such as 3 or more units, such as 4 or more units. Similarly, the products described as conjugates by SEQ ID NO.: 192 to 195 may have more than one unit of SEQ ID NO.: 184 to SEQ ID NO.: 187, such as 2 or more units, such as 3 or more units, such as 4 or more units.

[0861] Each antibody conjugated –L I -S L The number can be expressed as the average number of each antibody: N a In one embodiment, N a is an integer or decimal between 1 and 10. In one embodiment, the product described in SEQ ID NO.: 161 may have some -L of SEQ ID NO.: 175. I -S L The unit makes N a In one embodiment, the product described in any one of SEQ ID NOs.: 192 to 195 may have some -L of SEQ ID NOs.: 184 to 187. I -S L The unit makes N a From 1 to 10.

[0862] The bifunctional compounds according to the present invention are capable of sortilin binding through the Sortilin binding moiety (S L ) binds to sortilin and through the protein targeting moiety (T L ) binds to the target protein.

[0863] Thus, in one embodiment, the bifunctional compound of the present invention is capable of binding to Sortilin and the target protein simultaneously. This means that the bifunctional compound according to the present disclosure forms a ternary complex with Sortilin and the target protein. In the ternary complex, Sortilin and the target protein bind to the bifunctional compound simultaneously.

[0864] The formation of the ternary complex can be measured by various methods known in the art. For example, the formation of the ternary complex can be measured by Resonance energy transfer (FRET) assay or by time-resolved Resonance Energy Transfer (TR-FRET) assays. As measured by these assays, an increase in the homogeneous time-resolved fluorescence (HTRF) ratio indicates the formation of a ternary complex.

[0865] As demonstrated in the examples, the bifunctional compounds according to the present disclosure are able to bind to Sortilin and the target protein and form a ternary complex.

[0866] In one embodiment, the bifunctional compounds according to the present disclosure are capable of binding to Sortilin on the cell surface. In another embodiment, the bifunctional compounds are capable of forming a ternary complex with Sortilin and a target molecule on the cell surface.

[0867] In one embodiment, when S L Binds to sortilin on the cell surface and T L Upon binding to the target protein, the target protein is internalized into the cell. In one embodiment, the target protein is degraded after internalization into the cell. In one embodiment, degradation of the target protein occurs in the lysosomal compartment.

[0868] In one embodiment, the bifunctional compounds according to the present disclosure have a dissociation constant for binding to Sortilin of less than 50 μM, such as less than 40 μM, such as less than 30 μM, such as less than 20 μM, such as less than 10 μM, such as less than 5 μM, such as less than 4 μM, such as less than 3 μM, such as less than 2 μM, such as less than 1 μM, such as less than 0.8 μM, such as less than 0.6 μM, such as less than 0.5 μM, such as less than 0.4 μM, such as less than 0.3 μM, such as less than 0.2 μM, such as less than 0.1 μM, For example, less than 0.05μM, for example, less than 0.01μM; and the dissociation constant for binding to the target protein is less than 50μM, for example, less than 40μM, for example, less than 30μM, for example, less than 20μM, for example, less than 10μM, for example, less than 5μM, for example, less than 4μM, for example, less than 3μM, for example, less than 2μM, for example, less than 1μM, for example, less than 0.8μM, for example, less than 0.6μM, for example, less than 0.5μM, for example, less than 0.4μM, for example, less than 0.3μM, for example, less than 0.2μM, for example, less than 0.1μM.

[0869] In one embodiment, the target protein is TNFα. Thus, in one embodiment, the bifunctional compound according to the present disclosure is capable of forming a ternary complex between Sortilin and TNFα. In one embodiment, the bifunctional compound is capable of binding to Sortilin and TNFα simultaneously.

[0870] In one embodiment, the bifunctional compounds according to the present disclosure have S L The dissociation constant for binding to Sortilin is less than 50 μM, such as less than 2 μM, such as less than 0.5 μM, preferably less than 0.1 μM; and T L The dissociation constant for binding to TNFa is less than 100 μM, such as less than 0.5 μM, such as less than 0.1 μM.

[0871] In one embodiment, the bifunctional compounds according to the present disclosure have a dissociation constant for binding to Sortilin of less than 50 μM, such as less than 2 μM, such as less than 0.5 μM, preferably less than 0.1 μM, and a dissociation constant for binding to TNFα of less than 100 μM, such as less than 0.5 μM, such as less than 0.1 μM.

[0872] In one embodiment, the bifunctional compound provides, in S L Binds to sortilin on the cell surface and T LAfter binding to TNFα, TNFα is internalized into the cell. To assess whether the target protein is internalized, a method such as detecting the target protein in the cell culture supernatant can be used. Any suitable method can be used, such as ELISA, fluorescence detection of conjugates, HPLC, gel electrophoresis combined with staining, such as SDS-PAGE or immunoblotting, or other well-known techniques in the art. After cell lysis, the presence of the target protein or its fragment in the cell can also be assessed by similar methods.

[0873] This example demonstrates that bifunctional compounds according to the present disclosure can promote internalization of target proteins into cells through sortilin-mediated binding. These examples also demonstrate that internalization leads to degradation of the target protein.

[0874] In one embodiment, TNFα is degraded after internalization into the cell.

[0875] Preparation of bifunctional compounds:

[0876] The following abbreviations refer to the following definitions:

[0877] Ac (acetyl), aq (aqueous solution), h (hour), g (gram), L (liter), mg (milligram), MHz (megahertz), μM (micromole), min (minute), mm (millimeter), mmol (millimole), mM (millimole), mp (melting point), equiv (equivalent), mL (milliliter), μL (microliter), ACN (acetonitrile), AcOH (acetic acid), BINAP (2,2'-bis(diphenylphosphino)-1,1'-binaphthyl), BOC (tert-butyloxycarbonyl), CBZ (benzyloxycarbonyl), CDCl 3(deuterated chloroform), CD3OD (deuterated methanol), CH3CN (acetonitrile), c-hex (cyclohexane), DCC (dicyclohexylcarbodiimide), DCM (dichloromethane), DHP (O-(2,4-dinitrophenyl)-hydroxylamine), dppf (1,1'-bis(diphenylphosphino)ferrocene), DIC (diisopropylcarbodiimide), DIEA (diisopropylethylamine), DMF (dimethylformamide), DMSO (dimethyl sulfoxide), DMSO-d6 (deuterated dimethyl sulfoxide), EDC (1-(3-dimethyl-amino) -propyl)-3-ethylcarbodiimide), ESI (electrospray ionization), EtOAc (ethyl acetate), Et2O (ethyl ether), EtOH (ethanol), FMOC (fluorenylmethoxycarbonyl), HATU (dimethylamino-([1,2,3]triazolo[4,5-b]pyridin-3-yloxy)-methylene]-dimethyl-ammonium hexafluorophosphate), HPLC (high performance liquid chromatography), i-PrOH (2-propanol), K2CO3 (potassium carbonate), LC (liquid chromatography), m-CPBA (3-chloroperoxybenzoic acid), MD Autoprep (mass directed automated preparation), MeOH (methanol), MgSO4 (magnesium sulfate), MS (mass spectrometry), MSH (O-(2,4,6-trimethylbenzenesulfonyl)hydroxylamine), MTBE (methyl tert-butyl ether), Mtr. (4-methoxy-2,3,6-trimethylbenzenesulfonyl), MW (microwave), NBS (N-bromosuccinimide), NaHCO3 (sodium bicarbonate), NaBH4 (sodium borohydride), NMM (N-methylmorpholine), NMR (nuclear magnetic resonance), POA (phenoxyacetate), Py (pyridine), (benzotriazol-1-yl-oxy-tripyrrolidinophosphine hexafluorophosphate), RT (room temperature), Rt (retention time), SFC (supercritical fluid chromatography), SPE (solid phase extraction), T3P (propylphosphonic anhydride), TBAF (tetra-n-butylammonium fluoride), TBTU (2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate), TEA (triethylamine), TFA (trifluoroacetic acid), THF (tetrahydrofuran), TLC (thin layer chromatography), UV (ultraviolet light).

[0878] Generally, the compounds according to formula (I) and related formulas of the present invention can be prepared from readily available starting materials. If these starting materials are not commercially available, they can be prepared by standard synthesis techniques. Generally, the synthetic route of any single compound of formula (I) and related formulas will depend on the specific substituents of each molecule, and those of ordinary skill in the art can understand these factors. The following general methods and procedures described in the examples below can be used to prepare compounds of formula (I) and related formulas. The reaction conditions described in the following schemes, such as temperature, solvent or co-reagents, are given as examples only and are not restrictive. It should be understood that, in the case of giving typical or preferred experimental conditions (i.e., reaction temperature, time, reagent mole number, solvent, etc.), unless otherwise stated, other experimental conditions can also be used. The optimal reaction conditions may vary depending on the specific reactants or solvents used, but those skilled in the art can determine these conditions using conventional optimization procedures. For all protection and deprotection methods, see "Protecting Groups" by Philip J. Kocienski, Georg Thieme Verlag Stuttgart, New York, 1994 and "Protective Groups in Organic Synthesis" by Theodora W. Greene and Peter GM Wuts, Wiley Interscience, 3rd edition, 1999.

[0879] According to T L , L I and S L The properties of the compound of formula (I) can be synthesized by different synthetic strategies. L , L I and S L As described above in the specification, unless otherwise stated.

[0880] The compound of formula (I) (wherein T L , L I and S L As defined above) may be prepared from alternative compounds of formula (I) using a suitable interconversion procedure, for example as described in the Examples hereinafter, or conventional interconversion procedures well known to those skilled in the art.

[0881] exist Fig.23 In which R represents H or methyl and Q 1 Indicates –CH 2 -or key.

[0882] PG is a suitable protecting group which is compatible with the chemistry described in Schemes 1 to 19. Preferred groups PG are as follows: benzyloxycarbonyl (Cbz), p-methoxybenzylcarbonyl (Moz or MeOZ) group, tert-butyloxycarbonyl (BOC) group, 9-fluorenylmethoxycarbonyl (FMOC) group, alkanoyl group such as acetyl (Ac) group, benzoyl (Bz) group, benzyl (Bn) group, carbamate group, p-methoxybenzyl (PMB), 4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP) group, arylsulfonyl group such as toluenesulfonyl (Ts) or benzenesulfonyl group.

[0883] The compound of formula (I) (wherein T L , L I and S L As defined above) can be prepared by methods and reactions known to those skilled in the art comprising T L -L I Some compounds with S L Reaction or by containing L I -S L Some compounds and T L Such reactions may be, but are not limited to, amide bond formation, aromatic substitution, alkylation, and metal-catalyzed cross-coupling reactions using conditions known to those skilled in the art and described in the Examples (Scheme 1) below.

[0884] Solution 1

[0885] T L -L I +S L →T L -L I -S L

[0886] T L +L I -S L →T L -L I -S L

[0887] Alternatively, T L , L I or S L The precursor of isocyanate is synthesized by reacting with other moieties constituting the compound of formula (I). Further steps will produce the compound of formula (I) using methods and reactions known to those skilled in the art.

[0888] For example, but not limited to this case, when S L Corresponding to (IIIa), T L -LI or L I The moiety can react with a phenol derivative (Aa) to produce intermediates (Ba) or (Baa), respectively, where R La Corresponding to L I Partially reacted functional groups, producing R L With L I Connection ( Fig.23 ). The reaction of intermediate (Ba) or (Baa) with ester (Ca) by aromatic substitution yields ester (Da) or (Daa), respectively. After saponification conditions, acid (Ea) or (Eaa), respectively, is obtained. Alternatively, aromatic substitution with the corresponding acid (Ca, R=H) will directly produce acid (Ea) and (Eaa), respectively. Coupling of amide with ester (Fa) will provide intermediate (Ga) and (Gaa), respectively, which after saponification will produce a compound of formula (Ia) or intermediate (Haa). Under conditions described in the examples below or known to those skilled in the art, (Haa) and T L Further reaction of some will lead to compounds of formula (Ia).

[0889] Medical Uses:

[0890] The present invention is directed to the degradation of circulating extracellular proteins that mediate diseases, such as those involving immunity, inflammation, hematopoietic / hematological disorders (including those caused or exacerbated by angiogenesis), and abnormal cell proliferation, such as tumors and cancer.

[0891] The bifunctional compounds of the invention can be administered in any manner that allows the bifunctional compound to bind to extracellular proteins, typically in the bloodstream, and carry them to cells carrying sortilin for endocytosis and degradation. Thus, examples of methods of delivering the degraders of the invention include, but are not limited to, oral, intravenous, buccal, sublingual, subcutaneous, and nasal.

[0892] In one aspect, the invention provides bifunctional compounds for targeting sortilin-mediated lysosomal degradation of extracellular target molecules. In one embodiment, the target molecule is a disease or disorder-associated protein.

[0893] In another aspect, the present disclosure relates to the use of the bifunctional compounds described herein as medicaments. The present disclosure relates to pharmaceutical compositions comprising the bifunctional compounds described herein.

[0894] In another aspect, the present disclosure relates to a method of targeting lysosomal degradation of an extracellular target protein, comprising administering to a subject in need thereof a bifunctional compound described herein.

[0895] In another aspect, the present disclosure relates to a method of reducing plasma levels of a target molecule comprising administering to a subject in need thereof a bifunctional compound described herein.

[0896] In one aspect, the present disclosure provides a bifunctional compound described herein for use in treating a disorder or condition mediated by an extracellular protein in a subject in need thereof.

[0897] In one aspect, the disclosure relates to a bifunctional compound as described herein for use in removing an extracellular target molecule from the plasma of a subject.

[0898] In one embodiment, the condition or disease is associated with abnormal levels of an extracellular protein. In one embodiment, the condition or disease is associated with abnormally high levels of an extracellular target protein.

[0899] In one embodiment, the disorder or disease is associated with a mutated or misfolded extracellular protein.

[0900] In one embodiment, the extracellular target protein is selected from the group consisting of TNF-α, PCSK9, ANGPTL-3, antibody light chain, IgG, IgE, IgA IL-1, IL-2, IL-6, IFN-γ, VEGF, TFG-β1, IL-21, IL-22, IL-5, IL-10, IL-8, cholinesterase, human CCL2, carboxypeptidase B-2, neutrophil elastase, factor Xa, factor XI, factor XIa, factor XII, factor XIII, prothrombin, coagulation factor VII, coagulation factor IX, fibroblast growth factor 1, FGF-2, fibronectin 1, kallikrein-1, lipopolysaccharide, prothrombin, factor VII, coagulation factor IX, fibroblast growth factor 1, FGF-2, fibronectin 1, kallikrein-1, lipopolysaccharide, prothrombin, factor VII, factor IX, prothrombin ... Protein lipase, human matrix metallopeptidase 1, macrophage migration inhibitory factor, transforming growth factor-p (TGF-p), thrombospondin-1 (TSP-T), CD40 ligand, urokinase-type plasminogen activator, tissue-type plasminogen activator (TPA), plasminogen (PLG), plasminogen activator inhibitor-1, placental growth factor, phospholipase A2 group IB, phospholipase A2 group IIA, complement factor B, complement factor D, complement factor H, complement component 5, and complement C1s.

[0901] In one embodiment, the extracellular protein is PCSK9. In another embodiment, the extracellular target protein is PCSK9, and the disorder or condition is a lipoprotein metabolism disorder. In another embodiment, the lipoprotein metabolism disorder is associated with abnormal PCSK9 levels. In another embodiment, the disorder is selected from dyslipidemia, hypercholesterolemia and coronary heart disease.

[0902] In one embodiment, the extracellular protein is TNF-α. In another embodiment, the extracellular target protein is TNF-α, and the disorder or condition is selected from rheumatoid arthritis, inflammatory bowel disease, graft-versus-host disease, ankylosing spondylitis, psoriasis, hidradenitis suppurativa, refractory asthma, systemic lupus erythematosus, diabetes, and induction of cachexia. In another embodiment, the disorder is associated with abnormal TNF-α levels. In one embodiment, the disorder or condition is an inflammatory disease. In one embodiment, the disorder or condition is an autoimmune disease. In one embodiment, the disorder or condition is cancer.

[0903] Thus, in one aspect, the present disclosure provides a method for targeting lysosomal degradation of TNFα, comprising administering an effective amount of a bifunctional compound as described herein.

[0904] In another aspect, the present disclosure provides a method of removing TNFα from the plasma of a patient or subject in need thereof, comprising administering a bifunctional compound as described herein.

[0905] In another aspect, the present disclosure provides the use of a bifunctional compound described herein in the preparation of a medicament for treating a disease or condition.

[0906] In another aspect, the present disclosure provides the use of a bifunctional compound described herein in the preparation of a medicament for treating a disease or condition mediated by TNFα.

[0907] In one aspect, the present disclosure provides a method of treating a disease or condition comprising administering to a subject in need thereof a bifunctional compound described herein.

[0908] In one aspect, the present disclosure provides a method of treating a disorder or condition mediated by TNFα, comprising administering to a subject in need thereof a bifunctional compound according to the present disclosure.

[0909] The examples demonstrate that the bifunctional compounds targeting TNFα according to the present disclosure promote the removal of TNFα from animal plasma.

[0910] In one embodiment, the extracellular protein is an antibody light chain or IgG. In another embodiment, the extracellular target protein is IgG, and the disorder or condition is selected from type 1 autoimmune pancreatitis, interstitial nephritis, Riddle's thyroiditis, storiform fibrosis, Mikulicz's disease, Kuttner's tumor, inflammatory pseudotumor, mediastinal fibrosis, retroperitoneal fibrosis (Ormond's disease), aortitis, periaortitis, proximal bile duct stenosis, idiopathic hypocomplementary tubulointerstitial nephritis, multifocal fibrosclerosis, pachymeningitis, pancreatic enlargement, tumefactive lesions, pericarditis, rheumatoid arthritis (RA), inflammatory bowel disease, multiple sclerosis, myasthenia gravis, thyroid eye disease, disease), chronic inflammatory demyelinating polyneuropathy, warm autoimmune hemolytic anemia, ankylosing spondylitis, primary Sjogren's syndrome, psoriatic arthritis, systemic lupus erythematosus (SLE), sclerosing cholangitis, and IgG monoclonal gammopathy, monoclonal gammopathy of undetermined significance (MGUS).

[0911] In one embodiment, the subject is a mammal. In one embodiment, the mammal is a human. Example

[0912] Example 1: S with peptide derivatization L Bifunctional compounds directly interact with the extracellular domain of sortilin

[0913] Purpose

[0914] In this embodiment, S is described. LThe equilibrium dissociation constant (K) of the binary binding event between the bifunctional compound derived from the peptide and the soluble extracellular domain of Sortilin-6his D ) was measured by microscale thermophoresis (MST).

[0915] Materials and methods

[0916] In these experiments, a 12-point titration series of peptide solutions with a total volume of 1.2 uL was prepared in a TTP LVDS 384-well plate (SPT Labtech) using a Mosquito pipetting robot (SPT Labtech) in a buffer consisting of 16.7% DMSO (v / v) and 0.05% Tween 20 (v / v). The plate was spun down before adding 8.8 uL of a solution containing 114 nM sortilin-6his and 28.4 nM RED-tris-NTA (Nanotemper) in 57 mM Bis-Tris Propane pH 9, 57 mM NaCl, 0.05% Tween 20 (v / v) to each well. At this point, the plate was sealed with tape and placed on an Eppendorf thermomixer, shaken at 600 rpm for 3 minutes, then rotated on a Fisherbrand plate rotator for 15 seconds and further incubated at 19°C for 1 hour. MST was measured on Monolith NT. An automated device (Nanotemper) in standard treated capillaries at 5% LED power and high MST power. Dose responses were extracted from the original heat map at 20 seconds heat time, and dissociation constants were estimated by sigmoid curve fitting in MO. Affinity analysis program (Nanotemper). All experiments were repeated twice.

[0917] Figure 1 A shows an MST binding experiment using fluorescent sortilin-6his (100 nM) and the compound according to SEQ ID NO.: 147, resulting in the measured dissociation constant (K D ) is 703nM (N=2). Figure 1 B shows the same experimental setup as in Fig. A1, but titrating the compound according to SEQ ID NO.: 157 which is unable to bind to the fluorescent sortilin-6his due to a specific amidation at the C-terminus.

[0918] in conclusion

[0919] The compounds of the invention may bind to Sortilin.

[0920] Table 1: Binding to Sortilin (Kd) (K D<100nM:++; K D 100-2000nM: +)

[0921]

[0922]

[0923] Example 2: S with peptide derivatization L Bifunctional compounds promote ternary interaction between sortilin and target proteins Complex formation

[0924] Purpose

[0925] This example explores the bifunctional compounds (where S L The ability of a peptide derived from a peptide to induce the formation of a ternary complex consisting of streptavidin, a bifunctional compound and sortilin-6his. In this example, the bifunctional molecule includes a biotin molecule at one end, followed by a peptide linker of varying length and composition, and finally a sortilin binding peptide sequence. The induction of the ternary complex is measured by a proximity-based TR-FRET assay.

[0926] Materials and methods

[0927] In these experiments, target compounds were titrated at 24 points in 384-well TTP LVDS plates (SPT Labtech) using a Mosquito LV pipetting robot (SPT Labtech) to a final buffer composition of 50 mM Bis-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20, with or without 10% DMSO.

[0928] The titration series (4 uL) was transferred to a black 384-well plate (Corning) and the following were added: 4 μL of 10 nM MAb anti-6HIS Tb (Cisbio) in 50 mM Bis-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20; 4 μL of 250 nM streptavidin-d2 (Cisbio) in 50 mM Bis-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20; 4 μL of 200 nM sortilin-6His in 50 mM Bis-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20; 4 μL of 200 nM sortilin in 50 mM Bis-Tris propane pH 8. 50 mM NaCl, 0.05% Tween 20 solution. The reaction was incubated at room temperature for 2.5 hours, at which time the HTRF signal ratio at 665 / 620 nm was measured using a microplate reader (ClarioStar, BMG Labtech). All reactions were repeated three times, and the data are presented as a function of the HTRF ratio and the concentration of the bifunctional compound.

[0929] Figure 2 A shows the proximity-induced HTRF ratios of two peptide compounds as a function of compound concentration. For the compound according to SEQ ID NO.: 147, a bell-shaped dose response was observed, indicating that the concentration of ternary complexes formed at both low and high compound concentrations was relatively low, a common pharmacological property observed for bifunctional molecules, often referred to as the hook effect. The compound according to SEQ ID NO.: 157 was identical to the compound according to SEQ ID NO.: 147 in all respects except for the addition of a C-terminal amidation, so this peptide did not induce a dose-dependent increase in the HTRF ratio, indicating that this peptide was unable to mediate ternary complex formation between streptavidin and sortilin.

[0930] Table 2: AUC of TR-FRET measurements of ternary complex formation (AUC<5000: +; AUC>5000: ++)

[0931] SEQ ID NO.: AUC score 141 + 142 + 145 + 146 ++ 147 ++ 148 ++ 149 + 155 ++

[0932] in conclusion

[0933] The compounds of the invention can mediate ternary complex formation between sortilin and a target protein.

[0934] Example 3: Internalization of target protein

[0935] This example discusses the sortilin internalization bifunctional compound (where S LIn this example, the peptide degrader consists of a sortilin binding motif linked to a biotin warhead that is envisioned to target fluorescent NeutrAvidin-650 (NA650).

[0936] The cellular uptake of NA650 was studied in HEK293 cells expressing the full-length sortilin receptor (Petersen et al.,

[13] ). The cells were seeded in 50 μL of culture medium (DMEM (Lonza) + 10% FBS (Sigma Aldrich) + 1% penicillin-streptomycin (Sigma Aldrich) + 1% GlutaMAX (Gibco) + 100 μg / mL Zeocin (Invitrogen)) in a poly-L-lysine-coated 96-well plate (Perkin Elmer) (40K / well) and incubated in a cell incubator (37°C, humidified, 5% CO 2 ) and incubate overnight.

[0937] The medium was replaced with an assay medium (DMEM (Lonza) + 10% fetal bovine serum (SigmaAldrich) + 1% penicillin-streptomycin (SigmaAldrich) + 1% GlutaMAX (Gibco)) containing NA650 (Invitrogen) (100 nM) (Thermo Fischer) and a peptide bifunctional compound (9.8 nM to 10 μM). The cells were incubated in a cell incubator for 3 hours and then washed in dPBS (Bionordika). The DyLight650 signal in the cell layer was detected using a fluorescent plate reader (BMG Labtech Clariostar).

[0938] Figure 3 A shows cell-associated FI signals after incubation of HEK293 / sortilin with a range of concentrations of SEQ ID NO.: 147, SEQ ID NO.: 157 or SEQ ID NO.: 155 (9.8 nM to 10 μM) and NA650 (Invitrogen) (100 nM) for 3 hours (n=2). The bell-shaped response to increasing concentrations of SEQ ID NO.: 147 and SEQ ID NO.: 157 is consistent with the hook effect commonly observed in systems involving ternary complex formation. Data points are shown as mean + / - SEM.

[0939] Figure 3 B shows the small molecule sortilin binder AF38496 ( Cell-associated FI signals after 3 h of incubation of HEK293 / sortilin with SEQ ID NO.: 147 (300 nM) and NA650 (Invitrogen) (100 nM) in the presence of either SEQ ID NO.: 147 (300 nM) or NA650 (Invitrogen) (100 nM) in the presence of either SEQ ID NO.: 172 (a peptide without a biotin warhead) (0.1 nM-100 μM), SEQ ID NO.: 172 (a peptide without a biotin warhead) (0.025 nM-25 μM), or biotin alone (0.1 nM-100 μM) (n=2). Data show that degrader-induced NA650 internalization is inhibited by competitive binding of AF38469 (IC50589 nM) or RQLL peptide (IC50149 nM) to sortilin and by competitive binding of biotin (IC5056 nM) to the target. Data points are shown as mean + / - SEM.

[0940] in conclusion

[0941] This example demonstrates that bifunctional compounds consisting of a sortilin binding motif linked to a biotin warhead mediate the uptake of extracellular NA650. Furthermore, the data provided demonstrate that internalization of the target is dependent on the binding of the bifunctional compound to both the sortilin receptor and the target.

[0942] Example 4: Sorting of target molecules to lysosomes for degradation

[0943] Purpose

[0944] This example discusses the bifunctional compound (where S L The ability of IL-67 (derived from peptides) to mediate the trafficking of extracellular targets through the endo-lysosomal pathway for degradation in lysosomes. This was studied by fluorescence microscopic evaluation of the subcellular localization of internalized targets and on-blot evaluation of intracellular target levels after inhibition of lysosomal function.

[0945] Materials and methods

[0946] Cellular uptake of the target NA650 was analyzed by fluorescence microscopy. For this assay, coverslips in 4-well plates were coated with poly-L-lysine before HEK293 / sortilin cells (25K cells / coverslip) were seeded in culture medium (as described in Example 3) and incubated in a cell incubator (37°C, humidified, 5% CO 2) and incubated overnight. The medium was replaced with an assay medium (as described in Example 3) containing NA650 (Invitrogen) (500nM) (Thermo Fischer), a compound according to SEQ ID No.: 147 (2μM) and AF38469 (100μM, MedChemExpress). The cells were incubated in a cell incubator for 4 hours, then washed in dPBS (Bionordika), and then fixed with dPBS+4% PFA (SigmaAldrich) for 15 minutes. The cells were permeabilized in TBS+0.1% Triton X-100 (Sigma Aldrich) for 15 minutes, then washed with dPBS, and incubated overnight at 4°C with a primary antibody (LAMP-1mAb H4A3, Abcam Ab25630). Incubate with secondary antibody (goat anti-mouse Alexa-Fluor488 (ThermoFisher)) for 2 hours at room temperature, then wash in dPBS and incubate in Hoechst (1:10.000, Sigma Aldrich) for 10 minutes. Coverslips were briefly immersed in 70% ethanol (VWR) and mounted on slides with fluorescent mounting medium (Agilent Dako). Fluorescent signals were analyzed using a laser scanning confocal microscope (LSM-800).

[0947] Figure 4A Fluorescence microscopy imaging data are provided showing that the target NA650 (DyLight650) co-localizes with lysosomal markers in HEK293 / sortilin cells fixed and immunostained with anti-LAMP1 (Alexa-488) after incubation with the bifunctional molecule SEQ ID NO.: 147 (300 nM) and NA650 (100 nM) for 2 hours. The nuclei were stained with Hoechst. The scale bar is 20 uM.

[0948] The intracellular levels of NA650 after lysosomal function inhibition were investigated in HEK293 / sortilin cells. Cells were seeded in 4-well plates (Thermo Fisher) coated with poly-L-lysine (250K / well) in 250 μL of culture medium (as described in Example 3) and incubated overnight in a cell incubator.

[0949] The medium was replaced with assay medium (DMEM (Lonza), 10% fetal bovine serum (SigmaAldrich), 1% penicillin-streptomycin (SigmaAldrich), 1% GlutaMAX (Gibco)) with or without NA650 (Invitrogen) (100 nM) (Thermo Fischer), peptide bifunctional compounds (300 nM) and leupeptin (80 μM) (Sigma Aldrich). The cells were incubated in a cell incubator for 24 hours, then washed with dPBS (Bionordika) and lysed in lysis buffer (STE buffer (SigmaAldrich) + 1% Nonidet TM P 40 substitute (SigmaAldrich) + cOmplete (Roche)). The lysate was centrifuged and the supernatant was mixed with LDS sample buffer (Thermo Fisher) and DTT (Sigma Aldrich), boiled for 5 minutes, and then the proteins were separated on a 4-12% Bis-Tris gel (Thermo Fisher). The proteins were blotted onto a nitrocellulose membrane (Thermo Fisher) and the DyLight650 signal was detected using iBright 1500 (Thermo Fisher).

[0950] The membrane was blocked in blocking buffer (TBS (Fisher Scientific) containing 1% Tween 20 (Sigma Aldrich) and 5% milk powder) for 1 hour, and then incubated with primary anti-β-actin antibody (1:3500) (Sigma Aldrich) for 2 hours at room temperature. The blot was washed three times in washing buffer (TBS (Fisher Scientific) + 1% Tween 20 (Sigma Aldrich) + 0.5% milk powder) and incubated with secondary anti-mouse HRP antibody (1:3500) (Abcam) for 1 hour at room temperature, then washed three times in washing buffer, and the blot was developed and analyzed using ECL reagent (Cytiva) and iBright1500 (Thermo Fisher).

[0951] Figure 4BProvided is further evidence of intracellular accumulation of the target after inhibition of lysosomal cysteine ​​protease activity by addition of leupeptin (80 μM). In the example shown, cells were incubated with 100 nM of NA650 (Invitrogen), a peptide bifunctional compound (300 nM) and leupeptin (80 μM) (Sigma Aldrich) for 24 hours, then cell lysates were harvested and proteins were separated on SDS-PAGE gels. The bar graph shows quantification of the upper band of NA650 (mean with std).

[0952] in conclusion

[0953] This example demonstrates that targets of bifunctional compounds are sorted from the extracellular space to the lysosomal compartment for subsequent degradation.

[0954] Example 5: Depletion of target molecules in the extracellular medium

[0955] Purpose

[0956] This example studies the L Whether bifunctional compounds derived from peptides can deplete targets from the extracellular space was addressed by evaluating the fluorescence intensity (FI) in the cell culture supernatant after incubation of cells with bifunctional compounds and NA650.

[0957] Materials and methods

[0958] HEK293 / sortilin cells were cultured in DMEM (Lonza) supplemented with 10% fetal bovine serum (Sigma), 1% GlutaMax supplement (Gibco), penicillin-streptomycin (Thermo Fischer), and the selection antibiotic 100 μg / mL zeocin (Invitrogen). 35k cells / well were seeded in poly-L-lysine-coated 96-well plates (Perkin Elmer) and incubated in a cell incubator (37°C, humidified, 5% CO 2) and incubate for 16-24 hours. The culture medium was discarded and the cells were washed with assay medium containing FluoroBright DMEM (Gibco) supplemented with 10% fetal bovine serum (Sigma) and 1% GlutaMax supplement (Gibco). The culture medium was discarded and replaced with assay medium or assay medium containing 100nM NA650 (Invitrogen). For wells evaluating bifunctional compounds, the assay medium also contained concentrations of 20nM to 5μM. After incubation in a cell incubator for up to 72 hours, the culture medium was harvested and the presence of NA650 was evaluated by fluorescence intensity (FI) measurement of DyLight650 using a fluorescent microplate reader (BMG Labtech Clariostar).

[0959] Figure 5A NA650 signal in HEK293 / sortilin cell culture supernatant after incubation with SEQ ID NO.: 147 (20 nM to 10 μM) and NA650 (Invitrogen) (100 nM) up to 72 hours. Single addition of the bifunctional compound resulted in a significant decrease in signal after 24 hours (16%), 48 hours (46%), and 72 hours (61%) of incubation. For all time points, the highest uptake of the target was observed at 312 nM of the degrader.

[0960] Figure 6 The NA650 FI signal in HEK293 / Sortilin cell culture supernatant is shown after incubation with NA650 (Invitrogen) (100 nM) and SEQ ID NO.: 147 (20 nM-5 μM) or SEQ ID NO.: 155 (20 nM to 5 μM), a bifunctional compound with improved affinity for Sortilin, resulting in further reduction of target in the cell culture supernatant after 48 hours and 72 hours compared to SEQ ID NO.: 147.

[0961] in conclusion

[0962] This example demonstrates that target concentrations in the culture medium decrease over time, indicating bifunctional compound-mediated target depletion and further suggesting a sustainable mechanism of action.

[0963] Example 6: Peptide Binding

[0964] Purpose

[0965] In this example, the equilibrium dissociation constant (KD) describing the binary binding event between the peptide and the soluble extracellular domain of Sortilin-6his was measured by microscale thermophoresis (MST).

[0966] Materials and methods

[0967] MST was performed as described in Example 1.

[0968] Table 3: Binding to Sortilin (Kd) (K D Rating: K D <25nM:+++; K D 25-250 nM: ++; K D 250-2500: +; K D >2500nM:0).

[0969]

[0970]

[0971] in conclusion

[0972] In this example, it was assessed whether the monofunctional peptides bind directly to the soluble extracellular domain of Sortilin and the binding events were quantified by measuring the dissociation constant at equilibrium.

[0973] Example 7: Conjugation of Sortilin Binding Peptides to Antibodies

[0974] Purpose

[0975] In this example, the monoclonal antibody amorobumab (anti-PCSK9, light chain SEQ ID NO.: 67, heavy chain SEQ ID NO.: 66) or adalimumab (anti-TNFα, light chain SEQ ID NO.: 69, heavy chain SEQ ID NO.: 68) is covalently coupled to a peptide comprising an NHS group at one end linked to a C-terminal sortilin binding peptide sequence RQLL, thereby generating bifunctional amorobumab-linked-RQLL (SEQ ID NO.: 159) or adalimumab-linked-RQLL (SEQ ID NO.: 161). Negative control samples were generated by coupling amorobumab to a peptide identical in all respects to the above except lacking the C-terminal peptide sequence RQLL, thereby generating samples amorobumab-linked (SEQ ID NO.: 160) or adalimumab-linked (SEQ ID NO.: 162).

[0976] Materials and methods

[0977] The NHS-containing peptides NHS-PEG4-Mal-CGGSGGGGSGGGGSGGRQLL (SEQ ID NO.: 168) and NHS-PEG4-Mal-CGGSGGGGSGGGGSGG (SEQ ID NO.: 169) were dissolved to 5 mM in DMSO and added to a 4 mg / ml solution of the antibody in PBS at a 25 molar excess. The reaction was incubated at room temperature for 24 hours, at which time the unreacted NHS-containing peptide was removed by applying the sample to a 2 mL Zeba spin desalting column. Two separate samples were prepared according to this protocol, namely antibody-linked-RQLL and antibody-linked. The reactions were followed by SDS-PAGE analysis, as shown in FIG. Fig. 7A and Figure 7B shown.

[0978] Fig. 7A SDS-PAGE analysis of NHS-linker (SEQ ID NO.: 169) or NHS-linker-RQLL (SEQ ID NO.: 168) conjugated to amorobumab under non-reducing or reducing (2 mM DTT + heat) conditions is shown. Successful conjugation of amorobumab to NHS-linker and / or NHS-linker-RQLL was observed, as can be seen from the mass shift compared to the unconjugated antibody.

[0979] Figure 7B SDS-PAGE analysis of the coupling of NHS-linker or NHS-linker-RQLL to adalimumab under non-reducing or reducing (2mM DTT+heating) conditions is shown. Successful coupling of NHS-linker (SEQ ID NO.: 169) and / or NHS-linker-RQLL (SEQ ID NO.: 168) to adalimumab was observed, which can be seen from the mass shift compared to the uncoupled antibody.

[0980] in conclusion

[0981] The compounds of the invention may be covalently coupled to antibodies.

[0982] Example 8: Bifunctional compounds derived from antibody conjugates bind to Sortilin with nM affinity

[0983] Purpose

[0984] In this example, the equilibrium dissociation constant (KD) describing the binary binding event between a monoclonal antibody conjugated to a sortilin binder (SEQ ID NO.: 159) and the soluble extracellular domain of sortilin-6his was measured by microscale thermophoresis (MST).

[0985] Materials and methods

[0986] The MST binding experiment of Amorobumab-linked-RQLL (SEQ ID NO.: 159) or Amorobumab-linked (SEQ ID NO.: 160) solution was performed according to the protocol adapted from Example 1.

[0987] Figure 8 : shows the MST binding response as a function of antibody concentration. From this experiment, a clear sigmoidal dose response was observed for amorobumab conjugated to a peptide containing the sortilin binding sequence (SEQ ID NO.: 159, amorobumab-linked-RQLL), resulting in a dissociation constant of 540 nM, while amorobumab conjugated to a peptide lacking the C-terminal sortilin binding sequence (amorobumab-linked, SEQ ID NO.: 160) did not produce a sigmoidal binding curve, indicating that the antibody is unable to bind to sortilin with a measurable dissociation constant.

[0988] in conclusion

[0989] Covalent coupling of the compounds of the invention to antibodies results in sortilin binding.

[0990] Example 9: Antibody-derived bifunctional compounds retain binding to antigen

[0991] Purpose

[0992] In this example, we further evaluated whether amorobumab-linker-RQLL and amorobumab-link retained the ability to bind to PCSK9-6HIS by gel-filtration.

[0993] Materials and methods

[0994] Amorobumab-linked-RQLL (SEQ ID NO.: 159) or amorobumab-linked (SEQ ID NO.: 160) was mixed with PCSK9-6HIS (SEQ ID NO.: 171) at a molar ratio of 1:2.5 (1.67 μM amorobumab and 4.175 μM PCSK9-6HIS) in a final volume of 200 μL and incubated at 4°C for 24 hours. The next day, the samples were spun down at 13,400 rpm for 15 minutes at 4°C to remove potential aggregates and applied to the same PureHPLC system (Cytiva) connected to Superdex200increase 10 / 300 (Cytiva). In each experiment, 0.5mL fractions were collected and analyzed by SDS-PAGE (reducing and non-reducing). Negative control (PCSK9-6his alone) was prepared according to a similar procedure as above.

[0995] FIG. 9 shows the PCSK9-6HIS and amorobizumab-linker-RQLL ( Fig. 9A ) or Amorobismub-Linker ( Fig. 9B ) form a complex which can be purified by gel filtration. This is obvious because Fig. 9C SDS-PAGE analysis of the protein complex peak fractions showed a decrease in the peak size of PCSK-6HIS and co-elution with Amorobismumab-Linker-RQLL.

[0996] in conclusion

[0997] A complex is formed between the target and a) Amorobizumab-Linker-RQLL (SEQ ID NO.: 159) or b) Amorobizumab-Linker (SEQ ID NO.: 160), which can be purified by gel-filtration. This is obvious because the peak size reduction of PCSK-6HIS and co-elution with Amorobizumab-Linker-RQLL are shown in SDS-PAGE analysis of c) protein complex peak fractions.

[0998] Example 10: Bifunctional compounds derived from antibodies form ternary complexes

[0999] Purpose

[1000] In this example, the ability of a monoclonal antibody conjugated to a sortilin binding peptide to mediate the formation of a ternary protein complex with a target protein and sortilin was investigated.

[1001] Materials and methods

[1002] In these experiments, the bifunctional compounds Amorobizumab-linked-RQLL (SEQ ID NO.: 159) and Adalimumab-linked-RQLL (SEQ ID NO.: 161) were titrated in a total volume of 4 μL in 24 spots in a 384-well TTP LVDS plate (SPT Labtech) using a Mosquito LV pipetting robot (SPT Labtech) in a final buffer consisting of 50 mM Bis-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20. The same test was performed with antibody conjugates without a binding motif (Amorobizumab-linked SEQ ID NO.: 160 and Adalimumab-linked SEQ ID NO.: 162).

[1003] PCSK9 Targets:The following were added to a black 384-well plate (Corning): 4 μL of 10 nM mAb anti-6HIS Tb (Cisbio) in 50 mM Bis-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20; 4 μL of 250 nM streptavidin-d2 (Cisbio) in 50 mM Bis-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20; 4 μL of 200 nM sortilin-6His in 50 mM Bi-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20; 4 μL of 200 nM biotin-avi-PCSK9 in 50 mM Bis-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20; and 4 μL of a solution of Amorobizumab-linked-RQLL (SEQ ID NO.: 159) or Amorobizumab-linked (SEQ ID NO.: 160) in 50 mM Bis-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20.

[1004] TNF-α Target: The following were added to a black 384-well plate (Corning): 4 μL of 10 nM mAb anti-6HIS Tb (Cisbio) in 50 mM Bis-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20; 4 μL of 250 nM streptavidin-d2 (Cisbio) in 50 mM Bis-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20; 4 μL of 1000 nM sortilin-6His in 50 mM Bi-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20; 4 μL of 200 nM TNFα-biotin in 50 mM Bis-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20 solution; and 4 μL of adalimumab-linked-RQLL (SEQ ID NO.: 161) or adalimumab-linked (SEQ ID NO.: 162) in 50 mM Bis-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20 solution.

[1005] After incubation for 2.5 hours at room temperature, the HTRF signal ratio at 665 / 620 nm was measured on a ClarioStar microplate reader (BMG Labtech). All reactions were performed in duplicate and the data are presented as a function of the HTRF ratio and the concentration of the bifunctional compound.

[1006] Fig. 10A Shown is the HTRF (665 / 620 nm) ratio normalized to the amorobumab-linkage plotted against a concentration gradient (290 nM-3.5 fM) of amorobumab conjugated to a linker with or without RQLL after 2.5 h of incubation. This indicates that ternary complex formation depends on the RQLL motif as shown by the bell-shaped curve. (Error bars = SD; n = 2)

[1007] Fig. 10B The resulting HTRF (665 / 620 nm) signal baseline corrected to the lowest global signal is shown for a concentration gradient (400 nM-4.7 fM) of linker-conjugated adalimumab with or without RQLL after 2.5 hours of incubation. This indicates that ternary complex formation depends on the RQLL motif, as shown by the bell-shaped curve. (Error bars = SD; n = 3)

[1008] Fig. 10C The resulting HTRF (665 / 620 nm) signal baseline corrected to the minimum global signal is shown after 2.5 hours of incubation for a concentration gradient (400 nM-4.7 fM) of adalimumab conjugated to the sortilin binding peptides SEQ ID NO.: 184 to SEQ ID NO.: 187. This indicates the formation of a ternary complex, as can be seen in a bell-shaped curve (error bars = SD; n = 3).

[1009] in conclusion

[1010] The compounds of the invention mediate ternary complex formation between Sortilin and the target.

[1011] Example 11: Antibody-derived bifunctional compounds mediate uptake of extracellular targets

[1012] Purpose

[1013] In this example, the ability of sortilin to promote internalization of an extracellular target of a monoclonal antibody conjugated to a sortilin-binding peptide was explored.

[1014] Materials and methods

[1015] In this embodiment, a bifunctional antibody conjugate targeting PCSK9: amorobizumab-linked-RQLL (SEQ ID NO.: 159) and a bifunctional antibody conjugate targeting TNF-α: adalimumab-linked-RQLL (SEQ ID NO.: 161) are provided.

[1016] Cellular uptake was measured as described in Example 3 using Cy5 (Thermo Fisher) labeled targets (100-200 nM).

[1017] Fig.11 A shows the cell-associated FI signal after HEK293 / sortilin was incubated for 3 hours with a range of concentrations of amorobumab-linker-RQLL (SEQ ID NO.: 159) (1 nM to 0.5 μM) and Cy5-PCSK9 (200 nM). In a control experiment, cells were incubated with amorobumab with a peptide lacking the C-terminal sortilin binding sequence, amorobumab-linker (SEQ ID NO.: 160).

[1018] Fig.11 B shows the cell-associated FI signal after HEK293 / sortilin was incubated with a range of concentrations of Adalimumab-linked-RQLL (SEQ ID NO.: 161) (250 pM-250 nM) and TNFα (100 nM) for 3 hours. In a control experiment, cells were incubated with Adalimumab with a peptide lacking the C-terminal sortilin binding sequence, Adalimumab-linked (SEQ ID NO.: 162).

[1019] Fig.11 C shows the cell-associated FI signal after HEK293 / sortilin was incubated with a series of concentrations of adalimumab (250 pM-250 nM) coupled to the sortilin binding peptides of SEQ ID NO.: 184 to SEQ ID NO.: 187 and TNFα (100 nM) for 3 hours.

[1020] in conclusion

[1021] This example demonstrates that monoclonal antibodies conjugated to sortilin binding peptides can mediate the uptake of extracellular disease proteins such as PCSK9 or TNFα.

[1022] Example 12: Production of anti-LC VHHs

[1023] Purpose

[1024] In this example, a bifunctional nanobody coupled to a sortilin binding peptide was generated that was able to bind both a kappa light chain antibody and sortilin (NB-linker-RQLL, SEQ ID NO.: 158). A control without the RQLL motif was also prepared (NB-linker, SEQ ID NO.: 71).

[1025] Materials and methods

[1026] The plasmids expressing the bispecific nanobodies (Nanobody-pET11d:28-B09 (SEQ ID NO.: 177); Nanobody-delRQLL: pET11d:23-F06, Genscript (SEQ ID NO.: 173)) were transformed into E. coli strain Shuffle-T7-Express-Lys-Y (NEB) and incubated at 37°C for 18 hours before plating on plates containing 100 μg / ml ampicillin. Overnight cultures were grown in 100 ml of LB medium (Luria-Bertani) at 30°C and 100 μg / ml ampicillin and 175 RPM. The cell pellet from the overnight culture was resuspended in 1 liter of 2xYT medium (tryptone 16 gr / l, yeast extract 10 gr / l, NaCl 5 gr / l, 100 μg / ml ampicillin) and incubated at 30°C and 200 RPM. At OD600 of 0.75, IPTG (isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 0.4 mM. The culture was then incubated at 16°C and 200 RPM for 18 hours. The cell pellet was harvested at 6000 RPM for 15 minutes and kept at -20°C.

[1027] After harvest, cells are resuspended in 20mM Tris-HCl pH 8.0,500mM NaCl,20mM imidazoles,1mM PMSF, and cracked by ultrasonic treatment.By centrifugal clarification lysate, and supernatant is loaded on 5mL HisTrap FF Crude post (Cytiva), this post is balanced in 20mM Tris-HCl pH 8.0,500mM NaCl,20mM imidazoles.The loaded post is washed to baseline, eluted with the linear gradient of 20-300mM imidazoles.Protein is dialyzed (MWCO:6-8kDa) to the solution of sodium acetate pH 5.5,50mM NaCl containing 20mM at 4 ℃ overnight, then concentrated (MWCO:5kDa) in centrifugal concentrator. The concentrated sample was subjected to size exclusion chromatography on a Superdex75 Increase column (Cytiva) equilibrated in 25 mM Tris-HCl pH 7.4, 150 mM NaCl. The pure protein sample was concentrated, flash frozen and stored at -80°C.

[1028] Figure 12 shows the size exclusion chromatography elution profiles and final samples of A) NB-Linker-RQLL, SEQ ID NO.: 158 and B) NB-Linker, SEQ ID NO.: 71. Pooled and concentrated size exclusion fractions are indicated by grey boxes.

[1029] Example 13: Nanobody-derived bifunctional compounds bind to target molecules

[1030] Purpose

[1031] In this example, we used gel-filtration to evaluate the binding ability of NB-Linker-RQLL (SEQ ID NO.: 158) and NB-Linker (SEQ ID NO.: 71) to antibodies containing kappa light chains. In this example, amorobizumab was used as a tool antibody with a kappa light chain.

[1032] Materials and methods

[1033] NB-Linker-RQLL (SEQ ID NO.: 158) and NB-Linker (SEQ ID NO.: 71) were mixed with tool antibody at a molar ratio of 2.2:1 in a final volume of 250 uL and allowed to incubate at room temperature for 1 hour. The sample was spun down at 13400 rpm for 15 minutes at 4°C to remove potential aggregates and applied to the same Pure HPLC system (Cytiva) connected to Superdex75Increase 10 / 300 (Cytiva). In each experiment, 0.5 mL fractions were collected and analyzed by SDS-PAGE. Negative control (NB-linker alone) was prepared according to a similar procedure as above.

[1034] FIG. 13 shows the formation of IgG target and ( Fig.13A )NB-Linker-RQLL (SEQ ID NO.: 158) or ( Fig. 13B )NB-linker (SEQ ID NO.: 71) complexes are formed and can be purified by gel-filtration. SDS-PAGE analysis of the main peak confirmed the presence of a complex with the target protein. The pooled and concentrated size exclusion fractions are indicated by gray boxes.

[1035] in conclusion

[1036] NB-Linker-RQLL (SEQ ID NO.: 158) can bind to the target IgG and form a complex. The complex can be separated by size exclusion chromatography and confirmed by SDS-PAGE analysis, which demonstrates the co-elution of the target protein and the nanobody bifunctional compound.

[1037] Example 14: Lysosomal degradation of extracellular target IgG

[1038] Purpose

[1039] This example explores the cellular uptake and extracellular target degradation by a nanobody carrying the sortilin binding peptide sequence RQLL and the human IgG-κ light chain binding sequence (NB-linker-RQLL, SEQ ID NO.: 158).

[1040] Materials and methods

[1041] Using a similar approach as described in Example 3, the cellular uptake of Cy5-conjugated IgG was studied in HEK293 cells expressing the Sortilin receptor.

[1042] Fig.14A Shown are cell-associated FI signals after incubation of HEK293 / sortilin with a range of concentrations of NB-Linker-RQLL (SEQ ID NO.: 158) (8 nM to 8 μM) and Cy5-conjugated IgG (5 nM, 50 nM, or 500 nM) for 3 hours.

[1043] In different experiments, 35K or 250K cells were seeded in 4-well or 96-well plates (Thermo Fisher) with 100 μL or 250 μL of culture medium (as described in Example 3) and incubated in a cell incubator (37°C, humidified, 5% CO 2 ) and incubate overnight.

[1044] The medium was replaced with assay medium containing SEQ ID NO.: 158 or control SEQ ID NO.: 71 (0 μM to 2 μM) and Cy5-conjugated IgG (50 nM) as described in Example 3). In addition, leupeptin (80 μM) was added to a small group of samples to inhibit lysosomal proteases.

[1045] The cells were incubated in a cell incubator (until 72 hours), and then the cell supernatant was collected and the cells were harvested and lysed (as described in Example 4). The lysates and culture media were subjected to SDS-PAGE (as described in Example 4). The proteins were blotted onto nitrocellulose membranes (Thermo Fisher) and the Cy5 signal was detected using iBright 1500 (Thermo Fisher). Alternatively, IgG was detected by Western blotting (as described in Example 4) using anti-human IgG antibody (1:3500, Abcam) HRP-conjugated anti-rabbit IgG (1:3500, Abcam).

[1046] After anti-human-IgG western blotting, the membrane was probed with mouse anti-human β-actin (1:3500, Sigma-Aldrich) HRP-conjugated anti-mouse IgG (1:3500, Abeam). β-actin blot was used as a loading control.

[1047] Subsequently, the membranes were stripped in regeneration stripping buffer (Thermo Scientific) for 15 min at 37°C and reprobed with anti-human-NTR3 (sortilin) ​​antibody (1:3500, BD Bioscience), followed by incubation with HRP-conjugated anti-mouse IgG (1:3500, Abcam), and developed and analyzed using ECL reagents (Cytiva) and iBright 1500 (Thermo Fisher).

[1048] Fig. 14B Cy5 FI in cell culture supernatant (upper blot) and lysate (middle blot) of HEK293 / sortin cells after incubation for 72 hours with Cy5-conjugated IgG (κ light chain) (50 nM) and NB-linker-RQLL (SEQ ID NO.: 158) as shown. When the target was co-incubated with increasing concentrations of nanobodies, the intensity of the bands corresponding to the IgG heavy chain (Ig HC) and light chain (Ig LC) in the culture medium decreased. In the conditioned medium, no IgG bands could be detected in cells incubated with the highest nanobody concentration, indicating that the target had been completely ablated from the culture medium. Weak Ig LC signals and degradation products with lower molecular weight were detected in the corresponding lysate samples. No IgHC signal was observed in the cell lysate, indicating that the internalized IgG target was rapidly degraded after internalization. The lower blot confirmed the expression of sortin in the cells by Western blotting.

[1049] Fig. 14C Western blots of Cy5 FI (upper blot) and IgG signals in lysates of HEK293 / sortilin cells after incubation for 6:30 h with Cy5-conjugated IgG (50 nM) as indicated, NB-linker-RQLL (SEQ ID NO.: 158) or control nanobody (NB-linker, SEQ ID NO.: 71) (250 nM) and the lysosomal protease inhibitor leupeptin (80 μM) are shown (n=2). After incubation with leupeptin, an increase in the intensity of the bands corresponding to Ig HC and Ig LC was observed. In addition, no degradation products were observed. All of these together indicate that IgG is degraded in lysosomes after internalization. The two lower blots confirm the loading of the gel (anti-actin) and the expression of sortilin in the cells by Western blotting. The bar graph shows the quantification of Ig HC expressed as mean + / - SEM.

[1050] in conclusion

[1051] The anti-LC nanobody-derived bifunctional compounds mediated the cellular uptake of extracellular IgG as well as the protein degradation of extracellular IgG via lysosomal degradation.

[1052] Example 15: Binding of small molecule bifunctional compounds to sortilin

[1053] Purpose

[1054] In this example, the equilibrium dissociation constant (KD) describing the binary binding event between different compounds and the soluble extracellular domain of Sortilin-6his was measured by microscale thermophoresis (MST).

[1055] Materials and methods

[1056] MST was performed as described in Example 1.

[1057] Fig.15 Shown is the binding of BF025, BF023 and BF020 (highest concentration 12.5 μM) titrated with constant NTA sortilin-6His (100 nM) in an MST assay. (Error bars = SD; n = 2)

[1058] Table 4: Binding to Sortilin (Kd) (K D Rating - K D <1nM:++++; K D 1-10nM:+++; K D 10-250nM:++; K D >250nM: +; no binding: 0)

[1059]

[1060]

[1061] in conclusion

[1062] The small molecule compounds of the present invention bind to Sortilin.

[1063] Embodiment 16: wherein S L Bifunctional compounds derived from small molecules promote the formation of ternary complexes

[1064] Purpose

[1065] To demonstrate the ability of bifunctional compounds to form ternary complexes with sortilin and target molecules.

[1066] Materials and methods

[1067] In these experiments, bifunctional compounds were titrated in a total volume of 5 μL at 24 points in a 384-well TTP LVDS plate (SPT Labtech) using a Mosquito LV pipetting robot (SPT Labtech) in a final buffer consisting of 50 mM Bis-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20 and 8% DMSO.

[1068] To a black 384-well plate (Corning) was added the following: 5 μL of 8 nM MAb anti-6HIS Tb (Cisbio) in 50 mM Bis-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20; 5 μL of 200 nM streptavidin-d2 (Cisbio) in 50 mM Bis-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20; 5 μL of 400 nM sortilin-6His in 50 mM Bis-Tris propane pH 8, 50 mM NaCl, 0.05% Tween 20, Tween 20; and 5 μL of the bifunctional compound in 50 mM Bis-Tris propane pH 8. Solutions in 50 mM NaCl, 0.05% Tween 20, with or without 8% DMSO.

[1069] After incubation at room temperature for 2.5 hours, the HTRF signal ratio 665 / 620 nm was measured on a ClarioStar microplate reader (BMG Labtech). All reactions were performed in triplicate.

[1070] Fig.16 The resulting HTRF (665 / 620 nm) signal baseline corrected to the minimum local signal plotted against bifunctional compound concentrations (5 μM-0.6 pM) after 2.5 hours of incubation is shown. As shown by the bell-shaped curve, the bifunctional compound exhibits a binary interaction with Sortilin.

[1071] Table 5: AUC of ternary complex formation (AUC score - AUC < 25.000: +; AUC > 25.000: ++)

[1072]

[1073]

[1074] in conclusion

[1075] The compounds of the invention mediate ternary complex formation between sortilin and the warhead-bound target.

[1076] Example 17: Internalization of target extracellular proteins in cells mediated by bifunctional compounds

[1077] Purpose

[1078] This example explores the role of Sortilin in L The ability of bifunctional compounds derived from small molecules to promote internalization of extracellular targets.

[1079] Materials and methods

[1080] In this example, the small molecule degraders consisted of sortilin binders (BF017 to BF028) linked to a warhead biotin to target fluorescent NeutrAvidin-650 (NA650).

[1081] Using a method similar to that of Example 3, the cellular uptake of NA650 was studied in HEK293 cells expressing the full-length Sortilin receptor.

[1082] Fig.17 Shown are cell-associated FI signals after 3 h incubation of HEK293 / sortilin with a range of concentrations (2 nM to 2 μM) of compounds B025, B023 or B020 and NA650 (100 nM).

[1083] Table 6: AUC of target internalization (AUC score - AUC <100: +; AUC >100: ++)

[1084]

[1085]

[1086] in conclusion

[1087] The compounds of the invention mediate cellular internalization of the target.

[1088] Example 18: Bifunctional compounds mediate depletion of targets in extracellular cell culture media

[1089] Purpose

[1090] In this example, the ability of small molecule bifunctional compounds to deplete targets from the extracellular space was investigated by evaluating the fluorescence intensity (FI) in the cell culture supernatant after incubation of cells with the bifunctional compounds and NA650.

[1091] Materials and methods

[1092] HEK293 / sortin cells (35k cells / 96 wells) were seeded in culture medium (as described in Example 3) and incubated in a cell incubator for 24 hours. The culture medium was discarded, the cells were washed with assay medium containing FluoroBright DMEM (Gibco) and the supplements described in Example 3, and then incubated in assay medium containing 100 nM of NA650 and small molecule bifunctional compounds (20 nM to 5 μM). After an incubation time of up to 72 hours, the culture medium was harvested and the presence of NA650 was evaluated by FI measurement of DyLight650 using a fluorescent microplate reader (BMG Labtech Clariostar).

[1093] Fig.18Shown are NA650 FI signals in HEK293 / sortilin cell culture supernatants after 72 h incubation with NA650 (100 nM) and the bifunctional compounds BF025, BF023, or BF020 (20 nM to 5 μM) as indicated.

[1094] in conclusion

[1095] This example demonstrates that small molecule bifunctional compounds can induce depletion of a target in cell culture supernatants.

[1096] Example 19: Targets of bifunctional compounds are sorted from the extracellular space to lysosomes for degradation

[1097] Purpose

[1098] This example investigates the ability of small molecule bifunctional compounds to mediate target degradation in lysosomes. This was investigated by evaluating fluorescent target levels in blotted cell lysates after inhibition of lysosomal function.

[1099] Materials and methods

[1100] Intracellular levels of NA650 after inhibition of lysosomal function were investigated in HEK293 / sortilin cells. Cells were seeded in 4-well plates (250k cells / well) as described in Example 3 in 250 μL of culture medium and incubated overnight.

[1101] The medium was replaced with assay medium (as described in Example 3) containing or not containing the following components: NA650 (100 nM) (Thermo Fischer), B025 (300 nM), sortilin binder AF38469 (10 μM) and leupeptin (80 μM) (Sigma Aldrich). The cells were incubated in a cell incubator for 24 hours and then washed and lysed in lysis buffer (as described in Example 4). The lysate was centrifuged and the supernatant was mixed with LDS sample buffer (Thermo Fisher) and DTT (Sigma Aldrich), boiled for 5 minutes, and then the proteins were separated on a 4-12% Bis-Tris gel (Thermo Fisher). The proteins were blotted onto a nitrocellulose membrane (Thermo Fisher) and the DyLight650 signal was detected using iBright 1500 (Thermo Fisher). After evaluating FI, the membrane was blocked in blocking buffer (TBS (Fisher Scientific) containing 1% Tween 20 (Sigma Aldrich) and 5% milk powder) for 1 hour, and then incubated with primary anti-β-actin antibody (1:3500) (Sigma Aldrich) for 2 hours at room temperature. The blot was washed three times in washing buffer (TBS (Fisher Scientific) + 1% Tween 20 (Sigma Aldrich) + 0.5% milk powder) and incubated with secondary anti-mouse HRP antibody (1:3500) (Abcam) for 1 hour at room temperature, then washed three times in washing buffer, and the blot was developed and analyzed using ECL reagent (Cytiva) and iBright 1500 (Thermo Fisher).

[1102] Fig.19 FI in HEK293 / sortilin lysates harvested after 24 h incubation with NA650 (100 nM), B025 (300 nM), AF38469 (10 μM), and leupeptin (80 μM) as indicated is shown. A band corresponding to NA650 is visible in cells incubated with B025 alone, but is absent when cells are co-incubated with the competitive sortilin binder AF38469. In addition, this band is increased in cells incubated with leupeptin. Low molecular weight NA650 degradation products are most intense in cells whose lysosomal activity is not inhibited by leupeptin. Western blots against β-actin are shown as controls.

[1103] in conclusion

[1104] This example provides data demonstrating intracellular accumulation of the target after inhibition of lysosomal cysteine ​​protease activity by the addition of leupeptin. This suggests that internalization mediated by the small molecule bifunctional compound leads to lysosomal degradation of the target. In addition, the data show that internalization mediated by the small molecule bifunctional compound can be strongly inhibited by the addition of a competitive sortilin binder and is therefore promoted by sortilin.

[1105] Example 20: Internalization of extracellular targets

[1106] Purpose

[1107] This example explores the ability of sortilin to mediate the internalization of bifunctional small molecules into extracellular targets

[1108] Materials and methods

[1109] In this example, the small molecule degrader consists of a sortilin binder linked to a warhead DNP to target anti-DNP antibodies (BF001 to BF016)

[1110] In a manner similar to Example 3, the cellular uptake of AlexaFluor488-anti-DNP antibody (Thermo Fisher) was studied in HEK293 cells expressing the full-length sortilin receptor.

[1111] Fig. 20 Shown are cell-associated FI signals (n=2) after 3 h incubation of HEK293 / sortilin with a range of concentrations (1.9 nM to 2 μM) of compounds BF011, BF006 or BF005 and AlexaFluor488-anti-DNP antibody (100 nM). The bell-shaped response to increasing concentrations of bifunctional compounds is consistent with the hook effect observed in systems involving ternary complex formation.

[1112] Table 7: AUC of target internalization (AUC score - AUC < 10.000: +; AUC > 10.000: ++)

[1113] Compound AUC score BF004 + BF005 ++ BF006 + BF008 + BF010 ++ BF011 + BF014 ++

[1114] in conclusion

[1115] The compounds of the invention mediate cellular internalization of the target.

[1116] Example 21: Targets of bifunctional compounds are sorted from the extracellular space to lysosomes for degradation

[1117] Purpose

[1118] This example explores the ability of small molecule bifunctional compounds to mediate target degradation in lysosomes. This was investigated by evaluating fluorescent target levels in blotted cell lysates after inhibition of lysosomal function.

[1119] Materials and methods

[1120] The intracellular levels of AlexaFluor488-anti-DNP antibody (ThermoFisher) after internalization were investigated in HEK293 / sortilin cells. Cells (250k) were seeded in 4-well plates as described in Example 3, in 250 μL of culture medium, and incubated overnight.

[1121] The medium was replaced with an assay medium (as described in Example 3) containing AlexaFluor488-anti-DNP antibody (Thermo Fisher) (100 nM) and BF005 (30 nM). Selected samples were supplemented with sortilin inhibitor AF38469 (10 μM) or lysosomal protease inhibitor leupeptin (80 μM) (Sigma Aldrich). The cells were incubated in a cell incubator for 3-6:30 hours, then washed with dPBS (Bionordika), replaced with an assay medium without BF005 and AlexaFluor488-anti-DNP antibody and incubated in a cell incubator until 24 hours, or the cells were lysed. After incubation, the cells were lysed in lysis buffer, the lysate was subjected to SDS-PAGE, and the protein was then transferred to a nitrocellulose membrane as described in Example 4. AlexaFluor488 FI signal was detected using iBright 1500 (Thermo Fisher). After evaluating the FI signal, the membrane was blocked and incubated with primary anti-β-actin antibody (1:3500) (SigmaAldrich) and secondary anti-mouse HRP antibody (1:3500) (Abcam) as described in Example 4, and developed and analyzed using ECL reagent (Cytiva) and iBright1500 (Thermo Fisher). Subsequently, the membrane was stripped in regeneration stripping buffer and probed with anti-human NTR3 (sortilin) ​​antibody (1:3500, BD Bioscience) and HRP-conjugated anti-mouse IgG (1:3500, Abcam).

[1122] Fig.21AShown is the FI in HEK293 / sortilin lysates obtained after 3 hours of incubation with AlexaFluor488-anti-DNP antibody (100 nM) and BF005 (30 nM), removal of the target and degrader by replacing the medium with assay medium, further incubation from 0 to 24 hours, and then lysis of the cells as described above. The band corresponding to the Ig heavy chain (HC) is visible at the 0 hour time point, and the band intensity decreases with increasing incubation time. Western blots of anti-β-actin and anti-sortilin are shown as controls. The bar graph shows the quantification of the FI signal of the HC band normalized to the β-actin signal (mean + / - SEM) (n = 2).

[1123] In different experiments, HEK293 / sortilin cells were incubated with or without AlexaFluor488-anti-DNP antibody (100 nM), BF005 (30 nM), sortilin binder AF38469 (10 μM) and leupeptin (80 μM). Cells were incubated in a cell incubator for 6:30 hours, after which they were washed with dPBS and lysed to evaluate intracellular accumulation of targets as described above.

[1124] Fig.21B FI in lysates of HEK293 / sortilin incubated for 6:30 hours with AlexaFluor488-anti-DNP antibody (100 nM), BF005 (30 nM) and leupeptin (80 μM) as indicated are shown. SB001 (30 nM) and AF38469 (10 μM) were included as controls. A band corresponding to the Ig heavy chain (HC) is visible in lysates of cells co-incubated with target and BF005 degrader. The intensity of this band increases significantly after inhibition of lysosomal degradation with leupeptin and is absent in cells to which a competing sortilin binder (AF38469) or a control degrader without a DNP warhead (SB001) was added. The bar graph shows quantification of the FI signal of the HC band normalized to the β-actin signal (mean + / - SEM) (n=2).

[1125] in conclusion

[1126] The data provided in this example demonstrate rapid degradation of the internalized target. In addition, the data show intracellular accumulation of the target after inhibition of lysosomal cysteine ​​protease activity by the addition of leupeptin, indicating lysosomal degradation of the desired target. In addition, the data show that small molecule bifunctional compound-mediated internalization is strongly inhibited by competitive sortilin binding.

[1127] Example 22: Compounds of the invention mediate cellular uptake of target proteins via the Sortilin receptor.

[1128] Purpose

[1129] This example investigates the role of sortilin in the cellular internalization of extracellular targets mediated by the bifunctional compounds of the invention.

[1130] Materials and methods

[1131] In a manner similar to Example 3, cellular uptake of the target was studied in non-transfected HEK239 cells and HEK293 cells expressing the Sortilin receptor.

[1132] Fig. 22 Shown are cell-associated FI signals after 3 hours of incubation of HEK293 or HEK293 / Sortilin with a range of concentrations (2 nM to 2 μM) of three different bifunctional compounds: SEQ ID NO.: 155, NB-linked-RQLL (SEQ ID NO.: 158) or a small molecule bifunctional compound (BF005) and the corresponding targets as indicated. The data show that in all bifunctional compound formats, a bell-shaped FI response was shown in cells expressing Sortilin with increasing concentrations of the degrader. No response was observed in HEK293 cells.

[1133] in conclusion

[1134] This example demonstrates that cellular expression of Sortilin is required for bifunctional compound-mediated target uptake.

[1135] Example 23: Binding to TNFα

[1136] Purpose

[1137] The compounds according to the invention were evaluated for binding to TNFα.

[1138] Materials and methods

[1139] MST was measured similarly to Example 1 using TNFα-6his.

[1140] result

[1141] Fig.24 A Detection of binding of BF080, BF081 and BF082 titrated with constant NTA-labeled TNFα-6His (100 nM) in MST assay. (Error bars = SD; n = 2)

[1142] Table 8: Kd of TNFα binding (Kd score - K D <2μM:++; K D >2μM: +)

[1143] Compound <![CDATA[K D (Rating)]]> Compound <![CDATA[K D (Rating)]]> BF040 + BF081 ++ BF080 ++ BF082 + BF079 ++ BF077 ++ BF076 ++ BF0100 ++

[1144] Example 24: Bifunctional Compound-Mediated Cellular Uptake of TNFα

[1145] Purpose

[1146] Compounds according to the present disclosure were evaluated for binding to TNFα.

[1147] Materials and methods

[1148] Using a method similar to that of Example 3, the cellular uptake of TNFα was studied in HEK293 cells expressing the full-length Sortilin receptor using the TNFα target.

[1149] Fig.24 B shows a Western blot of mouse anti-TNFα (Invitrogen, MA5-23720) of cell lysates of HEK393 / sortilin incubated with a range of concentrations (100 nM-10 μM) of compounds BF040 and BF043 and TNFα (100 nM) for 24 hours. Control cells were incubated without addition of compounds and TNFα or with TNFα alone. A Western blot of anti-β-actin (Sigma, A5441) is shown as a control.

[1150] Fig.24 C shows the cell-associated FI signal after HEK293 / sortilin was incubated with a range of concentrations (0.5 nM to 20 μM) of BF040, BF043 and BF042 and Cy5-TNFα (100 nM) in the presence of 80 μM leupeptin for 24 hours. The efficacy of individual bifunctional compounds was evaluated by the AUC of TNFα internalization (Table 9)

[1151] Table 9: AUC of TNFα internalization (AUC score - AUC>25.000: ++++; AUC 10.000-25.000: +++; AUC1000-10.000: ++; AUC<1000: +)

[1152]

[1153]

[1154] Fig.24 D shows the cell-associated FI signal after HEK293 / sortilin cells were incubated with BF042 (0.5 nM to 20 μM) and Cy5-TNFα with or without the addition of leupeptin (80 μM) for 24 hours. Inhibition of lysosomal degradation by the addition of leupeptin resulted in a significant increase in the cell-associated signal.

[1155] In different experiments, HEK293 / sortilin cells were incubated with compound gradient series and 20 nM TNFα for up to 72 hours. After incubation, the remaining TNFα in the conditioned medium was assessed using the human TNFα Quantikine ELISA kit (R&D systems, DTA00D).

[1156] Fig.24 E shows the TNFα level in the conditioned medium of HEK293 / sortin cells 72 hours after adding 20 nM TNFα and the bifunctional molecules BF040, BF043 or BF042. TNFα is shown as a % normalized to the level measured in the conditioned medium of cells without the addition of the bifunctional molecules. After 72 hours, the maximum clearance rates (calculated by adding TNFα (100%) minus the remaining TNFα (%)) for BF040, BF043 and BF042 were 39.4%, 63.3% and 74.4%, respectively.

[1157] To monitor the cellular degradation of the target over time, a pulse-chase experiment was performed: HEK293 / sortilin cells were pre-incubated with bifunctional compounds (3 μM) and TNFα (100 nM) for 24 h, and then the culture supernatant was replaced with fresh medium without the target and bifunctional compounds. After replacing the medium, the cells were incubated for up to 24 h, and TNFα levels were assessed at different time points by Western blotting of cell lysates.

[1158] Fig.24 F shows anti-TNFα Western blot of HEK293 / sortilin cell lysates harvested at the indicated time points (0 hours to 24 hours) after preincubation with BF042 (3 μM) and TNFα (100 nM) for 24 hours. The preincubation medium was replaced at the 0 hour time point. A Western blot against β-actin is shown as a control. The cell lysate band representing TNFα is most intense at the 0 hour time point, significantly reduced at 1 hour, and almost undetectable after 7 hours of incubation, indicating that TNFα is rapidly degraded after internalization.

[1159] Fig.24 G shows cell-associated FI signals after HEK293 / sortilin were incubated for 24 h with a range of concentrations (0.5 nM to 20 μM) of BF042 and Cy5-TNFα (100 nM) with or without the addition of sortilin binder SB013 (1.25 μM, 5.0 μM, and 10 μM) or DMSO.

[1160] Fig.24H shows cell-associated FI signals after 24 h incubation of HEK293 / sortilin with BF077 (1 μM) and Cy5-TNFα (100 nM) in the presence of increasing concentrations of TF018 or TF005 as competitors for TNFα binding.

[1161] Conclusion The compounds of the present invention mediate the cellular internalization and lysosomal degradation of TNFα.

[1162] Example 25: In vivo efficacy in a mouse model of acute systemic inflammation

[1163] Purpose

[1164] To evaluate the ability to remove TNFα from plasma in animal models of systemic inflammation.

[1165] Materials and methods

[1166] The in vivo efficacy of the degraders was investigated in a mouse model of acute systemic inflammation induced by lipopolysaccharide (LPS). Female C57BL / 6J mice aged 8 weeks were used in this model and LPS was administered as an IP injection at a final concentration of 0.5 mg / kg in PBS in a volume of 10 ml / kg. Animals were dosed with test compounds (100-0.3 mg / kg / IP), dexamethasone (Dex) control (0.5 mg / kg / PO), or vehicle control (IP) 30 minutes to 3 hours before LPS administration. Untreated (normal) animals were included as controls for LPS-induced TNFα expression. One hour after LPS administration, blood samples were collected and TNFα levels were assessed by cytometric bead array assay.

[1167] result

[1168] Fig.25 TNFα plasma levels of animals treated with vehicle, Dex (0.5 mg / kg) and BF094 (10 mg / kg or 30 mg / kg) 1 hour after LPS administration are shown. Normal animals served as untreated controls. n=5 (normal / vehicle / Dex) or n=10 per group. A significant decrease in plasma TNFα was observed in animals treated with BF094 compared to the vehicle group. n=5 (normal / vehicle / Dex) or n=10 animals per group. ****p<0.0001 (one-way ANOVA).

[1169] in conclusion

[1170] The compounds according to the present invention can mediate targeted protein degradation in vivo. The bifunctional compounds can remove the target protein of interest from animal plasma. Specifically, the results show that the TNFα bifunctional compounds reduce the TNFα levels in the plasma of LPS-stimulated animals. The reduction of TNFα is comparable to the effect produced by dexamethasone, a known anti-inflammatory agent.

[1171] Example S1: Synthesis Scheme

[1172] General conditions

[1173] Compounds according to formula (I) can be prepared from readily available starting materials using solution phase and solid phase chemical schemes or mixed solution and solid phase schemes by several synthetic methods. Examples of synthetic routes are described in the following embodiments. All reported yields are non-optimized yields. Unless otherwise indicated, the compounds of formula (I) and related formulas obtained as racemic mixtures can be separated to provide enantiomerically enriched mixtures or pure enantiomers.

[1174] The standard conditions are as follows:

[1175] All temperatures are in degrees Celsius (°C) and are uncorrected.

[1176] Reagent grade chemicals and anhydrous solvents were purchased from commercial sources and used without further purification unless otherwise stated.

[1177] Use pre-packaged disposable SiO on Biotage instruments 2 The stationary phase column was subjected to silica gel chromatography with an eluent flow rate ranging from 15 mL / min to 200 mL / min and UV detection (254 nm and 280 nm).

[1178] Reverse phase preparative HPLC was performed using a C18 column with UV detection (215 nm, 220 nm and 254 nm) using MeCN in water (10 mM NH 4 HCO 3 ) or a gradient elution of MeCN in water (0.04% HCl) or MeCN in water (0.2% HCOOH).

[1179] Analytical HPLC chromatograms are performed using two main methods:

[1180] - Shimadzu 20AB was used (gradient was 10-80% B in 3.00 minutes, kept at 80% B for 1.0 minute, 80-10% B in 0.01 minutes, and then kept at 10% for 0.50 minutes (0.01-4.00 minutes: flow rate was 0.5 ml / min; 4.01-4.50 minutes: flow rate was 1.0 ml / min). Mobile phase A was 0.037% trifluoroacetic acid in water, and mobile phase B was 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was Kinetex 5um C18100A 50*2.1mm. The detection method was diode array (DAD).

[1181] - Shimadzu 20AD was used (gradient was 10-80% B in 3.00 minutes, kept at 80% B for 0.9 minutes, 80-10% B in 0.03 minutes, and then kept at 10% for 0.57 minutes (0.01-3.91 minutes: flow rate was 0.8 ml / min; 3.92-4.50 minutes: flow rate was 1.2 ml / min). Mobile phase A was 10 mM NH4HCO3 aqueous solution, and mobile phase B was 100% acetonitrile. The column used for chromatography was X-bridge Shield RP182.1*50mm column (5um particles). The detection method was diode array (DAD).

[1182] There are two main methods used to analyze the products by LCMS:

[1183] - Used on Agilent 1200 & 6110B (gradient was 5% B in 0.40 min, 5-95% B in 0.4-3.0 min, 95% B for 1.00 min, then 95-5% B in 0.01 min, flow rate was 1.0 ml / min. Mobile phase A was 0.037% trifluoroacetic acid in water, mobile phase B was 0.018% trifluoroacetic acid in acetonitrile. The column used for chromatography was Kinetex C18 50*2.1 mm column (5 um particles). The detection method was diode array (DAD) and positive electrospray ionization. The MS range was 100-1000.

[1184] - Used on Agilent 1200 & 6110B (gradient 5% in 0.40 min, 5-95% B in 0.40-3.0 min, hold 95% B for 0.45 min, then 95-5% in 0.01 min, flow rate 0.8 ml / min. Mobile phase A was H 2 O + 10 mM NH 4 HCO 3, mobile phase B is acetonitrile. The column used for chromatography is Xbridge Shield RP182.1*50mm column (5um particles). The detection method is diode array (DAD) and positive electrospray ionization. The MS range is 100-1000.

[1185] There are two main methods used for the analysis of intermediates by LCMS:

[1186] -The column used for chromatography on Shimadzu LC-20AD&MS2020 was Luna-C18 2.0*30 mm, (3 um particles). The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was 0.037% trifluoroacetic acid in water, and mobile phase B was 0.018% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 10-80% B in 4.30 minutes: 10% B in 0.01 minutes, 10-80% B (0.01-3.50 minutes), 80-10% B (3.50-3.80 minutes), and kept at 10% B for 0.50 minutes. The flow rate was 0.8 mL / min (0.01-3.80 minutes) and 1.2 mL / min (3.81-4.30 minutes).

[1187] - On Agilent 1200 & 6110B, the Agilent 1200 & 1956A column used for chromatography was XbridgeShield RP18 2.1*50 mm (5 μm particles). The detection method was diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was 10 mM ammonium bicarbonate in water, and mobile phase B was HPLC grade acetonitrile. The gradient was 10-80% B in 3.00 minutes: 10% B in 0.00 minutes, 10-80% B (0.00-2.00 minutes), kept at 80% B for 0.48 minutes, 80-10% B (2.48-2.50 minutes), kept at 10% B for 0.5 minutes. The flow rates were 1.0 mL / min (0.00-2.48 min) and 1.2 mL / min (2.50-3.00 min).

[1188] The 1H NMR spectra were recorded by 11 instruments with different data, as shown below:

[1189] NMR-A: Bruker AVANCE NEO 400MHz / 54mm instrument (MRCA 400 / 54 / ASC, 16971)

[1190] NMR-B: BrukerAVANCE III 400MHz / 54mm UltraShield Plus, long hold time, instrument (BZH 439'400'70I, D335 / 54-6776)

[1191] NMR-C: Bruker AVANCE III 400MHz / 54mm Ascend instrument (BZH 994'400'70I, D315'54-9223)

[1192] NMR-D: Varian 400MR 400MHz / 54mm instrument (MRCA 400 / 54 / ASC, MRYOO20874)

[1193] NMR-E: Bruker AVANCE III 400MHz / 54mm Ascend instrument (BZH 993'400'70I, D315'54-9213)

[1194] NMR-F: Bruker AVANCE III 400MHz / 54mm Ascend instrument (BZH 1157'400'70I, D315'54-9574)

[1195] NMR-H: Bruker AVANCE III 400MHz / 54mm Ascend instrument (BZH 1126'400'70I, D315'54-9527)

[1196] NMR-J: Bruker AVANCE NEO 400MHz / 54mm Ascend instrument (BZH 1396'400'70I, D315'54-10089)

[1197] NMR-K: Bruker AVANCE NEO 400MHz / 54mm Ascend instrument (BZH 1373'400'70I, D315'54-10026)

[1198] NMR-S: Varian 400MR 400MHz / 54mm instrument (MRCA 400 / 54 / ASC, 20609)

[1199] NMR-Y: Varian 400MR 400MHz / 54mm instrument (MRCA 400 / 54 / ASC, 20188)

[1200] Chemical shifts are referenced to the solvent peak, in 1H NMR, CDCl3 The solvent peak appears at 7.27 ppm, DMSO-d 6 The solvent peak appears at 2.50 ppm, D 2 The solvent peak of O appears at 4.79 ppm, CD 3 The solvent peak of OD appeared at 3.31 ppm.

[1201] SB001

[1202] (2S)-5,5-Dimethyl-2-[[6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl]amino]hexanoic acid

[1203]

[1204] Step 1: 5-Hydroxy-N-methyl-naphthalene-1-carboxamide

[1205] Trimethylaluminum (2M, 24.73mL, 10 equiv) was added dropwise to a solution of methylamine (2.67g, 39.56mmol, 8 equiv, HCl) in DCE (40mL) at 0°C. After addition, the mixture was stirred at this temperature for 15 minutes, and then 5-hydroxynaphthalene-1-carboxylic acid methyl ester (1g, 4.95mmol, 1 equiv) in DCE (10mL) was added dropwise at 0°C. The resulting mixture was stirred at 80°C for 16 hours. Then, the reaction mixture was cooled to 0°C, DCM (100mL) was added, and then 1N HCl solution was added until the aluminum salt was dissolved (100mL). The mixture was extracted with DCM (100mL x 3). The combined organic layers were washed with Na 2 SO 4 Drying, filtration and concentration in vacuo gave 5-hydroxy-N-methyl-naphthalene-1-carboxamide (500 mg, 50.24% yield, 100% purity) as a white solid.

[1206] Step 2: 6-[[5-(Methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylic acid methyl ester

[1207] A mixture of methyl 6-bromopyridine-3-carboxylate (697.35 mg, 3.23 mmol, 1.2 eq.), 5-hydroxy-N-methyl-naphthalene-1-carboxamide (541 mg, 2.69 mmol, 1 eq.), potassium phosphate (570.70 mg, 2.69 mmol, 1 eq.) and CuI (512.04 mg, 2.69 mmol, 1 eq.) in DMSO (10 mL) was degassed and heated with N 2 Purge three times, then 2 The mixture was stirred at 120°C for 16 h under a 24-hour atmosphere. 2O (40 mL) and extracted with EA (20 mL x 4). The organic layer was washed with Na 2 SO 4 Dry, filter and concentrate under reduced pressure.

[1208] The residue was purified by flash silica gel chromatography ( 12g The mixture was purified by silica flash column with a gradient of 0-60% ethyl acetate / petroleum ether as eluent at a flow rate of 25 mL / min. Methyl 6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylate (580 mg, 64.11% yield, 100% purity) was obtained as a white solid.

[1209] Step 3: 6-[[5-(Methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylic acid

[1210] To methyl 6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylate (250 mg, 743.29 μmol, 1 eq.) was dissolved in THF (15 mL) and H 2 O (5 mL) was added to the mixture of LiOH. 2 O (62.38 mg, 1.49 mmol, 2 eq.). The mixture was stirred at 25 °C for 2 h. The mixture was concentrated under reduced pressure and the residue was acidified to pH 2 with HCl (1 M). The residue was extracted with EA (20 mL x 5). The combined organic layers were washed with Na 2 SO 4 Dry, filter and concentrate in vacuo to afford 6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylic acid (200 mg, 83.48% yield) as a white solid.

[1211] Step 4: 6-[[5-(Methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl chloride

[1212] A mixture of 6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylic acid (200 mg, 620.51 μmol, 1 eq), oxalyl chloride (157.52 mg, 1.24 mmol, 108.63 μL, 2 eq), and DMF (4.54 mg, 62.05 μmol, 4.77 μL, 0.1 eq) in DCM (20 mL) was degassed at 0 °C and then heated under N 2 The mixture was stirred at 25°C for 5 hours under atmosphere. The reaction mixture was concentrated under reduced pressure to give 6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl chloride (211 mg, crude) as a colorless gum.

[1213] Step 5: (2S)-5,5-dimethyl-2-[[6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl]amino]hexanoic acid

[1214] To a mixture of (2S)-2-amino-5,5-dimethyl-hexanoic acid (98.59 mg, 619.20 μmol, 1 eq) and TEA (93.98 mg, 928.81 μmol, 129.28 μL, 1.5 eq) in DCM (5 mL) was added 6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl chloride (211 mg, 619.20 μmol, 1 eq). The mixture was stirred at 20 ° C for 16 hours. The mixture was filtered and concentrated in vacuo. The residue was purified by preparative HPLC (HCl conditions). (2S)-5,5-dimethyl-2-[[6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl]amino]hexanoic acid was obtained as a white solid (13.7 mg, 4.77% yield, 99.0% purity).

[1215] LCMS(ESI)[M+H] + m / z: Calculated 464.21, Found 464.1

[1216] 1 H NMR (400 MHz, DMSO-d 6 )δ:ppm 0.85(s,10H)1.14-1.36(m,2H)1.63-1.87(m,2H)2.87(d,J=4.52Hz,3H)4 .21-4.38(m,1H)7.27(d,J=8.53Hz,1H)7.38(d,J=7.28Hz,1H)7.52(dd,J= 8.41,7.15Hz,1H)7.58-7.67(m,2H)7.88(d,J=8.53Hz,1H)8.10(d,J=8.5 3Hz,1H)8.33(dd,J=8.78,2.51Hz,1H)8.45-8.58(m,2H)8.64-8.74(m,1H)

[1217] SB002

[1218] 2-[[6-Methyl-4-[4-(methylcarbamoyl)phenyl]-2-pyridyl]carbamoyl]-5-(trifluoromethyl)benzoic acid

[1219]

[1220] Step 1: 4-(2-amino-6-methyl-4-pyridinyl)-N-methyl-benzamide

[1221] In N 2 4-Bromo-6-methyl-pyridin-2-amine (1 g, 5.35 mmol, 1 eq.) and [4-(methylcarbamoyl)phenyl]boronic acid (1.05 g, 5.88 mmol, 1.1 eq.) were reacted in dioxane / H2O at 25 °C. 2 O (18 mL) was added to the mixture in one portion. 2 CO 3 (1.70 g, 16.04 mmol, 3.0 equiv) and Pd(dppf)Cl 2 (391.21 mg, 534.65 μmol, 0.1 eq.). The mixture was heated to 90 °C and stirred for 4 hours. The reaction was filtered and the filtrate was concentrated. The residue was purified by silica gel chromatography eluting with (DCM: MeOH = 91: 9 to 90: 10) to give a brown solid (100 mg, 7.75% yield, 100% purity).

[1222] Step 2: 4-[2-[1,3-dioxo-5-(trifluoromethyl)isoindolin-2-yl]-6-methyl-4-pyridinyl]-N-methyl-benzamide

[1223] In N 2 A mixture of 5-(trifluoromethyl)isobenzofuran-1,3-dione (89.57 mg, 414.44 μmol, 1 eq.) and 4-(2-amino-6-methyl-4-pyridyl)-N-methyl-benzamide (100 mg, 414.44 μmol, 1 eq.) in AcOH (3 mL) was added at 25° C. The mixture was heated to 120° C. and stirred at 120° C. for 2 hours, then concentrated in vacuo.

[1224] The residue was purified by column chromatography (SiO 2 , PE:EtOAc=2:1 to 1:1) and concentrated to obtain 4-[2-[1,3-dioxo-5-(trifluoromethyl)isoindolin-2-yl]-6-methyl-4-pyridinyl]-N-methyl-benzamide (120 mg, 273.11 μmol, 65.90% yield, 100% purity) as a yellow oil.

[1225] Step 3: 2-[[6-methyl-4-[4-(methylcarbamoyl)phenyl]-2-pyridyl]carbamoyl]-5-(trifluoromethyl)benzoic acid

[1226] In N 2To a mixture of 4-[2-[1,3-dioxo-5-(trifluoromethyl)isoindolin-2-yl]-6-methyl-4-pyridinyl]-N-methyl-benzamide (120 mg, 273.11 μmol, 1 eq.) in THF / water (3 mL) was added LiOH (19.62 mg, 819.33 μmol, 3 eq.) at 25 °C and stirred at 25 °C for 5 hours. The mixture was acidified to pH 2 using 2M HCl. DMF (0.5 mL) was added to help dissolve the product. The residue was purified twice by preparative HPLC (HCl conditions, column: Boston Green ODS150*30mm*5μm; mobile phase: [water (0.05% HCl)-ACN]; B%: 18%-58%, 9 minutes). After purification by preparative HPLC and lyophilization, (2-[[6-methyl-4-[4-(methylcarbamoyl)phenyl]-2-pyridinyl]carbamoyl]-5-(trifluoromethyl)benzoic acid (18.6 mg, 14.52% yield, 97.5% purity) was obtained as a white solid.

[1227] LCMS(ESI)[M+H] + m / z: Calculated 458.12, Found 458.0

[1228] 1 H NMR (400MHz, DMSO-d6) δ: ppm 2.50 (br s, 3H) 2.81 (d, J = 4.40Hz, 3H) 7.44 (s, 1H) 7.76-7.87 (m, 3H) 7.97-8.06 (m, 3H) 8.14 (s, 1H) 8.33 (br s,1H)8.58(br d,J=4.52Hz,1H)11.17(s,1H)

[1229] SB003

[1230] (2S)-5,5-Dimethyl-2-[[6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl]amino]hexanoic acid

[1231]

[1232] Step 1: 5-Hydroxynaphthalene-2-carboxylic acid

[1233] To a solution of 5-bromonaphthalene-2-carboxylic acid (5 g, 19.91 mmol, 1 eq.) in dioxane (50 mL) was added KOH (4.47 g, 79.66 mmol, 4 eq.) in H 2 O (50 mL). Then, 2tBuXphosPdG3 (395.94 mg, 497.86 μmol, 0.025 equiv) was added under N 2 The reaction was stirred at 110 °C for 16 hours. LCMS showed that the reaction was complete. The reaction mixture was acidified with 1M HCl until pH 4-5 and filtered. Water (50 mL) was added to the filtrate, and then extracted with EtOAc (2x50 mL). The combined organic layers were washed with brine (20 mL) and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate in vacuo. The residue is purified by silica gel chromatography eluting with (PE:EtOAc=100:1 to 1:1) to obtain 5-hydroxynaphthalene-2-carboxylic acid (3.2 g, 85.39% yield, 100% purity) as a yellow solid.

[1234] 1 H NMR (400 MHz, DMSO-d 6 )δ:ppm 13.01(1H,br s)10.32(1H,br s)8.49(1H,d,J=1.5Hz)8.20(1H,d,J=8.8Hz)7.90(1H,dd,J=8.8,1.6Hz)7.27-7.61(2H,m)7.00(1H,dd,J=7.5,0.8Hz)

[1235] Step 2: 5-Hydroxy-N-methyl-naphthalene-2-carboxamide

[1236] In N 2 To a solution of 5-hydroxynaphthalene-2-carboxylic acid (2.35 g, 12.49 mmol, 1 eq.) in DCM (30 mL) was added DIEA (4.84 g, 37.46 mmol, 6.53 mL, 3 eq.), EDCI (2.87 g, 14.99 mmol, 1.2 eq.), HOBt (2.02 g, 14.99 mmol, 1.20 eq.) and methylamine (843.17 mg, 12.49 mmol, 1 eq., HCl) at 20 °C. The reaction was stirred at 20 °C for 3 hours. The reaction mixture was poured into water (100 mL). The aqueous layer was extracted with DCM (3x50 mL). The combined organic layers were washed with brine (50 mL) and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate in vacuo.The residue is purified by silica gel chromatography eluting with (PE:EtOAc=20:1 to 1:3).

[1237] 5-Hydroxy-N-methyl-naphthalene-2-carboxamide was obtained as a red oil (2.4 g, 95.51% yield, 100% purity).

[1238] LCMS (ESI) [M+H] + m / z: calculated value 202.0, found value 202.2

[1239] 1 H NMR (400MHz, chloroform-d) δ: ppm 8.22-8.28 (2H, m) 7.80 (1H, dd, J = 8.8, 1.5Hz) 7.51 (1H, d, J = 8.3Hz) 7.37 (1H, t, J = 7.9Hz) 6.92 (1H, d, J = 7.3Hz) 6.31 (1H, br s)3.10(3H,d,J=5.0Hz)

[1240] Step 3: Methyl 6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylate

[1241] A mixture of 5-hydroxy-N-methyl-naphthalene-2-carboxamide (2.4 g, 11.93 mmol, 1 eq.), 6-bromopyridine-3-carboxylic acid methyl ester (3.09 g, 14.31 mmol, 1.2 eq.), copper (I) iodide (227.15 mg, 1.19 mmol, 0.1 eq.) and potassium phosphate (5.06 g, 23.85 mmol, 2 eq.) in DMSO (20 mL) was degassed and heated to 40 ℃ for 2 h. 2 Purge three times, then 2 The mixture was stirred at 120°C for 16 hours under a 4% CO atmosphere. The reaction mixture was poured into water (50 mL) and extracted with EA (3 x 30 mL). The combined organic layers were washed with brine (30 mL) and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate in vacuo. The residue was purified by silica gel chromatography eluting with (PE:EtOAc=100:1 to 1:2) to give methyl 6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylate (1.4 g, 33.96% yield, 97.3% purity) as a yellow solid.

[1242] LCMS (ESI) [M+H] + m / z: calculated value 336, found value 337

[1243] 1H NMR (400MHz, chloroform-d) δ: 8.70 (1H, d, J = 1.9Hz) 8.21-8.33 (2H, m) 7.87 (1H, d, J = 8.8Hz) 7.78 (1H, d, J = 8.3Hz) 7. 71(1H,dd,J=8.8,1.6Hz)7.51(1H,t,J=7.9Hz)7.29(1H,dd,J=7.6,0.8Hz)6.98(1H,d,J=8.6Hz)6.21(1H,br s)3.84(3H,s)3.00(3H,d,J=4.8Hz)

[1244] Step 4: 6-[[6-(Methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylic acid

[1245] To methyl 6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylate (1.57 g, 4.67 mmol, 1 eq.) was dissolved in THF (45 mL) and H 2 O (15 mL) was added to the mixture of LiOH. 2 O (391.76 mg, 9.34 mmol, 2 eq.). The mixture was stirred at 25 °C for 2 h. The mixture was concentrated under reduced pressure. The residue was acidified to pH 2 using HCl (1 M). The residue was extracted with EA (20 mL x 5). The combined organic layers were washed with Na 2 SO 4 Dry, filter and concentrate in vacuo to afford 6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylic acid (1.3 g, 82.09% yield, 95% purity) as a white solid.

[1246] LCMS(ESI)[M+H] + m / z: Calculated 323.10, Found 323.0

[1247] 1 H NMR (400 MHz, methanol-d 4 )δ:ppm 8.66(1H,d,J=2.26Hz)8.44(1H,d,J=1.51Hz)8.39(1H,dd,J=8.66,2.38Hz)7.90-7.96(2H,m)7. 83-7.88(1H,m)7.62(1H,t,J=7.91Hz)7.39(1H,d,J=6.78Hz)7.16(1H,d,J=8.53Hz)2.97(3H,s)

[1248] Step 5: 6-[[6-(Methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl chloride

[1249] A mixture of 6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylic acid (200 mg, 620.51 μmol, 1 eq), oxalyl chloride (118.14 mg, 930.77 μmol, 81.48 μL, 1.5 eq), and DMF (4.54 mg, 62.05 μmol, 4.77 μL, 0.1 eq) in DCM (10 mL) was degassed at 0 °C and then heated under N 2 The mixture was stirred at 25° C. for 2 hours under atmosphere. The reaction mixture was concentrated under reduced pressure to give 6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl chloride (215 mg, crude) as a yellow gum, which was used without further purification.

[1250] LCMS(ESI)[M+OMe] + m / z: Calculated 337.11, Found 337.1

[1251] Step 6: (2S)-5,5-dimethyl-2-[[6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl]amino]hexanoic acid

[1252]

[1253] To (2S)-2-amino-5,5-dimethyl-hexanoic acid (100 mg, 628.04 μmol, 1 eq) and Na 2 CO 3 (232.98 mg, 2.20 mmol, 3.5 equiv) in H 2 6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl chloride (214.01 mg, 628.04 μmol, 1 equivalent) was added to a mixture of 4-nitropropene (16 mL) and dioxane (6 mL). The mixture was stirred at 20 ° C for 2 hours. The reaction mixture was poured into water (40 mL) and extracted with EtOAc (40 mL x 3). The combined aqueous layer was lyophilized in vacuo to give a crude residue. The residue was purified by preparative HPLC (HCl). After purification, concentration and lyophilization, (2S)-5,5-dimethyl-2-[[6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl]amino]hexanoic acid (92.3 mg, 31.55% yield, 99.5% purity) was obtained in the form of a white solid.

[1254] LCMS(ESI)[M+H] + m / z: Calculated 464.21, Found 464.2

[1255] 1H NMR (400 MHz, DMSO-d 6 )δ:ppm 8.61-8.72(2H,m)8.48-8.56(2H,m)8.34(1H,dd,J=8.53,2.51Hz)7.96(1H,d,J=8.28Hz)7.80-7.93(2H,m)7.64(1H,t,J=7.91Hz)7.43(1H,d ,J=7.03Hz)7.29(1H,d,J=8.53Hz)4.16-4.41(1H,m)2.83(2H,d,J=4.52Hz)2.74-2.98(1H,m)1.62-1.90(2H,m)1.09-1.45(2H,m)0.86(9H,s)

[1256] SB004

[1257] 2-((6-methyl-4-(methylcarbamoyl)pyridin-2-yl)carbamoyl)-5-(trifluoromethyl)benzoic acid

[1258]

[1259] Step 1: 2-Chloro-N,6-dimethylisonicotinamide

[1260] To a solution of 2-chloro-6-methyl-pyridine-4-carboxylic acid (5 g, 29.14 mmol, 1 eq) in DMF (200 mL) was added methylamine (9.84 g, 145.70 mmol, 5 eq, HCl), TEA (14.74 g, 145.70 mmol, 20.28 mL, 5 eq) and HATU (22.16 g, 58.28 mmol, 2 eq). 2 The reaction was stirred at 20 °C for 2 h. 2 O (800 mL). The aqueous layer was extracted with EtOAc (3x200 mL). The combined organic layers were washed with brine (2000 mL) and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate in vacuo. The residue is purified by silica gel chromatography, eluting with (PE:EtOAc=20:1 to 1:1). 2-Chloro-N,6-dimethyl-pyridine-4-carboxamide (4.4 g, 66.90% yield, 81.8% purity) is obtained as a yellow solid.

[1261] LCMS(ESI)[M+H] + m / z: Calculated 185.0, Found 185.4

[1262] H NMR (400 MHz, chloroform-d) δ: ppm 7.47 (1H, s) 7.431H, s) 7.14 (1H, br s) 2.97 (3H, d, J = 4.9 Hz) 2.54 (3H, s)

[1263] Step 2: 2-((Biphenylmethylene)amino)-N,6-dimethylisonicotinamide

[1264] To a solution of 2-chloro-N,6-dimethyl-pyridine-4-carboxamide (3.4 g, 18.42 mmol, 1 eq.) and benzophenone imine (4.34 g, 23.94 mmol, 4.02 mL, 1.3 eq.) in toluene (85 mL) were added t-BuONa (4.42 g, 46.04 mmol, 2.5 eq.) and [1-(2-diphenylphosphanyl-1-naphthyl)-2-naphthyl]-biphenyl-phosphane (344.01 mg, 552.48 μmol, 0.03 eq.), Pd 2 (dba) 3 (168.64 mg, 184.16 μmol, 0.01 eq.) 2 The reaction was stirred at 90° C. for 16 hours. The reaction was concentrated in vacuo. The residue was purified by silica gel chromatography eluting with (PE:EtOAc=10:1 to 1:3). 2-(benzhydrylamino)-N,6-dimethyl-pyridine-4-carboxamide (600 mg, 90% yield, 90% purity) was obtained as a yellow solid.

[1265] LCMS(ESI)[M+H] + m / z: Calculated 330.1, Found 330.1

[1266] Step 3: 2-amino-N,6-dimethylisonicotinamide

[1267] 2-(Benzhydrylamino)-N,6-dimethyl-pyridine-4-carboxamide (600 mg, 1.82 mmol, 1 eq.) was dissolved in THF (5 mL) and HCl / H 2 O (5 mL, 10% purity) was stirred at 20 °C for 16 h. The reaction was washed with EtOAc (10 mL). The residue was washed with saturated NaHCO 3 The aqueous solution was adjusted to pH 10 and the aqueous layer was concentrated in vacuo. DCM (10 mL) was added to the residue and stirred for 10 minutes. The suspension was filtered and the filter cake was washed with DCM (10 mL × 3). The combined filtrate was concentrated to dryness to give 2-amino-N,6-dimethyl-pyridine-4-carboxamide (190 mg, 59.99% yield, 95% purity) in the form of a yellow solid.

[1268] LCMS(ESI)[M+H] + m / z: Calculated 166.2, Found 166.1

[1269] Step 4: 2-(1,3-dioxo-5-(trifluoromethyl)isoindolin-2-yl)-N,6-dimethylisonicotinamide

[1270] In N 2 A solution of 2-amino-N,6-dimethyl-pyridine-4-carboxamide (170 mg, 1.03 mmol, 1 eq.) and 5-(trifluoromethyl)isobenzofuran-1,3-dione (222.40 mg, 1.03 mmol, 1 eq.) in AcOH (4 mL) was stirred at 120 ° C for 16 hours. The reaction was concentrated and the residue was purified by silica gel chromatography eluting with (PE: EtOAc = 5: 1 to 1: 3). 2-[1,3-dioxo-5-(trifluoromethyl)isoindolin-2-yl]-N,6-dimethyl-pyridine-4-carboxamide (188 mg, 41.23% yield, 82% purity) was obtained in the form of a yellow solid.

[1271] LCMS(ESI)[M+H] + m / z: Calculated 364.1, Found 364.0

[1272] Step 5: 2-((6-methyl-4-(methylcarbamoyl)pyridin-2-yl)carbamoyl)-5-(trifluoromethyl)benzoic acid

[1273] 2-[1,3-dioxo-5-(trifluoromethyl)isoindolin-2-yl]-N,6-dimethyl-pyridine-4-carboxamide (210 mg, 578.05 μmol, 1 eq.) was dissolved in THF (4 mL) and H 2 O (4 mL) was added with LiOH. 2 O (72.77 mg, 1.73 mmol, 3 eq.), and in N 2 The mixture was stirred at 20°C for 1 hour. The reaction was concentrated and the residue was purified by preparative HPLC (column: Phenomenex Gemini-NX 150*30mm*5μm; mobile phase: [water (0.05% ammonium hydroxide v / v)-ACN]; B%: 4%-44%, 14 minutes). After preparative HPLC purification, the eluate was lyophilized to give 2-[[6-methyl-4-(methylcarbamoyl)-2-pyridyl]carbamoyl]-5-(trifluoromethyl)benzoic acid (14 mg, 5.88% yield, 96.7% purity) in the form of a white solid.

[1274] LCMS(ESI)[M+H] +m / z: Calculated 382.1, Found 382.0

[1275] 1 H NMR (400 MHz, DMSO-d 6 )δ:ppm 13.09(1H,br s)8.66(1H,br d,J=4.3Hz)8.39(1H,s)7.88-8.01(2H,m)7.74(1H,br d,J=8.0Hz)7.30(3H,s)2.79(3H,d,J=4.4Hz)2.45(3H,s)

[1276] SB007

[1277] (2S)-5,5-Dimethyl-2-[[6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carbonyl]amino]hexanoic acid

[1278]

[1279] Step 1: 6-[3-(Methylcarbamoyl)phenoxy]pyridine-3-carboxylic acid methyl ester

[1280] A mixture of 3-hydroxy-N-methyl-benzamide (400 mg, 2.65 mmol, 1 eq.), 6-bromopyridine-3-carboxylic acid methyl ester (571.66 mg, 2.65 mmol, 1 eq.), potassium phosphate (561.70 mg, 2.65 mmol, 1 eq.) and CuI (503.96 mg, 2.65 mmol, 1 eq.) in DMSO (10 mL) was degassed and heated to 40 ℃ with N 2 Purge three times, then 2 The mixture was stirred at 120°C for 16 h under a 24-hour atmosphere. 2 O (40 mL) and extracted with EA (20 mL x 4). The organic layer was washed with Na 2 SO 4 The residue was purified by flash silica gel chromatography ( 12g Purification was performed by silica flash column with a gradient of 0-100% ethyl acetate / petroleum ether as eluent at a flow rate of 25 mL / min. Methyl 6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carboxylate was obtained as a white solid (73.92% yield, 100% purity).

[1281] Step 2: 6-[3-(Methylcarbamoyl)phenoxy]pyridine-3-carboxylic acid

[1282] To methyl 6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carboxylate (460 mg, 1.61 mmol, 1 eq.) was dissolved in THF (15 mL) and H 2 O (5 mL) was added to the mixture of LiOH. 2 O (134.84 mg, 3.21 mmol, 2 eq.). The mixture was stirred at 25 °C for 2 h. The mixture was concentrated under reduced pressure. The residue was acidified to pH 2 with HCl (1 M) and extracted with EA (20 mL x 5). The combined organic layers were washed with Na 2 SO 4 Dry, filter and concentrate in vacuo to afford 6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carboxylic acid as a white solid (82.29% yield).

[1283] Step 3: 6-[3-(Methylcarbamoyl)phenoxy]pyridine-3-carbonyl chloride

[1284] A mixture of 6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carboxylic acid (0.1 g, 367.30 μmol, 1 eq), oxalyl chloride (69.93 mg, 550.95 μmol, 48.23 μL, 1.5 eq), and DMF (2.68 mg, 36.73 μmol, 2.83 μL, 0.1 eq) in DCM (5 mL) was degassed at 0 °C and then heated under N 2 The mixture was stirred at 25°C for 2 hours under atmosphere. The reaction mixture was concentrated under reduced pressure to obtain 6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carbonyl chloride (91.29 mg, crude) as a yellow gum which was used directly without further purification.

[1285] Step 4: (2S)-5,5-dimethyl-2-[[6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carbonyl]amino]hexanoic acid

[1286] To (2S)-2-amino-5,5-dimethyl-hexanoic acid (50 mg, 314.02 μmol, 1 eq) and Na 2 CO 3 (116.49 mg, 1.10 mmol, 3.5 equiv) in dioxane (8 mL), H 26-[3-(methylcarbamoyl)phenoxy]pyridine-3-carbonyl chloride (91.29 mg, 314.02 μmol, 1 equivalent) was added to the mixture in 40 mL of 1% paraformaldehyde (3 mL). The mixture was stirred at 20 ° C for 2 hours. The reaction mixture was poured into water (40 mL) and extracted with EtOAc (40 mL x 3). The combined aqueous layer was lyophilized in vacuo to obtain a crude residue. After purification, concentration and lyophilization, (2S)-5,5-dimethyl-2-[[6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carbonyl]amino]hexanoic acid (11 mg, 8.15% yield, 96.2% purity) was obtained as a white solid.

[1287] LCMS(ESI)[M+H] + m / z: Calculated 413.20, Found 414.1

[1288] 1 H NMR (400 MHz, DMSO-d 6 )δ:ppm 0.87(s,9H)1.17-1.35(m,2H)1.65-1.85(m,2H)2.77(d,J=4.5Hz,3H)4.31(td,J=8.4,5.3Hz,1H)7.17(d,J=8.5Hz,1H)7.34 (dd,J=8.0,1.8Hz,1H)7.53(t,J=7.9Hz,1H)7.61(t,J=1.9Hz,1H)7.72(d,J=7.8Hz,1H)8.31(dd,J=8.7,2.4Hz,1H)8.50(br d,J=4.5Hz,1H)8.63(d,J=2.3Hz,1H)8.71(d,J=7.8Hz,1H)

[1289] SB008

[1290] (2S)-2-[[6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[1291]

[1292] Step 1: 3-[1-(2-methoxyethyl)triazol-4-yl]phenol

[1293] To 3-ethynylphenol (576.00 mg, 4.88 mmol, 1.1 eq), 1-azido-2-methoxy-ethane (450 mg, 4.45 mmol, 1 eq), TEA (450.37 mg, 4.45 mmol, 619.48 μL, 1 eq) in tert-butyl alcohol (10 mL) and H 2O (10 mL) was added to the solution of CuSO 4 .5H 2 O (277.82 mg, 1.11 mmol, 0.25 eq), (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol sodium (440.86 mg, 2.23 mmol, 0.5 eq), and the mixture was stirred at 20 ° C for 2 hours. The reaction was filtered through celite and poured into water (40 mL), extracted with EtOAc (40 mL x3). The combined organic layer was washed with brine (40 mL) and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate in vacuo to give a crude residue. The residue is purified by silica gel chromatography eluting with (PE:EtOAc=20:1 to 1:3). 3-[1-(2-methoxyethyl)triazol-4-yl]phenol (1.0 g, 97.36% yield, 95% purity) is obtained as a white solid.

[1294] 1 H NMR (400 MHz, CDCl 3 )δ:ppm 7.87(1H,s)7.62(1H,br s)7.12-7.33(2H,m)6.82-6.98(1H,m)5.31(1H,s)4.57(2H,t,J=4.89Hz)3.79(2H,t,J=5.02Hz)3.37(3H,s)

[1295] Step 2: 6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylate

[1296] 3-[1-(2-methoxyethyl)triazol-4-yl]phenol (800 mg, 3.65 mmol, 1 equivalent), 6-bromopyridine-3-carboxylic acid methyl ester (788.00 mg, 3.65 mmol, 1 equivalent) and CuI (69.49 mg, 364.90 μmol, 0.1 equivalent), K 3 PO 4 A mixture of (1.55 g, 7.30 mmol, 2 eq.) in DMSO (10 mL) was degassed and heated with N 2 Purge three times, then 2 The mixture was stirred at 120°C for 16 hours under a C atmosphere. The reaction was poured into water (40 mL) and extracted with EtOAc (40 mL x 3). The combined organic layers were washed with brine (40 mL) and washed with anhydrous Na 2 SO 4The mixture was dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with (PE:EtOAc=20:1 to 1:3). Methyl 6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylate (660 mg, 51.04% yield) was obtained as a white solid.

[1297] 1 H NMR (400 MHz, CDCl 3 )δ:ppm 8.83(1H,d,J=2.01Hz)8.29(1H,dd,J=8.66,2.38Hz)7.91(1H,s)7.62-7.81(2H,m)7.49(1H,t,J=7.91H z)7.09-7.20(1H,m)6.98(1H,d,J=8.78Hz)4.50-4.66(2H,m)3.92(3H,s)3.73-3.85(2H,m)3.37(3H,s)

[1298] Step 3: 6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylic acid

[1299] To methyl 6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylate (170 mg, 479.74 μmol, 1 eq.) was dissolved in THF (3 mL) and H 2 O (1 mL) was added to the mixture of LiOH. 2 O (40.26 mg, 959.48 μmol, 2 eq.), and the mixture was stirred at 25°C for 2 hours. The mixture was concentrated under reduced pressure, and the residue was acidified to pH 2 with HCl (1 M). The residue was extracted with EA (20 mL×5). The combined organic layers were purified by Na 2 SO 4 Dry, filter and concentrate in vacuo to afford 6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylic acid (140 mg, 84.03% yield, 98% purity) as a yellow solid.

[1300] 1 H NMR (400 MHz, methanol-d 4)δ:ppm 8.74(1H,d,J=2.01Hz)8.31-8.39(2H,m)7.71-7.78(1H,m)7.61-7.67(1H,m)7.52(1H,t,J=7.91Hz)7.15( 1H,ddd,J=8.16,2.38,0.75Hz)7.07(1H,d,J=8.53Hz)4.61(2H,t,J=5.02Hz)3.76-3.99(2H,m)3.35(3H,s)

[1301] Step 4: 6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carbonyl chloride

[1302] A mixture of 6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylic acid (140 mg, 411.36 μmol, 1 eq), oxalyl chloride (78.32 mg, 617.04 μmol, 54.01 μL, 1.5 eq), and DMF (3.01 mg, 41.14 μmol, 3.17 μL, 0.1 eq) in DCM (5 mL) was degassed at 0 °C and then heated under N 2 The mixture was stirred at 25° C. for 2 hours under atmosphere. The reaction mixture was concentrated under reduced pressure to give 6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carbonyl chloride (150 mg, crude), which was used directly without further purification.

[1303] Step 5: (2S)-2-[[6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[1304] To (2S)-2-amino-5,5-dimethyl-hexanoic acid (50 mg, 314.02 μmol, 0.75 equiv) and Na 2 CO 3 (155.09 mg, 1.46 mmol, 3.5 eq.) in H 26-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carbonyl chloride (150 mg, 418.09 μmol, 1 equivalent) was added to a mixture of 2S (8 mL) and dioxane (3 mL). The mixture was stirred at 20 ° C for 2 hours. The reaction mixture was poured into water (40 mL) and extracted with EtOAc (40 mL x 3). The combined aqueous layer was lyophilized in vacuo. The residue was purified by preparative HPLC (HCl) to give (2S)-2-[[6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (42 mg, 20.65% yield, 99% purity) as a white solid.

[1305] LCMS(ESI)[M+H] + m / z: Calculated 482.2, Found 482.2

[1306] 1 H NMR (400 MHz, DMSO-d 6 )δ:ppm 8.70(1H,d,J=7.83Hz)8.65(1H,d,J=1.96Hz)8.61(1H,s)8.32(1H,dd,J =8.62,2.51Hz)7.73-7.80(1H,m)7.63-7.67(1H,m)7.52(1H,t,J=7.89H z)7.11-7.20(2H,m)4.57(2H,t,J=5.20Hz)4.23-4.43(1H,m)3.77(2H,t ,J=5.20Hz)3.26(3H,s)1.64-1.92(2H,m)1.16-1.38(2H,m)0.87(9H,s)

[1307] SB009

[1308]

[1309] Step 1: (3-Acetoxyphenyl)acetate

[1310] In N 2 Benzene-1,3-diol (1 g, 9.08 mmol, 1.52 mL, 1 eq.) was added to Ac at 20 °C. 2 Toluenesulfonyl chloride (173.14 mg, 908.18 μmol, 0.1 eq.) was added to 14.83 g, 145.31 mmol, 13.61 mL, 16 eq. The reaction was stirred at 20 °C for 1.5 h. The reaction was heated by 10% NaOH / H 2O (45 mL) and extracted with DCM (3 x 30 mL). The combined organic layers were washed with brine (50 mL) and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate in vacuo. The residue was purified by silica gel chromatography eluting with (PE:EtOAc=50:1 to 10:1). (3-acetoxyphenyl)acetate (1.6 g, 90.73% yield) was obtained as a colorless oil.

[1311] LCMS(ESI)[M+H] + m / z: Calculated 195.0, Found 194.8

[1312] 1 H NMR (400 MHz, chloroform-d) δ: ppm 7.38 (1H, t, J = 8.1 Hz) 6.99 (2H, dd, J = 8.2, 2.2 Hz) 6.85-6.95 (1H, m) 2.22-2.36 (6H, m)

[1313] Step 2: (3-Hydroxyphenyl)acetate

[1314] In N 2 To a mixture of (3-acetoxyphenyl)acetate (1.6 g, 8.24 mmol, 1 eq.) and benzene-1,3-diol (907.27 mg, 8.24 mmol, 1.37 mL, 1 eq.) in DMSO (10 mL) was added K in one portion at 20 °C. 2 CO 3 (1.14 g, 8.24 mmol, 1 eq.). The mixture was stirred at 20 °C for 4 hours. TLC showed that the reaction was complete. The mixture was poured into water (20 mL) and the aqueous phase was extracted with ethyl acetate (10 mL x 6). The combined organic phases were washed with brine (20 mL) and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate in vacuo. The residue is purified by silica gel chromatography (petroleum ether / ethyl acetate=10 / 1 to 2 / 1). (3-Hydroxyphenyl)acetate (1.9 g, 75.78% yield) is obtained as a colorless oil.

[1315] 1 H NMR (400 MHz, chloroform-d) δ: ppm 7.21 (1H, t, J = 8.1 Hz) 6.61-6.72 (2H, m) 6.56 (1H, t, J = 2.3 Hz) 2.27-2.36 (3H, m)

[1316] Step 3: 3-(2-methoxyethoxy)phenol

[1317] In N2 To a mixture of (3-hydroxyphenyl)acetate (1.7 g, 11.17 mmol, 1 eq.) and 2-methoxyethyl 4-methylbenzenesulfonate (2.32 g, 10.06 mmol, 0.9 eq.) in DMF (30 mL) was added K in one portion at 20 °C. 2 CO 3 (3.40 g, 24.58 mmol, 2.2 eq.). The mixture was stirred at 80°C for 4 hours. To the reaction mixture was added NaOH (20%, 32 mL), and the mixture was stirred at 0°C for 15 minutes. The aqueous layer was washed with HCl / H 2 O (1 M, 240 mL) until pH 5 and extracted with DCM (3 x 30 mL). The combined organic layers were washed with brine (100 mL) and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate in vacuo. Combine the residue with the residue of ES17843-53-P1A and purify by silica gel chromatography eluting with (PE:EA=100:1 to 3:1). Obtain 3-(2-methoxyethoxy)phenol (1.04 g, 55.34% yield) in the form of a yellow oil.

[1318] 1 H NMR (400 MHz, chloroform-d) δ: ppm 7.12 (1H, t, J = 8.0 Hz) 6.22-6.55 (3H, m) 5.24 (1H, br s) 4.10 (2H, dd, J = 5.4, 3.9 Hz) 3.70-3.82 (2H, m) 3.41-3.51 (3H, m)

[1319] Step 4: 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carboxylic acid methyl ester and 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carboxylic acid

[1320] In N 2 To a mixture of 3-(2-methoxyethoxy)phenol (916 mg, 5.45 mmol, 1 eq.) and methyl 6-bromopyridine-3-carboxylate (1.18 g, 5.45 mmol, 1 eq.) in DMSO (12 mL) was added CuI (103.72 mg, 544.62 μmol, 0.1 eq.) and K 3 PO 4 (2.31 g, 10.89 mmol, 2 eq.) The reaction was stirred at 120 °C for 16 h. 100 mL of H 2O, then the mixture was filtered and the filter cake was washed with 30 mL of EtOAc. The filtrate was extracted with EtOAc (3x30 mL). The combined organic layers were washed with brine (20 mL) and washed with anhydrous Na 2 SO 4 The crude product was purified by silica gel chromatography using (PE:EtOAc=20:1 to 1:1) as eluent to give methyl 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carboxylate (260 mg, 857.21 μmol, 15.74% yield) as a yellow oil. The residual aqueous layer was washed with 1M HCl / H 2 O until pH 2, then extracted with EtOAc (3 x 30 mL). The combined organic layers were washed with brine (30 mL) and washed with anhydrous Na 2 SO 4 Drying, filtration and concentration in vacuo gave crude 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carboxylic acid (500 mg, 31.74% yield).

[1321] 1 H NMR (400MHz, chloroform-d) δ: ppm 8.84(1H,dd,J=2.3,0.6Hz)8.27(1H,dd,J=8.6,2.4Hz)7.30-7.38(1H,m)7.05-7.19(1H,m)6.81-6.97(1H,m)6. 70-6.78(1H,m)6.37-6.56(1H,m)4.05-4.19(2H,m)3.93(2H,s)3.75(2H,td,J=4.7,2.2Hz)3.46(3H,d,J=1.7Hz)

[1322] Step 5: 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carboxylic acid

[1323] To methyl 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carboxylate (260 mg, 857.21 μmol, 1 eq.) was dissolved in THF (4.5 mL) and H 2 To the mixture in 2% CO (1.5 mL) was added LiOH. 2 O (71.94 mg, 1.71 mmol, 2 eq.). The reaction was stirred at 20 °C for 2 h. The mixture was concentrated under reduced pressure and the residue was acidified to pH 2 using HCl (1 M). The residue was extracted with EtOAc (20 mL x 5). The combined organic layers were washed with Na 2 SO 4Dry, filter and concentrate in vacuo. The residue was purified by silica gel chromatography eluting with (DCM:MeOH=50:1 to 10:1) to give 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carboxylic acid (247.9 mg, 99.97% yield) as a white oil.

[1324] LCMS(ESI)[M+H] + m / z: Calculated 290.1, Found 289.9

[1325] 1 H NMR (400 MHz, chloroform-d) δ: ppm 8.94 (1H, d, J = 2.3 Hz) 8.34 (1H, dd, J = 8.7, 2.4 Hz) 7.30-7.41 (1H, m) 6.97 (1H, d, J = 8.7 Hz) 6.85-6.91 (1H, m) 6.71-6.83 (2H, m) 4.10-4.17 (2H, m) 3.78 (2H, dd, J = 5.4, 4.0 Hz) 3.43-3.51 (3H, m)

[1326] Step 6: 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carbonyl chloride

[1327] In N 2 , DMF (2.53 mg, 34.57 μmol, 2.66 μL, 0.1 eq) and oxalyl chloride (65.82 mg, 518.52 μmol, 45.39 μL, 1.5 eq) were added to 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carboxylic acid (100 mg, 345.68 μmol, 1 eq) in DCM (4 mL) at 0°C. The reaction was stirred at 20°C for 2 hours. The residue was concentrated in vacuo to give 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carbonyl chloride (106 mg, 99.65% yield) as a colorless oil.

[1328] LCMS(ESI)[M-Cl+OMe] + m / z: calculated 304.0, found 304.0

[1329] Step 7: (2S)-2-[[6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[1330] To (2S)-2-amino-5,5-dimethyl-hexanoic acid (50.00 mg, 314.02 μmol, 0.912 equiv) and Na 2 CO 3(127.78 mg, 1.21 mmol, 3.5 equiv) in dioxane (3 mL) and H 2 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carbonyl chloride (106 mg, 344.46 μmol, 1 equivalent) was added to the solution in 4% paraformaldehyde (1% paraformaldehyde) (8 mL). The reaction was stirred at 20 ° C for 2 hours. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (10 mL x 3). The combined aqueous layer was lyophilized in vacuo to give a crude residue. The crude product was combined with the crude product of (ES17843-67-P1A) and purified by preparative HPLC (column: Boston Green ODS150*30 mm*5 μm; mobile phase: [water (0.05% HCl)-ACN]; B%: 29%-69%, 9 minutes). After preparative HPLC purification, the residue was lyophilized to give (2S)-2-[[6-[3-(2-methoxyethoxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (12 mg, 8.00% yield, 98.8% purity) as a white solid.

[1331] LCMS(ESI)[M+H] + m / z: calculated 431.2, found 431.2

[1332] 1 H NMR (400 MHz, DMSO-d 6 )δ:ppm 8.68(1H,d,J=7.8Hz)8.64(1H,d,J=2.3Hz)8.29(1H,dd,J=8.6,2.4Hz)7.32(1H, t,J=8.2Hz)7.08(1H,d,J=8.7Hz)6.83(1H,dd,J=8.2,2.1Hz)6.76(1H,t,J=2.3H z)6.72(1H,dd,J=7.8,1.8Hz)4.23-4.38(1H,m)4.23-4.38(1H,m)4.06-4.11(2H ,m)3.64-3.66(2H,m)3.29(3H,s)1.62-1.85(2H,m)1.13-1.38(2H,m)0.87(9H,s)

[1333] SB010

[1334] (2S)-2-[[6-[3-[1-(2-acetylaminoethyl)triazol-4-yl]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[1335]

[1336] Step 1: tert-Butyl N-[2-[4-(3-hydroxyphenyl)triazol-1-yl]ethyl]carbamate

[1337] To 3-ethynylphenol (600 mg, 5.08 mmol, 1.1 eq), tert-butyl N-(2-azidoethyl)carbamate (859.80 mg, 4.62 mmol, 1 eq), TEA (467.22 mg, 4.62 mmol, 642.67 μL, 1 eq) in tert-butyl alcohol (10 mL) and H 2 CuSO4 was added to the solution in O (10 mL) 4 .5H 2 O (288.22 mg, 1.15 mmol, 0.25 eq), (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-ol sodium (457.37 mg, 2.31 mmol, 0.5 eq), the mixture was stirred at 20 ° C for 12 hours. The reaction was filtered through celite and poured into water (40 mL) and extracted with EtOAc (40 mL x3). The combined organic layer was washed with brine (40 mL) and washed with anhydrous Na 2 SO 4 The residue was purified by column chromatography (SiO 2 , petroleum ether / ethyl acetate=20 / 1 to 3 / 1) to obtain tert-butyl N-[2-[4-(3-hydroxyphenyl)triazol-1-yl]ethyl]carbamate (1.2 g, 85.39% yield) as a white solid.

[1338] LCMS(ESI)[M+H] + m / z: Calculated 304.1, Found 305.1

[1339] Step 2: 3-[1-(2-aminoethyl)triazol-4-yl]phenol

[1340] To a solution of tert-butyl N-[2-[4-(3-hydroxyphenyl)triazol-1-yl]ethyl]carbamate (1 g, 3.29 mmol, 1 eq) in DCM (10 mL) was added TFA (3.75 g, 32.86 mmol, 2.43 mL, 10 eq). The reaction mixture was stirred at 20 ° C for 17 hours under nitrogen. The reaction mixture was directly concentrated under reduced pressure. 3-[1-(2-aminoethyl)triazol-4-yl]phenol was obtained as a white solid.

[1341] Step 3: N-[2-[4-(3-Hydroxyphenyl)triazol-1-yl]ethyl]acetamide

[1342] In N 2, 3-[1-(2-aminoethyl)triazol-4-yl]phenol (700 mg, 3.43 mmol, 1 eq) and TEA (1.73 g, 17.14 mmol, 2.39 mL, 5 eq) in DCM (10 mL) were added dropwise acetyl chloride (269.05 mg, 3.43 mmol, 244.59 μL, 1 eq) at 0°C. The reaction mixture was warmed to 20°C and stirred for 2 hours. The reaction was poured into water (40 mL) and extracted with DCM (40 mL x 3). The combined organic layers were washed with brine (40 mL) and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate in vacuo. The residue was purified by preparative HPLC (basic conditions) and the residual aqueous solution was lyophilized to give N-[2-[4-(3-hydroxyphenyl)triazol-1-yl]ethyl]acetamide (150 mg, 17.77% yield) as a pale yellow solid.

[1343] 1H NMR(400MHz,DMSO-d6)δ:ppm 8.26-8.67(m,1H)8.05(br t,J=5.5Hz,1H)7.27(d,J=1.0Hz,1H)7.20-7.24(m,2H)6.63-6.76(m,1H)4.42(t,J=5.9Hz,2H)3.51(q,J=5.8Hz,2H)1.67-1.88(m,3H).

[1344] Step 4: 6-[3-[1-(2-acetylaminoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylic acid methyl ester

[1345] A mixture of methyl 6-bromopyridine-3-carboxylate (105.27 mg, 487.28 μmol, 1.2 eq), N-[2-[4-(3-hydroxyphenyl)triazol-1-yl]ethyl]acetamide (100 mg, 406.07 μmol, 1 eq), potassium phosphate (86.19 mg, 406.07 μmol, 1 eq) and CuI (77.34 mg, 406.07 μmol, 1 eq) in DMSO (4 mL) was degassed and heated with N 2 Purge three times, then 2 The mixture was stirred at 120°C for 16 hours under a 4% CO atmosphere. The reaction mixture was extracted with EA (20 mL x 4). The organic layer was washed with Na 2 SO 4 The residue was purified by flash silica gel chromatography ( 12g The mixture was purified by silica flash column with a gradient of 0-100% ethyl acetate in petroleum ether at a flow rate of 25 mL / min to afford methyl 6-[3-[1-(2-acetylaminoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylate as a white solid (90.40% yield).

[1346] 1 H NMR (400MHz, DMSO-d6) δ: ppm 8.71 (d, J=2.0Hz, 1H) 8.61 (s, 1H) 8.34 (dd, J=8.5, 2.5Hz, 1H) 8.04 (br t,J=5.8Hz,1H)7.76(d,J=7.8Hz,1H)7.63-7.66(m,1H)7.53(t,J=7.9Hz,1H)7.13-7 .21(m,2H)4.43(t,J=6.0Hz,2H)3.86(s,3H)3.50(q,J=6.0Hz,2H)1.76-1.79(m,3H)

[1347] Step 5: 6-[3-[1-(2-acetylaminoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylic acid

[1348] To methyl 6-[3-[1-(2-acetylaminoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylate (100 mg, 262.20 μmol, 1 eq.) was dissolved in THF (0.6 mL) and H 2 O (0.2 mL) was added to the mixture of LiOH. 2 O (22.01 mg, 524.41 μmol, 2 eq.) was added, and the mixture was stirred at 25 °C for 2 h. The mixture was concentrated under reduced pressure and acidified to pH 2 with HCl (1 M). The residue was extracted with EA (20 mL x 5). The combined organic layers were purified by evaporation with Na 2 SO 4 Drying, filtration and concentration in vacuo afforded 6-[3-[1-(2-acetylaminoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylic acid as a white solid (93.44% yield, 90% purity).

[1349] 1H NMR (400MHz, DMSO-d6) δ: ppm 8.68 (d, J=2.0Hz, 1H) 8.61 (s, 1H) 8.31 (dd, J=8.7, 2.4Hz, 1H) 8.05 (br s,1H)7.75(d,J=8.0Hz,1H)7.63(s,1H)7.53(t,J=7.9Hz,1H)7.16(br d,J=8.8Hz,2H)4.43(br t,J=5.8Hz,2H)3.51(q,J=5.8Hz,2H)1.78(s,3H)

[1350] Step 6: (2,5-Dioxopyrrolidin-1-yl)6-[3-[1-(2-acetylaminoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylate

[1351] To a solution of 6-[3-[1-(2-acetylaminoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylic acid (80 mg, 217.77 μmol, 1 eq) in DMF (2 mL) were added 1-hydroxypyrrolidine-2,5-dione (62.66 mg, 544.43 μmol, 2.5 eq) and EDCI (62.62 mg, 326.66 μmol, 1.5 eq), and the mixture was degassed three times and stirred under N 2 The mixture was stirred at 20 °C for 3 hours. Water (4 mL) and ethyl acetate (5 mL) were added. The organic layer was washed with water (3 ml), brine (5 mL x 2), dried over anhydrous sodium sulfate, filtered and concentrated. (2,5-dioxopyrrolidin-1-yl) 6-[3-[1-(2-acetylaminoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylate (100 mg, crude) was obtained as a yellow oil.

[1352] Step 7: (2S)-2-[[6-[3-[1-(2-acetylaminoethyl)triazol-4-yl]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[1353] (2,5-Dioxopyrrolidin-1-yl)-6-[3-[1-(2-acetylaminoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylate (80 mg, 172.25 μmol, 1 eq), (2S)-2-amino-5,5-dimethyl-hexanoic acid (27.43 mg, 172.25 μmol, 1 eq), and DIEA (33.39 mg, 258.38 μmol, 45.01 μL, 1.5 eq) were dissolved in DCM (0.4 mL), DMF (2.4 mL), and H 2 The mixture was degassed with N 2 Purge three times, then 2The mixture was stirred at 20 ° C for 4 hours under atmosphere. The reaction mixture was concentrated in vacuo to give a crude residue, which was purified by preparative HPLC (HCl conditions). The residual aqueous solution was lyophilized to give (2S)-2-[[6-[3-[1-(2-acetylaminoethyl)triazol-4-yl]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (22 mg, 25.01% yield, 99.6% purity) as a white solid.

[1354] LCMS(ESI)[M+H] + m / z: Calculated 508.24, Found 509.3

[1355] 1 H NMR(400MHz, DMSOd6)δ:8.71(d,J=7.8Hz,1H)8.65(d,J=2.5Hz,1H)8.62(s,1H)8.32(dd,J=8.5,2.5Hz,1H)8.07(br t,J=5.6Hz,1H)7.74(d,J=8.0Hz,1H)7.60-7.65(m,1H)7.52(t,J=7.9Hz,1H)7.11-7.19(m,2H)4.43(t, J=5.9Hz,2H)4.31(td,J=8.4,5.0Hz,3H)3.50(q,J=6.0Hz,2H)1.78(s,3H)1.16-1.36(m,2H)0.86(s,9H)

[1356] SB011

[1357] (2S)-2-[[6-[3-(2-Acetylaminoethoxy)phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[1358]

[1359] Step 1: [3-[2-(tert-Butyloxycarbonylamino)ethoxy]phenyl]acetate

[1360] (3-Hydroxyphenyl)acetate (1.7 g, 11.17 mmol, 1.52 mL, 1 equivalent), tert-butyl N-(2-hydroxyethyl)carbamate (1.80 g, 11.18 mmol, 1.73 mL, 1 equivalent), PPh 3 A mixture of 2-nitropropene (3.52 g, 13.41 mmol, 1.2 eq.) in THF (30 mL) was degassed and washed with N 2 Purge three times, add DIAD (2.71 g, 13.41 mmol, 2.61 mL, 1.2 eq.) at 0 °C, and then stir under N 2The mixture was stirred at 20°C for 16 hours under a C atmosphere. The reaction mixture was poured into water (40 mL) and extracted with EtOAc (40 mL x 3). The combined organic layers were washed with brine (40 mL) and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate in vacuo. The residue was purified by silica gel chromatography eluting with (PE:EtOAc=20:1 to 3:1) to give [3-[2-(tert-butoxycarbonylamino)ethoxy]phenyl]acetate (1.9 g, 54.70% yield, 95% purity) as a colorless oil.

[1361] 1 H NMR (400 MHz, CDCl 3 )δ:ppm 7.29(1H,t,J=8.28Hz)6.78(1H,dd,J=8.16,2.13Hz)6.71(1H,dd,J=8.16,1.38Hz)6.65(1H,t,J=2.26Hz)4.01(2H,t,J=5.02Hz)3.54(2H,br d,J=4.27Hz)2.31(3H,s)1.47(9H,s)

[1362] Step 2: tert-Butyl N-[2-(3-hydroxyphenoxy)ethyl]carbamate

[1363] To a mixture of [3-[2-(tert-butoxycarbonylamino)ethoxy]phenyl]acetate (1.7 g, 5.76 mmol, 1 eq) in MeOH (30 mL) was added K 2 CO 3 (1.59 g, 11.51 mmol, 2 eq.) and H 2 O (6 mL), and the mixture was stirred at 20 ° C for 2 hours. The reaction mixture was poured into 10% HCl aqueous solution (80 mL) and extracted with EtOAc (40 mL x 3). The combined organic layer was washed with brine (40 mL) and washed with anhydrous Na 2 SO 4 The residue was purified by column chromatography (SiO 2 , petroleum ether / ethyl acetate=50 / 1 to 1 / 3) to give tert-butyl N-[2-(3-hydroxyphenoxy)ethyl]carbamate (900 mg, 61.73% yield) as a colorless oil.

[1364] 1 H NMR (400 MHz, CDCl 3)δ:ppm 7.12(1H,t,J=8.13Hz)6.40-6.54(3H,m)3.98(2H,t,J=5.07Hz)3.52(2H,br d,J=5.01Hz)1.47(9H,s)

[1365] Step 3: 6-[3-[2-(tert-Butyloxycarbonylamino)ethoxy]phenoxy]pyridine-3-carboxylic acid methyl ester

[1366] Tert-butyl N-[2-(3-hydroxyphenoxy)ethyl]carbamate (100 mg, 394.80 μmol, 1 equivalent), methyl 6-fluoropyridine-3-carboxylate (74.00 mg, 477.03 μmol, 1.21 equivalent), Cs 2 CO 3 A mixture of 2-nitropropene (385.90 mg, 1.18 mmol, 3 eq.) in DMF (3 mL) was degassed and heated with N 2 Purge three times, then 2 The mixture was stirred at 20°C for 16 hours under a 4% CO atmosphere. The reaction mixture was poured into water (50 mL) and extracted with EA (3 x 30 mL). The combined organic layers were washed with brine (30 mL) and washed with anhydrous Na 2 SO 4 Dry, filter and concentrate in vacuo. The residue was purified by silica gel chromatography eluting with (PE:EtOAc=100:1 to 1:2) to give methyl 6-[3-[2-(tert-butoxycarbonylamino)ethoxy]phenoxy]pyridine-3-carboxylate (120 mg, 70.43% yield, 90% purity) as a yellow solid.

[1367] LCMS (ESI) [M+H-100] + m / z: Calculated 289.1, Found 289.0

[1368] 1 H NMR (400 MHz, CDCl 3 )δ:ppm 8.84(1H,d,J=2.20Hz)8.29(1H,dd,J=8.62,2.38Hz)7.24-7.40(1H,m)6.95(1H,d,J=8.68Hz)6.78(2H,ddd ,J=12.93,8.22,1.96Hz)6.69-6.73(1H,m)4.02(2H,t,J=5.07Hz)3.93(3H,s)3.47-3.58(2H,m)1.46(9H,s)

[1369] Step 4: 6-[3-(2-aminoethoxy)phenoxy]pyridine-3-carboxylic acid methyl ester

[1370] A mixture of methyl 6-[3-[2-(tert-butoxycarbonylamino)ethoxy]phenoxy]pyridine-3-carboxylate (120 mg, 308.95 μmol, 1 eq.), HCl / dioxane (4 M, 772.37 μL, 10 eq.) in dioxane (3 mL) was stirred at 20° C. for 2 hours. The mixture was concentrated in vacuo to give methyl 6-[3-(2-aminoethoxy)phenoxy]pyridine-3-carboxylate (90 mg, 89.70% yield, HCl salt) as a white solid, which was used directly without purification.

[1371] 1 H NMR (400 MHz, DMSO-d 6 )δ:ppm 8.70(1H,d,J=2.26Hz)8.32(4H,br dd,J=8.66,2.38Hz)7.39(1H,t,J=8.16Hz)7.14(1H,d,J=8.78Hz)6.91(1H,dd,J=8 .16,1.88Hz)6.77-6.86(2H,m)4.20(2H,t,J=5.14Hz)3.86(3H,s)3.15-3.26(2H,m)

[1372] Step 5: 6-[3-(2-acetylaminoethoxy)phenoxy]pyridine-3-carboxylic acid methyl ester

[1373] To a mixture of methyl 6-[3-(2-aminoethoxy)phenoxy]pyridine-3-carboxylate (90 mg, 312.18 μmol, 1 eq.) in THF (3 mL) was added Ac 2 O (38.24 mg, 374.61 μmol, 35.09 μL, 1.2 equiv) and Et 3 N (94.77 mg, 936.53 μmol, 130.35 μL, 3 eq.) and the mixture was stirred at 20 °C for 12 hours. The reaction mixture was concentrated and poured into water (40 mL), then extracted with EtOAc (40 mL x 3). The combined organic layers were washed with brine (40 mL) and washed with anhydrous Na 2 SO 4 Drying, filtration and concentration in vacuo gave methyl 6-[3-(2-acetamidoethoxy)phenoxy]pyridine-3-carboxylate (80 mg, 77.58% yield) as a yellow oil which was used directly without purification.

[1374] 1 H NMR (400 MHz, DMSO-d 6)δ:ppm 8.71(1H,d,J=1.96Hz)8.31(1H,dd,J=8.56,2.45Hz)8.04-8.22(1H,m)7.35(1H,t,J=8.13Hz)7.12(1H,d,J=8.68Hz) 6.86(1H,dd,J=8.31,1.83Hz)6.73-6.82(2H,m)3.98(2H,t,J=5.69Hz)3.86(3H,s)3.39(2H,q,J=5.62Hz)1.82(3H,s)

[1375] Step 6: 6-[3-(2-Acetylaminoethoxy)phenoxy]pyridine-3-carboxylic acid

[1376] To methyl 6-[3-(2-acetylaminoethoxy)phenoxy]pyridine-3-carboxylate (80 mg, 242.18 μmol, 1 eq.) was dissolved in THF (3 mL) and H 2 O (1 mL) was added to the mixture of LiOH. 2 O (20.32 mg, 484.36 μmol, 2 eq.) was added and the mixture was stirred at 25 °C for 12 h. The mixture was concentrated under reduced pressure and the residue was acidified to pH 2 using HCl (1 M). The residue was extracted with EA (20 mL x 5) and the combined organic layers were washed with Na 2 SO 4 Drying, filtration and concentration in vacuo gave 6-[3-(2-acetamidoethoxy)phenoxy]pyridine-3-carboxylic acid (70 mg, 24% yield, 90% purity) as a yellow oil.

[1377] 1 H NMR (400 MHz, DMSO-d 6 )δ:ppm 8.68(1H,d,J=2.26Hz)8.29(1H,dd,J=8.66,2.38Hz)8.02-8.15(1H,m)7.35(1H,t,J=8.16Hz)7.09(1H,d, J=8.53Hz)6.85(1H,dd,J=8.28,2.01Hz)6.73-6.80(2H,m)3.96-4.01(2H,m)3.40-3.42(2H,m)1.82(3H,s)

[1378] Step 7: (2,5-Dioxopyrrolidin-1-yl)6-[3-(2-acetylaminoethoxy)phenoxy]pyridine-3-carboxylate

[1379] To a solution of 1-hydroxypyrrolidine-2,5-dione (63.67 mg, 553.26 μmol, 2.5 eq.) in DMF (5 mL) were added 6-[3-(2-acetamidoethoxy)phenoxy]pyridine-3-carboxylic acid (70 mg, 221.30 μmol, 1 eq.) and EDCI (63.64 mg, 331.95 μmol, 1.5 eq.), the mixture was degassed three times and heated under N 2 The mixture was stirred at 20°C for 3 hours. The reaction mixture was poured into water (40 mL) and extracted with EtOAc (40 mL x 3). The combined organic layers were washed with brine (40 mL) and washed with anhydrous Na 2 SO 4 Drying, filtration and concentration in vacuo gave (2,5-dioxopyrrolidin-1-yl)-6-[3-(2-acetamidoethoxy)phenoxy]pyridine-3-carboxylate (85 mg, 74.33% yield, 80% purity) as a yellow oil which was used directly without purification.

[1380] LCMS(ESI)[M+H] + m / z: Calculated 414.1, Found 414.0

[1381] Step 8: (2S)-2-[[6-[3-(2-acetylaminoethoxy)phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[1382] (2,5-Dioxopyrrolidin-1-yl)-6-[3-(2-acetamidoethoxy)phenoxy]pyridine-3-carboxylate (85 mg, 205.62 μmol, 1 eq.), (2S)-2-amino-5,5-dimethyl-hexanoic acid (33.15 mg, 208.20 μmol, 1.01 eq.) and DIEA (26.58 mg, 205.62 μmol, 35.82 uL, 1 eq.) were dissolved in DCM (0.5 mL), H 2 A mixture of O (1.5 mL) and DMF (3 mL) was degassed and washed with N 2 Purge three times, then 2 The mixture was stirred at 25° C. for 16 hours under atmosphere. The reaction was concentrated in vacuo, and the crude product was purified by preparative HPLC (HCl) column: Boston Green ODS 150*30mm*5μm; mobile phase: [water (0.05% HCl)-ACN]; B%: 24%-64%, 9 minutes. (2S)-2-[[6-[3-(2-acetylaminoethoxy)phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (16.5 mg, 15.79% yield, 90% purity) was obtained as a white solid.

[1383] LCMS(ESI)[M+H] + m / z: Calculated 458.22, Found 458.2

[1384] 1 H NMR (400 MHz, DMSO-d 6 )δ:ppm 8.68(1H,d,J=7.83Hz)8.64(1H,d,J=2.08Hz)8.29(1H,dd,J=8.62,2.51Hz)8.03-8.13(1H,m)7.33(1H,t,J=8.13Hz)7.09(1H,d,J=8.93H z)6.80-6.92(1H,m)6.70-6.77(2H,m)4.31(1H,td,J=8.47,5.20Hz)3.97(2H,t,J=5.69Hz)3.39(2H,q,J=5.62Hz)1.81(3H,s)1.77(1H,br t,J=5.14Hz)1.11-1.37(3H,m)0.87(9H,s)

[1385] SB012

[1386] (2S)-2-[[6-[3-(Acetylaminomethyl)phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[1387]

[1388] Step 1: N-[(3-Hydroxyphenyl)methyl]acetamide

[1389] In N 2 , 3-(aminomethyl)phenol (800 mg, 6.50 mμoλ, 1 eq) and Et 3 Acetyl chloride (509 mg, 6.50 mmol, 463 μL, 1.0 equiv) was added dropwise to a solution of N (1.64 g, 16.2 mmol, 2.26 mL, 2.5 equiv) in DCM (20 mL). The reaction mixture was warmed to 20 ° C and stirred for 2 hours. The reaction mixture was filtered and the filter cake was washed with 25 mL of ethyl acetate. The combined organic layers were concentrated in vacuo and the residue was purified by reverse phase HPLC (water (0.05% ammonium hydroxide v / v)-ACN; column: YMC Triart C18250*50mm*7μm). The product was lyophilized to give N-[(3-hydroxyphenyl)methyl]acetamide (720 mg, 65% yield, 97% purity) in the form of a colorless oil.

[1390] 1H NMR (400MHz, CDCl3) δ: 7.10 (t, J = 7.82Hz, 1H), 6.76 (s, 1H), 6.66-6.74 (m, 2H), 5.96 (br s, 1H), 4.30 (d, J = 6.00Hz, 2H), 1.95 (s, 3H).

[1391] Step 2: 6-[3-(Acetylaminomethyl)phenoxy]pyridine-3-carboxylic acid methyl ester

[1392] A mixture of N-[(3-hydroxyphenyl)methyl]acetamide (620 mg, 3.75 mmol, 1 eq), methyl 6-bromopyridine-3-carboxylate (810 mg, 3.75 mmol, 1 eq), potassium phosphate (796.7 mg, 3.75 mmol, 1 eq) and CuI (714 mg, 3.75 mmol, 1.0 eq) in DMSO (15 mL) was degassed and heated with N 2 Purge three times, then 2 The mixture was stirred at 120°C for 16 h under a CO atmosphere. 2 O (40 mL), and extracted with ethyl acetate (20 mL x 4). The organic layer was washed with Na 2 SO 4 The residue was purified by flash silica gel chromatography ( 12g Purification by silica flash column with a 0-100% ethyl acetate / petroleum ether gradient at a flow rate of 25 mL / min) gave methyl 6-[3-(acetylaminomethyl)phenoxy]pyridine-3-carboxylate as a yellow oil (54% yield, 86% purity).

[1393] 1 H NMR (400MHz, CDCl3) δ: 8.70-8.77 (m, 1H), 8.22 (dd, J = 8.63, 2.38Hz, 1H), 7.33 (t, J = 7. 75Hz,1H),7.11(d,J=7.63Hz,1H),6.97-7.05(m,2H),6.89(d,J=8.75Hz,1H),5.83(br s,1H),4.40(d,J=5.75Hz,2H),3.85(s,3H),2.89(s,2H),2.81(s,2H)1.97(s,3H).

[1394] Step 3: 6-[3-(Acetylaminomethyl)phenoxy]pyridine-3-carboxylic acid

[1395] To methyl 6-[3-(acetylaminomethyl)phenoxy]pyridine-3-carboxylate (610 mg, 2.0 mmol, 1 eq.) was dissolved in THF (15 mL) and H 2 O (5 mL) was added to the mixture of LiOH. 2 O (170 mg, 4.0 mmol, 2.0 equiv). The mixture was stirred at 25 °C for 2 hours. The mixture was concentrated under reduced pressure and the residue was acidified to pH 2 using HCl (1 M). The residue was extracted with ethyl acetate (20 mL x 5) and the combined organic layers were washed with Na 2 SO 4 Drying, filtration and concentration in vacuo gave 6-[3-(acetylaminomethyl)phenoxy]pyridine-3-carboxylic acid (500 mg, 86% yield, 100% purity) as a yellow oil.

[1396] 1 H NMR (400MHz, methanol-d4) δ: 8.84 (d, J=1.75Hz, 1H), 8.31 (dd, J=8.63, 2.38Hz, 1H), 7.40 (t, J= 7.82Hz,1H),7.18(d,J=7.63Hz,1H),7.04-7.11(m,2H),6.98(d,J=8.76Hz,1H),5.95(br s, 1H), 4.47 (d, J = 5.75Hz, 2H), 2.05 (s, 3H), 1.26 (t, J = 7.13Hz, 2H).

[1397] Step 4: (2,5-dioxopyrrolidin-1-yl)6-[3(acetylaminomethyl)phenoxy]pyridine-3-carboxylate

[1398] To a solution of 6-[3-(acetylaminomethyl)phenoxy]pyridine-3-carboxylic acid (50 mg, 174 μmol, 1 eq.) in DMF (2 mL) were added 1-hydroxypyrrolidine-2,5-dione (50 mg, 436 μmol, 2.5 eq.) and EDCI (50 mg, 261 μmol, 1.5 eq.), the mixture was degassed three times, and heated under N 2 , stirred at 20°C for 3 hours. The reaction mixture was poured into water (4 mL) and extracted with ethyl acetate (5 mL). The organic layer was washed with water (3 ml), brine (5 mL x 2), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give (2,5-dioxopyrrolidin-1-yl)-6-[3-(acetylaminomethyl)phenoxy]pyridine-3-carboxylate (50 mg, 68% yield, 92% purity) as a yellow oil.

[1399] LCMS(ESI)[M+H] +m / z: Calculated 383.1, Found 384.0

[1400] Step 5: (2S)-2-[[6-[3-(Acetylaminomethyl)phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[1401] (2,5-Dioxopyrrolidin-1-yl) 6-[3-(acetylaminomethyl)phenoxy]pyridine-3-carboxylate (50 mg, 130 μmol, 1 eq.), (2S)-2-amino-5,5-dimethyl-hexanoic acid (21 mg, 130 μmol, 1 eq.) and DIEA (25 mg, 195 μmol, 34 μL, 1.5 eq.) were dissolved in DCM (0.2 mL), DMF (1.2 mL), H 2 The mixture was degassed with N 2 Purge three times, then 2 The mixture was stirred at 20° C. for 4 hours under an atmosphere. The reaction mixture was concentrated in vacuo to give a residue, which was purified by preparative HPLC (water (0.225% FA)-ACN, YMC Triart C18250*50mm*7μm). The residual aqueous solution was lyophilized to give (2S)-2-[[6-[3-(acetylaminomethyl)phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (41 mg, 71% yield, 96% purity)) in the form of a white solid.

[1402] 1 H NMR (400MHz, CDCl3) δ: 8.59 (br s, 1H), 8.13 (br d, J = 7.75Hz, 1H), 7.32-7.41 (m, 1H), 7.21 (br s, 1H), 7.14 (br d,J=7.25Hz,1H),6.99-7.09(m,2H),6.90(br d,J=8.00Hz,1H),6.51(br s,1H),4.70(br d,J=5.00Hz,1H),4.44(br s,2H),1.91-2.10(m,4H),1.79(br d,J=6.25Hz,1H),1.14-1.39(m,3H),0.87(s,9H).

[1403] BF021

[1404] (2S)-2-[[6-[[6-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[1405]

[1406] Step 1: Synthesis of (2,5-dioxopyrrolidin-1-yl) 5-bromonaphthalene-2-carboxylate

[1407] To a solution of 5-bromonaphthalene-2-carboxylic acid (1.0 g, 3.98 mmol, 1 eq.) in DMF (10 mL) were added 1-hydroxypyrrolidine-2,5-dione (1.15 g, 9.96 mmol, 2.5 eq.) and EDCI (1.15 g, 5.97 mmol, 1.5 eq.), the mixture was degassed three times and stirred under N 2 , and stirred at 20°C for 3 hours. The reaction mixture was poured into H 2 O (20 mL) and filtered. 2 O (3 x 20 mL) and concentrated in vacuo to give (2,5-dioxopyrrolidin-1-yl) 5-bromonaphthalene-2-carboxylate (1.39 g, 100.00% yield, 100% purity) as a white solid.

[1408] 1 H NMR (400MHz, DMSO) δ: ppm 8.93 (1H, d, J = 0.8Hz) 8.33 (1H, t, J = 8.0Hz) 8.22 (1H, d, J = 0.8Hz) 8.14 (1H, d, J = 4.8Hz) 7.62 (1H, t, J = 8.0Hz) 2.90 (4H, s)

[1409] Step 2: Synthesis of N-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethyl]-5-bromo-naphthalene-2-carboxamide

[1410] A mixture of 5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]-N-(2-aminoethyl)pentanamide (600 mg, 2.10 mmol, 1 eq), (2,5-dioxopyrrolidin-1-yl) 5-bromonaphthalene-2-carboxylate (729.38 mg, 2.10 mmol, 1 eq) and DIEA (812.28 mg, 6.29 mmol, 1.09 mL, 3 eq) in DMF (20 mL) was degassed and heated with N 2 Purge three times, then 2 The mixture was stirred at 20° C. for 16 hours under a 4% CO atmosphere. The precipitate was filtered and dried under reduced pressure to give N-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethyl]-5-bromo-naphthalene-2-carboxamide (1 g, crude) as a white solid, which was used directly without purification.

[1411] Step 3: Synthesis of N-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethyl]-5-hydroxy-naphthalene-2-carboxamide

[1412] To a mixture of N-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethyl]-5-bromo-naphthalene-2-carboxamide (1 g, 1.93 mmol, 1 eq) in dioxane (30 mL) was added KOH (432.07 mg, 7.70 mmol, 4 eq) in H 2 O (30 mL). Then, 2 tBuXPhos Pd G3 (76.46 mg, 96.26 μmol, 0.05 eq) was added under N 2 The reaction was stirred at 110 °C for 16 hours. The reaction was extracted with EtOAc (50 mL x 3). The aqueous layer was adjusted to pH 3-4 with 1M HCl aqueous solution and extracted with EtOAc (150 mL x 3). The organic layer was washed with Na 2 SO 4Dried and concentrated to give N-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethyl]-5-hydroxy-naphthalene-2-carboxamide (220 mg, 385.49 μmol, 20.02% yield, 80% purity) as a yellow solid. The aqueous layer was concentrated and the crude product was triturated with MeOH (30 mL), filtered and concentrated to give N-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethyl]-5-hydroxy-naphthalene-2-carboxamide (350 mg, crude) as a light red solid.

[1413] Step 4: Synthesis of 6-[[6-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylate

[1414] To N-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethyl]-5-hydroxy-naphthalene-2-carboxamide (120 mg, 262.84 μmol, 1 eq) and Cs 2 CO 3 (128.46 mg, 394.26 μmol, 1.5 eq) was added to a solution of 6-fluoropyridine-3-carboxylic acid methyl ester (44.85 mg, 289.12 μmol, 1.1 eq) in DMF (3 mL). The reaction was stirred at 25 °C for 16 h. 2 O (10 mL) was added to the solution, and the solution was extracted with EtOAc (50 mL x 8). The organic layer was washed with Na 2 SO 4 The crude product was triturated with MeOH (5 mL), filtered and dried under reduced pressure to give methyl 6-[[6-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylate (160 mg, 47.47% yield, 92.28% purity) as an off-white solid.

[1415] LCMS(ESI)[M+H] + m / z: Calculated 591.28, Found 592.3

[1416] Step 5: Synthesis of 6-[[6-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylic acid

[1417] To methyl 6-[[6-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylate (110 mg, 185.91 μmol, 1 eq) was dissolved in THF (3 mL), MeOH (2 mL) and H 2 O (1 mL) was added to the solution of LiOH. 2 O (15.60 mg, 371.82 μmol, 2 eq.). The reaction was stirred at 25 °C for 2 h. MeOH and THF were removed in vacuo, the aqueous layer was adjusted to pH 4-5 with 1N HCl aqueous solution and extracted with EtOAc (20 mL x 2). The organic layer was washed with Na 2 SO 4 Drying and concentration gave 6-[[6-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylic acid (160 mg, 88.29% yield, 94.5% purity, HCl salt) as a brown solid, which was used directly without purification.

[1418] LCMS(ESI)[M+Na] + m / z: Calculated 577.2, Found 578.1

[1419] 1 H NMR (400 MHz, DMSO-d 6)δ:ppm 8.81(1H,br t,J=5.3Hz)8.58(2H,d,J=1.8Hz)8.34(1H,dd,J=8.7,2.4Hz)8.09(1H,br t,J=5.0Hz)7.91-7.99(2H,m)7.83(1H,d,J=8.8Hz)7.65(1H,t,J=7.9Hz)7.46(1H,d,J=7.5Hz)7.29(1H,d,J=8.5Hz)6.42(1H,br d,J=2.0Hz)4.23(1H,dd,J=7.7,5.1Hz)4.04(1H,dd,J=7.7,4.4Hz)3.36(2H,br d,J=6.0Hz)3.24-3.29(2H,m)2.97-3.03(1H,m)2.71-2.77(1H,m)2.08(2H,br t,J=7.4Hz)1.38-1.58(4H,m)1.21-1.35(3H,m)

[1420] Step 6: Synthesis of (2,5-dioxopyrrolidin-1-yl)6-[[6-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylate

[1421] To a solution of 6-[[6-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylic acid (110 mg, 190.43 μmol, 1 eq) and EDCI (54.76 mg, 285.64 μmol, 1.5 eq) in DMF (5 mL) was added 1-hydroxypyrrolidine-2,5-dione (54.79 mg, 476.07 μmol, 2.5 eq). The reaction was stirred at 25 °C for 3 h. The reaction was concentrated to give (2,5-dioxopyrrolidin-1-yl) 6-[[6-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylate (128.4 mg, crude) as a yellow oil which was used without purification.

[1422] Step 7: (2S)-2-[[6-[[6-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[1423] (2,5-Dioxopyrrolidin-1-yl)6-[[6-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylate (128.4 mg, 190.30 μmol, 1 eq), (2S)-2-amino-5,5-dimethyl-hexanoic acid (30.30 mg, 190.30 μmol, 1 eq) and DIEA (36.89 mg, 285.45 μmol, 49.72 μL, 1.5 eq) were dissolved in DCM (0.5 mL), DMF (3.0 mL) and H 2 The mixture was degassed with N 2 Purge three times, then 2 The mixture was stirred at 25° C. for 3 hours under atmosphere. The reaction was concentrated and the residue was purified by preparative HPLC (column: Boston Green ODS 150*30mm*5μm; mobile phase: [water (0.05% HCl)-ACN]; B%: 21%-61%, 9 minutes) to give (2S)-2-[[6-[[6-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (18 mg, 13.16% yield, 100% purity) as a white solid.

[1424] LCMS(ESI)[M+H] + m / z: calculated 718.3, found 719.1

[1425] 1 H NMR (400 MHz, DMSO-d 6)δ:ppm 8.60-8.71(2H,m)8.47-8.55(2H,m)8.34(1H,dd,J=8.6,2.5Hz)7.81-7.99(4H,m )7.64(1H,t,J=7.9Hz)7.43(1H,dd,J=7.5,0.8Hz)7.25-7.30(1H,m)6.39(2H,br s)4.30(1H,td,J=8.5,5.2Hz)4.24(1H,dd,J=7.7,4.5Hz)4.04(1H,dd,J=7.6,4.5Hz)3.36(2H,br d,J=6.0Hz)3.25(2H,q,J=6.0Hz)2.96-3.04(1H,m)2.74(1H,dd,J=12.4,5.1Hz)2.52-2.58(1H, m)2.07(2H,t,J=7.3Hz)1.67-1.85(2H,m)1.40-1.61(4H,m)1.19-1.36(4H,m)0.83-0.87(9H,m)

[1426] BF027

[1427] (2S)-2-[[6-[[5-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[1428]

[1429] Step 1: Synthesis of (2,5-dioxopyrrolidin-1-yl) 5-bromonaphthalene-1-carboxylate

[1430] To a solution of 5-bromonaphthalene-1-carboxylic acid (1 g, 3.98 mmol, 1 eq.) in DMF (10 mL) were added 1-hydroxypyrrolidine-2,5-dione (1.15 g, 9.96 mmol, 2.5 eq.) and EDCI (1.15 g, 5.97 mmol, 1.5 eq.), the mixture was degassed three times and stirred under N 2 The mixture was stirred at 20 °C for 3 h. The reaction mixture was poured into H 2 O (20 mL) and filtered. 2 O (3 x 20 mL) and concentrated in vacuo to give (2,5-dioxopyrrolidin-1-yl) 5-bromonaphthalene-1-carboxylate (1.39 g, 100.00% yield) as a white solid.

[1431] 1H NMR (400 MHz, DMSO-d 6 )δ:ppm 8.64-8.60(2H,m)8.43(1H,m)8.10(1H,m),7.90(1H,m),7.70-7.65(1H,m),2.95(4H,s).

[1432] Step 2: N-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethyl]-5-bromo-naphthalene-1-carboxamide

[1433] To a solution of 5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]-N-(2-aminoethyl)pentanamide (1 g, 3.49 mmol, 1 eq) in DMF (60 mL) was added DIPEA (902.54 mg, 6.98 mmol, 1.22 mL, 2 eq) and (2,5-dioxopyrrolidin-1-yl)-5-bromonaphthalene-1-carboxylate (1.22 g, 3.49 mmol, 1 eq). The mixture was stirred at 20 °C for 16 h. The reaction was filtered and the filter cake was purified by H 2 O (3 x 20 mL) and concentrated in vacuo to give N-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethyl]-5-bromo-naphthalene-1-carboxamide (1.38 g, 76.08% yield) as a white solid.

[1434] LCMS(ESI)[M+H] + m / z: calculated 520.5, found 521.2

[1435] Step 3: N-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethyl]-5-hydroxy-naphthalene-1-carboxamide

[1436] N-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethyl]-5-bromo-naphthalene-1-carboxamide (1 g, 1.93 mmol, 1 eq) was stirred in dioxane (20 mL) and H 2tBuXPhos Pd G3 (76.46 mg, 96.26 μmol, 0.05 eq) and KOH (432.04 mg, 7.70 mmol, 4 eq) were added to a solution in 4% 4% HCl (20 mL). The mixture was stirred at 110 ° C for 16 hours. The reaction mixture was concentrated under reduced pressure and washed with MTBE (30 mL x2). The solid was acidified to pH 5 with 1M HCl, collected and dried in vacuo to give N-[2-[5-[(3aS, 4S, 6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethyl]-5-hydroxy-naphthalene-1-carboxamide (800 mg, crude) in the form of a brown solid, which was used directly without further purification.

[1437] LCMS(ESI)[M+H] + m / z: calculated 457.5, found 457.3

[1438] Step 4: 6-[[5-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylic acid methyl ester

[1439] To a solution of N-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethyl]-5-hydroxy-naphthalene-1-carboxamide (800 mg, 1.75 mmol, 1 eq) in DMF (10 mL) was added methyl 6-fluoropyridine-3-carboxylate (271.82 mg, 1.75 mmol, 1 eq) and Cs 2 CO 3 (1.14 g, 3.50 mmol, 2 eq.). The reaction mixture was stirred at 20 °C for 16 h. 2 O (40 mL) and extracted with EA (40 mL x 3). The organic layer was washed with Na 2 SO 4 Drying, filtration and concentration under reduced pressure gave methyl 6-[[5-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylate (800 mg, 38.58% yield) as a grey solid which was used without purification.

[1440] LCMS(ESI)[M+H] +m / z: calculated 591.7, found 592.1

[1441] Step 5: 6-[[5-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylic acid

[1442] To methyl 6-[[5-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylate (800 mg, 1.35 mmol, 1 eq) in THF (12 mL) and H 2 O (4 mL) was added to the mixture of LiOH. 2 O (113.48 mg, 2.70 mmol, 2 eq). The mixture was stirred at 25 ° C for 4 hours. The mixture was acidified to pH 5 with HCl (1 M) and concentrated under reduced pressure to give 6-[[5-[2-[5-[(3aS, 4S, 6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylic acid (100 mg, crude) as a yellow solid.

[1443] LCMS(ESI)[M+Na] + m / z: calculated 577.2, found 578.3

[1444] Step 6:

[1445] (2,5-Dioxopyrrolidin-1-yl)-6-[[5-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylate

[1446] To a solution of 6-[[5-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylic acid (100 mg, 173.12 μmol, 1 eq) in DMF (2 mL) were added 1-hydroxypyrrolidine-2,5-dione (49.81 mg, 432.79 μmol, 2.5 eq) and EDC (49.78 mg, 259.67 μmol, 1.5 eq), and the mixture was degassed three times and heated under N 2 The mixture was stirred at 20° C. for 4 hours. The reaction mixture was concentrated under reduced pressure to give (2,5-dioxopyrrolidin-1-yl) 6-[[5-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylate (100 mg, crude) as a pale yellow gum.

[1447] Step 7: (2S)-2-[[6-[[5-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[1448] (2,5-Dioxopyrrolidin-1-yl)-6-[[5-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carboxylate (100 mg, 148.21 μmol, 1 eq), (2S)-2-amino-5,5-dimethyl-hexanoic acid (23.60 mg, 148.21 μmol, 1 eq) and DIEA (28.73 mg, 222.31 μmol, 38.72 uL, 1.5 eq) were dissolved in DCM (0.4 mL), DMF (2.4 mL) and H 2 The mixture was degassed with N 2 Purge three times, then 2The mixture was stirred at 20° C. for 4 hours under an atmosphere of 4% CO. The reaction mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC (HCl conditions) to give (2S)-2-[[6-[[5-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethylcarbamoyl]-1-naphthyl]oxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (5 mg, 4.53% yield, 96.6% purity) as a white solid.

[1449] 1 H NMR (400 MHz, DMSO-d 6 )δ:ppm 8.65-8.70(1H,m)8.58-8.64(1H,m)8.55(1H,m)8.33(1H,dd,J=8.8,2.4Hz)8.13(1H,d,J=8.4Hz)7.90(1H,m)7 .88(1H,d,J=8.Hz)7.61–7.68(2H,m)7.52–7.56(1H,m)7.38(1H,d,J=6.8Hz)7.26(1H,d,J=8.4Hz)6.40(1H,br s)6.33(1H,br s)4.24–4.31(2H,m)4.05-4.09(1H,m)3.25–3.35(4H,m)3.04-3.08(1H,m)2.75(1H,dd ,J=12.4,5.1Hz)2.07(2H,t,J=7.3Hz)1.32-1.81(5H,m)1.15-1.30(5H,m)0.85(9H,s)

[1450] LCMS(ESI)[M+H] + m / z: calculated 718.31, found 719.2

[1451] BF022

[1452] (S)-5,5-dimethyl-2-(6-(3-(2-(2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoylamino)ethoxy)ethoxy)ethoxy)phenoxy)nicotinylamino)hexanoic acid

[1453]

[1454] Step 1: 2-[2-[2-(tert-Butoxycarbonylamino)ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate

[1455] To a solution of tert-butyl N-[2-[2-(2-hydroxyethoxy)ethoxy]ethyl]carbamate (1.5 g, 6.02 mmol, 1 eq) in DCM (30 mL) was added p-toluenesulfonyl chloride (2.29 g, 12.03 mmol, 2.0 eq) and Et 3 N (1.83 g, 18.05 mmol, 2.51 mL, 3.0 equiv). The mixture was stirred at 20 °C for 16 h. The mixture was poured into water (100 mL) and the aqueous phase was extracted with DCM (20 mL x 3). The combined organic phases were washed with brine (20 mL) and washed with anhydrous Na 2 SO 4 The mixture was dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate = 20 / 1 to 5 / 1) to give 2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (2.3 g, 94.74% yield) as a brown oil.

[1456] LCMS(ESI)[M+Na] + m / z: Calculated 426.1, Found 426.1

[1457] 1 H NMR (400MHz, chloroform-d) δ: ppm 7.81 (2H, d, J = 8.3Hz) 7.35 (2H, d, J = 8.0Hz) 4.94 (1H, br s)4.13-4.23(2H,m)3.68-3.73(2H,m)3.48-3.61(6H,m)3.30(2H,br s)2.36-2.55(3H,m)1.44(9H,s)

[1458] Step 2: tert-Butyl (2-(2-(2-(3-hydroxyphenoxy)ethoxy)ethoxy)ethyl)carbamate

[1459] To a solution of 2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (2.0 g, 4.96 mmol, 1.0 eq) and (3-hydroxyphenyl)acetate (904.99 mg, 5.95 mmol, 1.2 eq) in DMF (40 mL) was added K 2 CO 3 (2.06 g, 14.87 mmol, 3.0 equiv.). The mixture was stirred at 100 °C for 16 h. The mixture was poured into water (100 mL) and extracted with EtOAc (50 mL x 2). The combined organic phases were washed with brine (50 mL) and washed with anhydrous Na 2SO 4 The mixture was dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate=10 / 1 to 1 / 1) to give tert-butyl N-[2-[2-[2-(3-hydroxyphenoxy)ethoxy]ethoxy]ethyl]carbamate (1.0 g, 59.1% yield) in the form of a pale yellow oil.

[1460] LCMS(ESI)[M+Na] + m / z: calculated 341.2, found 364.2

[1461] Step 3: 6-[3-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylic acid methyl ester

[1462] To a mixture of tert-butyl N-[2-[2-[2-(3-hydroxyphenoxy)ethoxy]ethoxy]ethyl]carbamate (1.0 g, 2.93 mmol, 1 eq.) and methyl 6-fluoropyridine-3-carboxylate (545.26 mg, 3.51 mmol, 1.2 eq.) in DMF (20 mL) was added Cs 2 CO 3 (2.86 g, 8.79 mmol, 3.0 equiv.). The mixture was stirred at 80 °C for 16 h. The mixture was cooled to 20 °C and poured into water (100 mL). The aqueous phase was extracted with ethyl acetate (60 mL x 3). The combined organic phases were washed with brine (60 mL) and washed with anhydrous Na 2 SO 4 The mixture was dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, petroleum ether / ethyl acetate=10 / 1 to 2 / 1) to give methyl 6-[3-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate (900 mg, 64.4% yield) in the form of a pale yellow oil.

[1463] LCMS(ESI)[M+Na] + m / z: calculated 477.2, found 499.3

[1464] Step 4: 6-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)phenoxy)nicotinate

[1465] To a mixture of methyl 6-[3-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate (1.0 g, 2.10 mmol, 1 eq) in DCM (20 mL) was added TFA (11.96 g, 104.93 mmol, 7.77 mL, 50 eq). The mixture was stirred at 20 °C for 3 h. The mixture was concentrated in vacuo and dissolved in DCM (200 mL) and washed with saturated NaHCO 3 The aqueous solution was basified until pH 8 and then extracted with DCM (3 x 50 mL). The combined organic layers were washed with brine (50 mL) and washed with anhydrous Na 2 SO 4 Drying, filtration and concentration in vacuo gave methyl 6-[3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate (900 mg, 94.9% yield) as a yellow oil.

[1466] LCMS(ESI)[M+H] + m / z: calculated 377.2, found 377.1

[1467] Step 5: 6-(3-(2-(2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoylamino)ethoxy)ethoxy)phenoxy)nicotinate

[1468] In N 2 To a mixture of 5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid (493.29 mg, 2.02 mmol, 0.95 eq) in DMF (15 mL) was added HATU (1.21 g, 3.19 mmol, 1.5 eq) and DIEA (824.07 mg, 6.38 mmol, 1.11 mL, 3.0 eq). The mixture was stirred at 20 °C for 30 minutes, then 6-[3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]phenoxy]pyridine-3-carboxylic acid methyl ester (800 mg, 2.13 mmol, 1 eq) was added. The reaction was stirred for 1 hour, then the mixture was poured into water (100 mL). The aqueous phase was extracted with ethyl acetate (50 mL x 3). The combined organic phases were washed with brine (50 mL) and washed with anhydrous Na 2 SO 4The mixture was dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, MeOH / ethyl acetate = 0 / 1 to 1 / 10) to give methyl 6-[3-[2-[2-[2-[5-[(3aS, 4S, 6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate (900 mg, 70.3% yield) in the form of a yellow solid.

[1469] LCMS (ESI) [M+H] + m / z: calculated value 603.2, found value 603.4

[1470] 1H NMR (400MHz, chloroform-d) δ: ppm 8.82 (1H, d, J = 1.8Hz) 8.28 (1H, dd, J = 8.5, 2.3Hz) 7.33 (1H, t, J = 8.2Hz) 6.94 (1H, d, J = 8.3Hz) 6.83 (1H, d, J = 1.8Hz) 6.69-6.80 (2H, m) 6.63 (1H, br t,J=5.4Hz)6.19(1H,br s)4.49(1H,dd,J=7.7,4.6Hz)4.29(1H,dd,J=7.7,4.6Hz)4.12-4.16(3H,m)3.93(3H,s)3.82-3.88(2H,m)3.69-3.74(2H,m)3.63-3.68(2H,m)3 .54-3.61(2H,m)3.40-3.47(2H,m)3.08-3.14(1H,m)2.89(1H,dd,J=12.8,5.0Hz)2.73(1H,d,J=12.8Hz)2.21(2H,td,J=7.3,2.5Hz)1.99(1H,br s)1.60-1.75(4H,m)1.34-1.50(4H,m)

[1471] Step 6:

[1472] 6-(3-(2-(2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoylamino)ethoxy)ethoxy)ethoxy)phenoxy)nicotinic acid

[1473] To methyl 6-[3-[2-[2-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate (900 mg, 1.49 mmol, 1 eq) was dissolved in THF (10 mL), MeOH (5 mL), H 2 LiOH.H O (5 mL) was added to the mixture 2 O (187.99 mg, 4.48 mmol, 3.0 equiv). The mixture was stirred at 20 °C for 2 hours. The mixture was concentrated in vacuo to remove MeOH, then the aqueous layer was acidified with 1M aqueous HCl until pH 1-2 and extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine (50 mL) and washed with anhydrous Na 2 SO 4 The mixture was dried, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (column height: 250 mm, diameter: 100 mm, 100-200 mesh silica gel, MeOH / ethyl acetate = 0 / 1 to 1 / 10) to give 6-[3-[2-[2-[2-[5-[(3aS, 4S, 6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylic acid (450 mg, 51.2% yield) as a white gum.

[1474] LCMS (ESI) [M+H] + m / z: calculated value 589.2, found value 589.1

[1475] Step 7: 2,5-dioxopyrrolidin-1-yl-6-(3-(2-(2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoylamino)ethoxy)ethoxy)ethoxy)phenoxy)nicotinate

[1476] In N 2To a mixture of 6-[3-[2-[2-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylic acid (500 mg, 849.37 μmol, 1 eq) and 1-hydroxypyrrolidine-2,5-dione (146.63 mg, 1.27 mmol, 1.5 eq) in DMF (10 mL) was added EDC (244.24 mg, 1.27 mmol, 1.5 eq). The mixture was stirred at 20 °C for 2 h. The mixture was poured into water (50 mL) and saturated NaHCO 3 The aqueous phase was extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with brine (30 mL) and washed with anhydrous Na 2 SO 4 Drying, filtration and concentration in vacuo gave (2,5-dioxopyrrolidin-1-yl) 6-[3-[2-[2-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate (500 mg, 85.8% yield) as a light yellow oil which was used without further purification.

[1477] LCMS (ESI) [M+H] + m / z: calculated value 686.3, found value 686.5

[1478] Step 8: (S)-5,5-dimethyl-2-(6-(3-(2-(2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoylamino)ethoxy)ethoxy)ethoxy)phenoxy)nicotinylamino)hexanoic acid

[1479] In N 2To a mixture of (2,5-dioxopyrrolidin-1-yl)6-[3-[2-[2-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate (300 mg, 437.48 μmol, 1 eq) and (2S)-2-amino-5,5-dimethyl-hexanoic acid (69.66 mg, 437.48 μmol, 1 eq) in DMF (10 mL) was added DIEA (169.62 mg, 1.31 mmol, 228.60 μL, 3.0 eq). The mixture was stirred at 20° C. for 16 h. The mixture was poured into water (100 mL) and the aqueous phase was extracted with ethyl acetate (50 mL x 3). The combined organic phases were washed with brine (50 mL) and washed with anhydrous Na 2 SO 4 The mixture was dried, filtered and concentrated in vacuo. The residue was purified by preparative HPLC (column: Boston Green ODS150*30mm*5μm; mobile phase: [water (0.05% HCl)-ACN]; B%: 22%-62%, 9 minutes) to give (2S)-2-[[6-[3-[2-[2-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (90 mg, 26.84% yield, HCl salt) as a white solid.

[1480] LCMS (ESI) [M+H] + m / z: calculated value 730.3, found value 730.3

[1481] 1 H NMR (400 MHz, DMSO-d 6)δ: ppm 8.69 (1H, d, J = 7.8 Hz), 8.64 (1H, d, J = 2.3 Hz), 8.28 (1H, dd, J = 8.5, 2.5 Hz), 7.83 (1H, t, J = 5.4 Hz), 7.33 (1H, t, J = 8.2 Hz), 7.09 (1H, d, J = 8.5 Hz), 6.84 (1H, dd, J = 7.9, 2.1 Hz), 6.70 - 6.80 (2H, m), 6.41 (1H, br s), 4.26 - 4.38 (2H, m), 4.07 - 4.17 (3H, m), 3.71 - 3.77 (2H, m), 3.56 - 3.61 (2H, m), 3.50 - 3.54 (3H, m), 3.18 (2H, q, J = 5.9 Hz), 3.06 - 3.11 (1H, m), 2.81 (1H, dd, J = 12.4, 5.1 Hz), 2.51 - 2.56 (3H, m), 2.01 - 2.09 (3H, m), 1.68 - 1.83 (2H, m), 1.55 - 1.65 (1H, m), 1.39 - 1.53 (3H, m), 1.20 - 1.35 (4H, m), 0.87 (9H, s)

[1482] 1 H NMR (400 MHz, chloroform - d) δ: ppm 8.67 (1H, s), 8.27 (1H, br d, J = 7.1 Hz), 7.78 (1H, brd, J = 7.4 Hz), 7.30 - 7.38 (2H, m), 6.93 - 7.05 (1H, m), 6.80 (2H, br t, J = 7.8 Hz), 6.74 (1H, br s), 6.41 (1H, br s), 4.73 (1H, br d, J = 3.9 Hz), 4.54 (1H, br s), 4.36 (1H, br s), 4.13 - 4.21 (2H, m), 3.85 (2H, br s), 3.58 - 3.75 (4H, m), 3.50 - 3.57 (2H, m), 3.36 (2H, br s), 3.19 (1H, br s), 2.94 (1H, br d, J = 9.8 Hz), 2.74 (1H, br d, J = 12.6 Hz), 2.12 (3H, br d, J = 7.1 Hz), 1.96 - 2.05 (5H, m), 1.32 - 1.45 (5H, m), 0.89 (9H, s)

[1483] BF025

[1484] 2-[[6-[3-[2-[2-[2-[2-[2-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid

[1485]

[1486] Step 1: 2-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate

[1487] tert-Butyl N-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate (100 mg, 262.15 μmol, 1 eq) was dissolved in THF (1 mL). 2 The mixture was cooled to 0°C and 4-methylbenzenesulfonyl chloride (59.97 mg, 314.58 μmol, 1.2 eq.) dissolved in THF (1 mL) was added dropwise. The reaction mixture was allowed to warm to 20°C and stirred for 16 hours. TLC (KMnO 4 , DCM: MeOH = 10:1) showed that the reaction was complete. THF was evaporated and 50 mL of H 2 O, followed by 5 mL of CH 2 Cl 2 The product was extracted twice and washed with 5 mL of brine. 4 The residue was purified by silica gel chromatography eluting with (DCM: MeOH = 50: 1 to 10: 1) to give 2-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (100 mg, 71.21% yield, 100% purity) as a colorless oil.

[1488] 1 H NMR (400MHz, chloroform-d) δ: ppm 7.82 (2H, d, J = 8.1Hz) 7.36 (2H, d, J = 8.0Hz) 5.06 (1H, br s) 4.11-4.26 (2H, m) 3.50-3.80 (21H, m) 3.33 (2H, br s)2.47(3H,s)1.46(9H,s)

[1489] Step 2: tert-Butyl N-[2-[2-[2-[2-[2-[2-(3-hydroxyphenoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate

[1490] To a solution of 2-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (800 mg, 1.49 mmol, 1 eq) and (3-hydroxyphenyl)acetate (272.68 mg, 1.79 mmol, 1.2 eq) in DMF (10 mL) was added Cs 2 CO 3 (1.46 g, 4.48 mmol, 3 eq.) 2 The reaction mixture was concentrated to a residue, and H 2 O (20 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (25 mL) and washed with anhydrous Na 2 SO 4 The crude product was purified by ...

Claims

1. A bifunctional compound comprising a structure of formula (I): T L –L I –S L (I) in S L is the part that binds to sortilin; L I is a joint or a bond; and T L A portion that binds to an extracellular target molecule; or a pharmaceutically acceptable salt thereof.

2. The bifunctional compound according to claim 1, wherein S L Having a structure according to Formula III: Where Q 1 is a bond or -CH2-; and R 3 Having formula (IIIa), formula (IIIb) or formula B; in, R 3a is selected from H, halogen, alkoxy, -CF3 and optionally substituted C1-C4 alkyl; and in * represents a connection with formula (III), and R L Indicates that L I connection.

3. A bifunctional compound according to any one of the preceding claims, wherein S L According to any one selected from formula IIIc, IIId, IIIe, IIIf, IIIg and IIIh: Where R 3a is selected from H, halogen, alkoxy, -CF3 and optionally substituted C 1-4 alkyl; and R L Indicates that L I connection.

4. The bifunctional compound according to any one of claims 2 to 3, wherein R 3a For H.

5. The bifunctional compound according to any one of claims 2 to 3, wherein R 3a It is halogen or -CF3.

6. The bifunctional compound according to any one of claims 2 to 3, wherein R 3a It is an alkoxy group.

7. The bifunctional compound according to any one of claims 2 to 3, wherein R 3a is an optionally substituted C 1-4 alkyl.

8. The bifunctional compound according to any one of claims 2 to 3, wherein S L Selected from: Where R L Indicates that L I connection.

9. The bifunctional compound according to any one of claims 2 to 3 and 8, wherein S L According to formula (IIIi): Where R L Indicates that L I connection.

10. The bifunctional compound according to any one of claims 2 to 3 and 8, wherein S L According to formula (IIIj): Where R L Indicates that L I connection.

11. A bifunctional compound according to any one of claims 2 to 3 and 8, wherein S L According to formula (IIIk): Where R L Indicates that L I connection.

12. A bifunctional compound according to any one of claims 2 to 3 and 8, wherein S L According to formula (IIIm): Where R L Indicates that L I connection.

13. A bifunctional compound according to any one of claims 2 to 3 and 8, wherein S L According to formula (IIIn): Where R L Indicates that L I connection.

14. A bifunctional compound according to any one of claims 2 to 3 and 8, wherein S L According to formula (IIIn): Where R L Indicates that L I connection.

15. A bifunctional compound according to any one of the preceding claims, wherein S L is the S-stereoisomer of formula III or any one of formulas IIIa to IIIo.

16. A bifunctional compound according to any one of the preceding claims, wherein S L is the R-stereoisomer of formula III or any one of formulas IIIa to IIIo.

17. The bifunctional compound according to any one of claims 1 and 2, wherein S L According to formula (DI): Formula (DI) Where R L Indicates that L I connection.

18. A bifunctional compound according to any one of claims 1, 2 and 17, wherein S L According to formula (D-II): Formula (D-II) Where R L Indicates that L I connection.

19. The bifunctional compound according to claim 1, wherein S L Having a structure according to formula (II): in R 1 Selected from heteroaryl, aryl, heterocyclic, C3-C 10 Cycloalkyl, -(C1-C5 alkyl)-aryl and C1-C 10 alkyl, -BO-aryl, each of which is optionally substituted by one or more identical or different substituents R IIa substituted; wherein B is an optionally substituted C1-C5 alkyl; R 2 Selected from C1-C 10 Alkyl and C2-C 10 alkenyl, each of which is optionally substituted by one or more identical or different substituents R IIb replace; R IIa is selected from -O-aryl, -CH2-aryl, Cl, Br, F, C1-C3 alkoxy and C1-C3 alkyl, wherein each of -O-aryl, -CH2-aryl, C1-C3 alkoxy and C1-C3 alkyl is optionally substituted with one or more identical or different substituents selected from H, -O-CH2-C(=O)O-, -O-CH2-C(=O)NH, halogen, alkoxy, -CF3 and optionally substituted C1-C4 alkyl; R IIb Selected from C1-C 10 Alkoxy, C3-C 10 Cycloalkyl, C1-C 10 Alkyl and -CH2-aryl; Where S L By R 1 or R IIa With L I Coupling.

20. The bifunctional compound according to claim 19, wherein R 1 is a heteroaryl group, which is optionally substituted by one or more identical or different substituents R IIa replace.

21. The bifunctional compound according to any one of claims 19 or 20, wherein R 1 is pyridyl, which is optionally substituted by one or more identical or different substituents R IIa replace.

22. A bifunctional compound according to any one of claims 19 to 21, wherein R IIa is -O-aryl which is optionally substituted by one or more identical or different substituents selected from H, halogen, alkoxy, -CF3 and optionally substituted C1-C4 alkyl.

23. A bifunctional compound according to any one of claims 19 to 22, wherein R IIa is -O-phenyl or -O-naphthyl which is optionally substituted by one or more identical or different substituents selected from H, halogen, alkoxy, -CF3 and optionally substituted C1-C4 alkyl.

24. A bifunctional compound according to any one of claims 19 to 23, wherein R 1 Having formula (IIIa) or formula (IIIb): in R 3a is selected from H, halogen, alkoxy, -CF3 and optionally substituted C1-C4 alkyl; in * represents a connection with formula (II), and R L Indicates that L I connection.

25. The compound according to claim 19, wherein R 1 According to formula B: Where R L Indicates that L I connection, and * Represents the connection with formula II.

26. A bifunctional compound according to any one of claims 19 to 25, wherein R 2 is optionally substituted by one or more identical or different substituents R IIb Substituted C1-C 10 Alkyl, such as C1, C2, C3, C4, C5, C6, C7, C8, C9 or C 10 alkyl.

27. A bifunctional compound according to any one of claims 19 to 26, wherein R 2 Having formula (IIa): Where Q 1 is a bond or –CH2-; and in * It indicates the connection with formula (II).

28. The bifunctional compound according to claim 1, S L Having a structure according to Formula IV: in R 4 is H or F; R 4’ is H; R 5 is halogen, H, C1-C6 alkyl, C 2-6 Alkenyl or C1-C6 haloalkyl; R 6 is halogen, H, C1-C6 alkyl or C1-C6 haloalkyl; Q 2 is a bond or CH2; R 7 is a 5-6 membered heteroaromatic monocyclic ring having one or two heteroatoms, wherein the heteroaromatic ring is optionally substituted by one or two substituents independently selected from -CN; C1-C3 alkyl; halo-C1-C3 alkyl; C1-C3 alkoxy, halogen; -C(O)NH-C1-C3 alkyl; aryl optionally substituted by -C(O)NH-C1-C3 alkyl; and optionally substituted heteroaryl, or a pharmaceutically acceptable salt thereof, wherein S L By R 7 With L I Coupling.

29. The bifunctional compound according to claim 28, wherein R 4 For H.

30. The bifunctional compound according to any one of claims 28 or 29, wherein R 4’ For H.

31. A bifunctional compound according to any one of claims 28 to 30, wherein R 5 It is halogen, H or C1-C6 haloalkyl.

32. A bifunctional compound according to any one of claims 28 to 31, wherein R 6 It is halogen, H or C1-C6 haloalkyl.

33.

34. A bifunctional compound according to any one of claims 28 to 32, wherein R 5 is halogen, H or C1-C6 haloalkyl; and R 6 It is halogen, H, C1-C6 alkyl or C1-C6 haloalkyl.

35. A bifunctional compound according to any one of claims 28 to 34, wherein R 5 It is H or -CF3.

36. A bifunctional compound according to any one of claims 28 to 35, wherein R 6 It is H or -CF3.

37. The bifunctional compound according to claims 28 to 36, wherein R 5 and R 6 At least one of them is -CF3.

38. The bifunctional compound according to claims 28 to 36, wherein R 5 or R 6 One of them is -CF3, and R 5 or R 6 One of them is H.

39. A bifunctional compound according to any one of claims 28 to 38, wherein R 4 For H, R 5 is -CF3 or H; R 6 is -CF3 or H; and R 4 'For H.

40. A bifunctional compound according to any one of claims 28 to 39, wherein Q 2 is the key.

41. A bifunctional compound according to any one of claims 28 to 40, wherein R 7 is a 6-membered heteroaromatic monocyclic ring having one heteroatom substituted by a C1 alkyl group, wherein the heteroaromatic ring is optionally further substituted by an optionally substituted aryl group, And among them S L By R 7 With L I Coupling.

42. The bifunctional compound according to claims 28 to 41, wherein Q 2 is the key, R 4 For H, R 5 is -CF3 or H; R 6 is -CF3 or H; R 4 'For H, Q 2 is the key, R 7 is a 6-membered heteroaromatic monocyclic ring having one heteroatom substituted by a C1 alkyl group, wherein the heteroaromatic ring is optionally further substituted by an optionally substituted aryl group, And among them S L By R 7 With L I Coupling.

43. A bifunctional compound according to any one of claims 28 to 42, wherein R 7 is a pyridinyl group substituted by a C1 alkyl group, wherein the pyridinyl group is optionally further substituted by an optionally substituted aryl group.

44. A bifunctional compound according to any one of claims 28 to 43, wherein R 7 It is a pyridyl group substituted by a C1 alkyl group.

45. A bifunctional compound according to any one of claims 28 to 45, wherein R 7 It is a pyridyl group substituted by a C1 alkyl group and a phenyl group.

46. ​​A bifunctional compound according to any one of claims 28 to 45, wherein R 7 Having a structure according to Formula (IVa) or Formula (IVb): in * represents a connection with formula (IV), and wherein R L Indicates that L I connection.

47. A bifunctional compound according to any one of claims 28 to 46, wherein the S L Selected from any one of Formula IVc, IVd, IVe and IVf: Where R L Indicates that L I connection.

48. The bifunctional compound of claim 1, wherein S L is a substituent derived from a compound of formula VI: in R 8 C1-C 10 Alkyl; and R 9 Selected from C1-C 10 Alkyl, aryl and heteroaryl, each of which is optionally substituted by one or more identical or different substituents R VIa replace; R VIa is selected from the group consisting of aryl, heteroaryl, -O-aryl, -O-heteroaryl and halogen, wherein each of aryl, heteroaryl, -O-aryl and -O-heteroaryl is optionally substituted with one or more halogens.

49. The bifunctional compound of claim 1, wherein S L is a substituent derived from a compound of formula (VI): in R 10 for and R 11 is an optionally substituted heteroaryl group or an optionally substituted aryl group.

50. The bifunctional compound of claim 1, wherein S L is a substituent derived from a compound selected from the group consisting of:

51. The bifunctional compound of claim 50, wherein S L is a substituent derived from the following compound:

52. The bifunctional compound of claim 50, wherein S L is a substituent derived from the following compound:

53. The bifunctional compound of claim 1, wherein S L is a substituent derived from a compound of formula (VIII) or formula (IX): in, R VIIIa is a carboxylic acid or an ester thereof, wherein any of the carbon atoms of the norbornene ring may be replaced by C 1-6 Alkyl substitution, or the carboxylic acid can be condensed to C 1-5 ester. Where R IXa is an optionally substituted divalent C1-C5 alkyl group, wherein one or more methylene groups are optionally replaced by -CO(NH)-, R IXb is a group selected from hydroxy, alkoxy, amino or aminoalkyl.

54. The bifunctional compound of claim 53, wherein the compound of formula (VIII) or (IX) is selected from 2-methyl-3,5-dioxo-4-azatricyclo[5.2.1.0(2,6)]dec-8-en-4-yl)acetic acid, 2-(1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-methanoisoindol-2-yl)propanoic acid methyl ester, 2-(1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-methanoisoindol-2-yl)-4-(methylthio)butanoic acid, and 2-(3,5-Dioxo-4-azatricyclo[5.2.1.0(2,6)]dec-8-en-4-yl)-4-methylpentanoic acid.

55. The bifunctional compound of claim 1, wherein S L is a substituent derived from a compound of formula (X): in R Xa and R Xb Independently for C 1-5 alkyl, acyl, amino, sulfonyl, chlorine, bromine, iodine or fluorine; and R Xc C 1-5 The alkyl group, or the acid is replaced by a tetrazole.

56. The bifunctional compound of claim 55, wherein the compound of formula (X) is 4-[(3,4-dichlorophenyl)amino]-3-(3-methylbenzyl)-4-oxobutanoic acid or 3-benzyl-4-[(3-chloro-2-methylphenyl)amino]-4-oxobutanoic acid.

57. The bifunctional compound of claim 1, wherein S L is a substituent derived from formula (XI): in R XIa C 1-5 Alkyl, acyl, amino, sulfonyl, chlorine, bromine, iodine or fluorine; R XIb is a bond or a C optionally substituted by a C1-C5 alkyl group 1- C3 alkyl, and R XIc It is a carboxylic acid, a C1-C5 ester of a carboxylic acid or a tetrazole.

58. The bifunctional compound of claim 56, wherein the compound of formula (XI) is 3-oxo-1,2,3,4-tetrahydro-2-quinoxalinyl)acetic acid.

59. The bifunctional compound of claim 1, wherein S L is a substituent derived from formula (A): in A 1 , A 2 and A 3 Each is independently selected from halogen, H, C1-C4 alkyl, C1-C4 haloalkyl, C2-C5 alkenyl and C2-C5 haloalkenyl; A 4 is selected from H, C1-C3 alkyl, C1-C3 haloalkyl, C3-C8 aryl, C3-C8 aryl having one or more halogen substituents, C3-C8 heteroaryl and C3-C8 heteroaryl having one or more halogen substituents; A 5 Selected from C3-C 20 Aryl, C3-C 20 heteroaryl and 3 to 12 membered heterocycle; wherein aryl, heteroaryl or heterocycle is optionally substituted with one or more substituents independently selected from halogen, -OH, cyano, carbonyl, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, acetyl, C1-C4 alkoxy, C1-C4 haloalkoxy, C3-C8 aryl and C3-C8 heteroaryl; or A 4 and A 5 together to form a 6- to 20-membered heterocyclic ring, wherein Wherein the heterocycle is monocyclic, bicyclic or tricyclic, and is optionally substituted by one or more substituents independently selected from halo, -OH, cyano, carbonyl, C1-C4 alkyl, C1-C4 haloalkyl, acetyl, C1-C4 haloalkoxy, C1-C4 haloalkoxy.

60. The bifunctional compound of claim 1 or 59, wherein S L According to any one of formulas AI to A-III: or its derivatives, wherein R L Indicates that L I connection.

61. The bifunctional compound of claim 1, wherein S L Including peptides.

62. The bifunctional compound of claim 61, wherein S L Included are peptides having a length of no more than 50 amino acid residues, such as no more than 45, such as no more than 40, such as no more than 35, such as no more than 32, such as no more than 30, such as no more than 28, such as no more than 26, such as no more than 24, such as no more than 22, such as no more than 20, such as no more than 19, such as no more than 18, such as no more than 17, such as no more than 16, such as no more than 15, such as no more than 14, such as no more than 13, such as no more than 12, such as no more than 11, such as no more than 10, such as no more than 9, such as no more than 8, such as no more than 7, such as no more than 6, such as no more than 5, such as no more than 4 amino acid residues.

63. A bifunctional compound according to any one of claims 61 or 62, wherein S L Included are peptides comprising at least 4 amino acid residues, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 12, such as at least 14, such as at least 16, such as at least 18, such as at least 20, such as at least 22, such as at least 24, such as at least 26, such as at least 28 amino acid residues.

64. A bifunctional compound according to any one of claims 61 to 63, wherein S L It comprises or consists of peptides comprising 4 to 32 amino acids.

65. A bifunctional compound according to any one of claims 61 to 63, wherein S L Peptides comprising 4 or 5 amino acids are included.

66. A bifunctional compound according to any one of claims 1 or claims 61 to 65, wherein S L Included are peptides of 4 to 50 amino acids in length, said peptides comprising fragments of the amino acid sequence of a protein selected from: a. Progranulin (SEQ ID NO.: 3), b. Neurotensin (SEQ ID NO.: 4), c. Brain-derived neurotrophic factor (BDNF) (SEQ ID NO.: 177) d. Apolipoprotein B (ApoB) (SEQ ID NO.: 176) e. Nerve Growth Factor (NGF) (SEQ ID NO.: 178) or a variant of said fragment, which has at least 60% sequence identity, such as at least 80%, such as at least 90%, such as at least 95% sequence identity with the corresponding original protein fragment of any one of a) to e).

67. A bifunctional compound according to any one of claims 1 or claims 61 to 66, wherein S L A peptide fragment comprising the amino acid sequence of progranulin (SEQ ID NO.: 3) or a variant of said fragment or consisting thereof, wherein the variant of said fragment has at least 60% sequence identity, such as at least 80% sequence identity, such as at least 90%, such as at least 95%, such as at least 99% sequence identity with the corresponding original fragment of SEQ ID NO.:

3.

68. A bifunctional compound according to any one of claims 1 or claims 61 to 66, wherein S L A peptide fragment comprising the amino acid sequence of progranulin (SEQ ID NO.: 3) or a variant of said fragment or consisting thereof, wherein the variant of said fragment has up to 5 amino acid substitutions, such as up to 4 amino acid substitutions, such as up to 3 amino acid substitutions, such as up to 2 amino acid substitutions, such as up to 1 amino acid substitution compared to the corresponding original fragment of SEQ ID NO.:

3.

69. A bifunctional compound according to any one of claims 1 or claims 61 to 66, wherein S L A peptide fragment comprising the amino acid sequence of neurotensin (SEQ ID NO.: 4) or a variant of said fragment or consisting thereof, wherein the variant of said fragment has at least 60% sequence identity, such as at least 80% sequence identity, such as at least 90%, such as at least 95%, such as at least 99% sequence identity with the corresponding original fragment of SEQ ID NO.:

4.

70. A bifunctional compound according to any one of claims 1 or claims 61 to 66, wherein S L A peptide fragment comprising the amino acid sequence of neurotensin (SEQ ID NO.: 4) or a variant of said fragment or consisting thereof, wherein the variant of said fragment has up to 5 amino acid substitutions, such as up to 4 amino acid substitutions, such as up to 3 amino acid substitutions, such as up to 2 amino acid substitutions, such as up to 1 amino acid substitution compared to the corresponding original fragment of SEQ ID NO.:

4.

71. A bifunctional compound according to any one of claims 1 or claims 61 to 66, wherein S L It comprises or consists of a sequence selected from the following: H-PYMKLAPGELTIIL-OH (SEQ ID NO.:44), H-NEKLSQLQTYMI-OH(SEQ ID NO.:45), H-KDADLYTSRVMLSSQVP-OH (SEQ ID NO.:46), H-ITVDPRLFKRRLRRSPRVLFSTQPPR-OH (SEQ ID NO.:49), H-WSGPIGVSWGLRAAAAGGAFP-OH (SEQ ID NO.:50), H-WSGPIGVSWGLRAAAAGGAFPRGGRWRR-OH (SEQ ID NO.:51), H-GVSWGLR-OH (SEQ ID NO.: 52) H-WSGPIGVSWGLRAAAAGFQLL-OH (SEQ ID NO.:179).

72. The bifunctional compound of claim 1, wherein S L Included are peptides comprising the sequence: X5-X1X2X3X4 (SEQ ID NO.: 16) in, X5 is an optional amino acid residue or a peptide comprising 2 to 30 amino acid residues or a bond, X1 is R, P, F, Y, L, K, G or H; X2 is Q, Y, L, E or G; X3 is Y, F, L, I, Q, E or N; X4 is M, K, L or a conservative substitution of L; and L I Coupled to the N-terminus.

73. The bifunctional compound of claim 72, wherein X5 is a peptide comprising at least 2 amino acid residues, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 11, such as at least 12, such as at least 13, such as at least 14, such as at least 15, such as at least 20, such as at least 25 amino acid residues, such as at least 28 amino acid residues, such as at least 30 amino acid residues.

74. The bifunctional compound of claim 72, wherein X5 is a peptide comprising no more than 32 amino acid residues, such as no more than 29, such as no more than 28, such as no more than 27, such as no more than 26, such as no more than 25, such as no more than 24, such as no more than 23, such as no more than 22, such as no more than 21, such as no more than 20, such as no more than 15, such as no more than 10, such as no more than 5 amino acid residues.

75. The bifunctional compound according to any one of claims 72 to 74, wherein X5 is a fragment of the amino acid sequence of a protein selected from: a. Progranulin (SEQ ID NO.: 3), b. Neurotensin (SEQ ID NO.: 4), c. Brain-derived neurotrophic factor (BDNF) d. Apolipoprotein B (ApoB) e. Nerve Growth Factor (NGF) or a variant of said fragment, which has at least 60% sequence identity, such as at least 80%, such as at least 90%, such as at least 95% sequence identity with the corresponding original protein fragment of any one of a) to e).

76. The bifunctional compound according to any one of claims 72 to 75, wherein X5 comprises or consists of a peptide fragment of the amino acid sequence of progranulin (SEQ ID NO.: 3) or a variant of said fragment, which variant of said fragment has at least 60% sequence identity, such as at least 80% sequence identity, such as at least 90%, such as at least 95%, such as at least 99% sequence identity with the corresponding original fragment of SEQ ID NO.:

3.

77. The bifunctional compound according to any one of claims 72 to 75, wherein X5 comprises or consists of a peptide fragment of the amino acid sequence of progranulin (SEQ ID NO.: 3) or a variant of said fragment, which variant of the fragment has at most 5 amino acid substitutions, such as at most 4 amino acid substitutions, such as at most 3 amino acid substitutions, such as at most 2 amino acid substitutions, such as at most 1 amino acid substitution compared to the corresponding original fragment of SEQ ID NO.:

3.

78. The bifunctional compound according to any one of claims 72 to 75, wherein X5 comprises or consists of a peptide fragment of the amino acid sequence of neurotensin (SEQ ID NO.: 3) or a variant of said fragment, which has at least 60% sequence identity, such as at least 80% sequence identity, such as at least 90%, such as at least 95%, such as at least 99% sequence identity with the corresponding original fragment of SEQ ID NO.:

3.

79. The bifunctional compound according to any one of claims 72 to 75, wherein X5 comprises or consists of a peptide fragment of the amino acid sequence of neurotensin (SEQ ID NO.: 4) or a variant of said fragment, which has at most 5 amino acid substitutions, such as at most 4 amino acid substitutions, such as at most 3 amino acid substitutions, such as at most 2 amino acid substitutions, such as at most 1 amino acid substitution compared to the corresponding original fragment of SEQ ID NO.:

4.

80. The bifunctional compound according to any one of claims 72 to 75, wherein X5 is selected from: key an amino acid residue selected from L, T or Y; REAPRWDAPLRDPAL(SEQ ID NO.:17); REALRWDAPLRDPAP(SEQ ID NO.:18); PYILKRQLYENKPRR(SEQ ID NO.:19); LYENKPR (SEQ ID NO.: 20); and APLRDPAP (SEQ ID NO.: 21) WSGPIGVSWGLRAAAAG(SEQ ID NO.:180) CREAPRWDAPLRDPAL(SEQ ID NO.:181) or a variant thereof having 1 to 5 amino acid substitutions.

81. The bifunctional compound of any one of claims 72 to 80, wherein X5 is a bond.

82. The bifunctional compound according to any one of claims 72 to 80, wherein X5 is APLRDPAP (SEQ ID NO.: 21).

83. The bifunctional compound according to any one of claims 72 to 80, wherein X5 is REAPRWDAPLRDPAL (SEQ ID NO.: 17).

84. The bifunctional compound according to any one of claims 72 to 80, wherein X5 is REALRWDAPLRDPAP (SEQ ID NO.: 18).

85. A bifunctional compound according to any one of claims 72 or 84, wherein X1 is selected from Y, F, R, P, L or H, X2 is selected from Q, Y or L, X3 is selected from Y, F, L, I or Q, and X4 is L.

86. A bifunctional compound according to any one of claims 72 or 85, wherein X1 is selected from Y, F, R or P, X2 is selected from Q or Y, X3 is selected from L, Y, F or I, and X4 is L.

87. A bifunctional compound according to any one of claims 72 to 86, wherein X1 is selected from Y, F or R, X2 is selected from Q or Y, X3 is L, and X4 is L.

88. The bifunctional compound of any one of claims 72 to 87, wherein X1 is F or R.

89. A bifunctional compound according to any one of claims 72 to 88, wherein X2 is Q.

90. The bifunctional compound of any one of claims 72 to 89, wherein X3 is Q.

91. The bifunctional compound of any one of claims 72 to 90, wherein X4 is L.

92. The bifunctional compound of any one of claims 72 to 91, wherein X5 is a bond.

93. A bifunctional compound according to any one of claims 72 to 92, wherein X1 is selected from F or R, X2 is Q, X3 is L, X4 is L.

94. A bifunctional compound according to any one of claims 72 to 92, wherein the peptide according to SEQ ID NO.: 16 is located at S L The C-terminus of 95. A bifunctional compound according to any one of claims 1 or 72 to 94, wherein S L Comprising or consisting of a peptide selected from: RQLL-OH (SEQ ID NO.: 22), FQLL-OH (SEQ ID NO.: 23), KQLL-OH (SEQ ID NO.: 24), PQLL-OH (SEQ ID NO.: 25), PYIL-OH (SEQ ID NO.: 26), RYLL-OH (SEQ ID NO.: 27), FYLL-OH (SEQ ID NO.: 28), YQLL-OH (SEQ ID NO.: 29), LLQL-OH (SEQ ID NO.: 30), FYIL-OH (SEQ ID NO.: 31), RYIL-OH (SEQ ID NO.: 32), PYLL-OH (SEQ ID NO.: 33), PYYL-OH (SEQ ID NO.: 34), PYFL-OH (SEQ ID NO.: 35), RYYL-OH (SEQ ID NO.: 36), RYFL-OH (SEQ ID NO.: 37), RQFL-OH (SEQ ID NO.: 38), RQYL-OH (SEQ ID NO.: 39), LQLL-OH (SEQ ID NO.: 40), HQLL-OH (SEQ ID NO.: 41), IQLL-OH (SEQ ID NO.: 42), FYYL-OH (SEQ ID NO.: 43), PYMKLAPGELTIIL-OH (SEQ ID NO.: 44), NEKLSQLQTYMI-OH (SEQ ID NO.: 45), KDADLYTSRVMLSSQVP-OH (SEQ ID NO.: 46), ITVDPRLFKKRRLRSPRVLFSTQPPR-OH (SEQ ID NO.: 49), WSGPIGVSWGLRAAAAGGAFP-OH (SEQ ID NO.: 50), WSGPIGVSWGLRAAAAGGAFPRGGRWRR-OH (SEQ ID NO.: 51), GVSWGLR-OH (SEQ ID NO.: 52), REAPRWDAPLRDPALRQLL-OH (SEQ ID NO.: 53), REALRWDAPLRDPAPRQLL-OH (SEQ ID NO.: 54), PYILKRQLYENKPRRPYIL-OH (SEQ ID NO.: 55), LYENKPRRPYIL-OH (SEQ ID NO.: 56), APLRDPAPRQLL-OH (SEQ ID NO.: 57), REAPRWDAPLRDPALFQLL-OH (SEQ ID NO.: 58), REAPRWDAPLRDPALRYLL-OH (SEQ ID NO.: 59), REAPRWDAPLRDPALRQYL-OH (SEQ ID NO.: 60), REAPRWDAPLRDPALRYYL-OH (SEQ ID NO.: 61), TGGFM-OH (SEQ ID NO.: 62), and YGGFL-OH (SEQ ID NO.: 63), WSGPIGVSWGLRAAAAGFQLL-OH (SEQ ID NO.: 182), WSGPIGVSWGLRAAAAGRQLL-OH (SEQ ID NO.: 183), CREAPRWDAPLRDPALRQLL-OH (SEQ ID NO.: 184), CREAPRWDAPLRDPALFQLL-OH (SEQ ID NO.: 186), and Where L I Attached at the N-terminus.

96. A bifunctional compound according to any one of claims 1 or 72 to 94, wherein S L A peptide comprising, according to or consisting of any of the following sequences: RQLL-OH (SEQ ID NO.: 22), FQLL-OH (SEQ ID NO.: 23), RYLL-OH (SEQ ID NO.: 27), FYYL-OH (SEQ ID NO.: 28), YQLL-OH (SEQ ID NO.: 29), REAPRWDAPLRDPALRQLL-OH(SEQ ID NO.:53), REALRWDAPLRDPAPRQLL-OH(SEQ ID NO.:54), REAPRWDAPLRDPALFQLL-OH(SEQ ID NO.:58), REAPRWDAPLRDPALRYLL-OH(SEQ ID NO.:59), REAPRWDAPLRDPALRQYL-OH(SEQ ID NO.:60), REAPRWDAPLRDPALRYYL-OH(SEQ ID NO.:61), APLRDPAPRQLL-OH (SEQ ID NO.:57), PYILKRQLYENKPRRPYIL-OH(SEQ ID NO.:55), LYENKPRRPYIL-OH (SEQ ID NO.:56), Where L I Attached at the N-terminus.

97. The bifunctional compound of claim 1, wherein S L Comprising an amino acid sequence selected from the group consisting of: IKLSGGVQAKAGVINMDKSESM(SEQ ID NO.:7) IKLSGGVQAKAGVINMFKSESY(SEQ ID NO.:8) IKLSGGVQAKAGVINMFKSESYK(SEQ ID NO.:9) GVQAKAGVINMFKSESY(SEQ ID NO.:10) GVRAKAGVRNMFKSESY(SEQ ID NO.:11) GVRAKAGVRN(Nle)FKSESY(SEQ ID NO.:12) YKSLRRKAPRWDAPLRDPALRQLL(SEQ ID NO.:13) YKSLRRKAPRWDAYLRDPALRQLL(SEQ ID NO.:14) YKSLRRKAPRWDAYLRDPALRPLL (SEQ ID NO.:15).

98. According to any one of claims 61 to 97, the bifunctional compound, wherein S L One to four amino acid residues are conservatively substituted.

99. A bifunctional compound according to any one of claims 61 to 98, wherein S L One to four amino acid residues in the protein are replaced by non-naturally occurring amino acids.

100. A bifunctional compound according to any one of claims 61 to 97, wherein S L One to four amino acid residues in the protein are modified amino acid residues.

101. The bifunctional compound according to any one of claims 61 to 100, wherein the peptide is a modified peptide, for example a peptide modified by acylation, amidation, acetylation, esterification and / or alkylation.

102. A bifunctional compound according to any one of the preceding claims, wherein S L For a peptide capable of binding to Sortilin, the dissociation constant between the peptide and Sortilin is less than 50 μM, such as less than 2 μM, such as less than 0.5 μM, preferably less than 0.1 μM.

103. A peptide comprising or consisting of a sequence selected from the group consisting of: RQLL-OH (SEQ ID NO.: 22), FQLL-OH (SEQ ID NO.: 23), KQLL-OH (SEQ ID NO.: 24), PQLL-OH (SEQ ID NO.: 25), PYIL-OH (SEQ ID NO.: 26), RYLL-OH (SEQ ID NO.: 27), FIL-OH(SEQ ID NO.:28), YQLL-OH(SEQ ID NO.:29), LLQL-OH(SEQ ID NO.:30), FYIL-OH(SEQ ID NO.:31), RYIL-OH(SEQ ID NO.:32), PYLL-OH(SEQ ID NO.:33), PYYL-OH(SEQ ID NO.:34), PYFL-OH(SEQ ID NO.:35), RYYL-OH(SEQ ID NO.:36), RYFL-OH(SEQ ID NO.:37), RQFL-OH(SEQ ID NO.:38), RQYL-OH(SEQ ID NO.:39), LQLL-OH(SEQ ID NO.:40), HQLL-OH(SEQ ID NO.:41), IQLL-OH(SEQ ID NO.:42), FYYL-OH(SEQ ID NO.:43), PYMKLAPGELTIYL-OH(SEQ ID NO.:44), NEKLSQLQTYMI-OH(SEQ ID NO.:45), KDADLYTSRVMLSSQVP-OH(SEQ ID NO.:46), RLFKKRRRLRSPRVLF-NH2(SEQ ID NO.:47), ITVDPRLFKKRRLRSRPRVLF-NH2(SEQ ID NO.:48), ITVDPRLFKKRRRLRSPRVLFSTQPPR-OH(SEQ ID NO.:49), WSGPIGVSWGLRAAAAGGAFP-OH(SEQ ID NO.:50), WSGPIGVSWGLRAAAAGGAFPRGGRWRR-OH(SEQ ID NO.:51), GVSWGLR-OH(SEQ ID NO.:52), REAPRWDAPLRDPALRQLL-OH(SEQ ID NO.:53), REALRWDAPLRDPAPRQLL-OH(SEQ ID NO.:54), PYILKRQLYENKPRRPYIL-OH(SEQ ID NO.:55), LYENKPRRPYYL-OH(SEQ ID NO.:56), APLRDPAPRQLL-OH(SEQ ID NO.:57), REAPRWDAPLRDPALFQLL-OH(SEQ ID NO.:58), REAPRWDAPLRDPALRYLL-OH(SEQ ID NO.:59), REAPRWDAPLRDPALRQYL-OH(SEQ ID NO.:60), REAPRWDAPLRDPALRYYL-OH(SEQ ID NO.:61), TGGFM-OH (SEQ ID NO.: 62), and YGGFL-OH (SEQ ID NO.: 63), LYEN-NH3 (SEQ ID NO.: 64), and LYENK-NH3 (SEQ ID NO.: 65) WSGPIGVSWGLRAAAAGFQLL-OH (SEQ ID NO.:182); WSGPIGVSWGLRAAAAGRQLL-OH (SEQ ID NO.:183); CREAPRWDAPLRDPALRQLL-OH (SEQ ID NO.:184); and CREAPRWDAPLRDPALFQLL-OH (SEQ ID NO.:186).

104. The peptide according to claim 103, wherein the peptide comprises or consists of the sequence RQLL-OH (SEQ ID NO.: 22).

105. The peptide according to claim 103, wherein the peptide comprises or consists of the sequence FQLL-OH (SEQ ID NO.: 23).

106. The peptide of claim 103, wherein the peptide comprises or consists of the sequence RYLL-OH (SEQ ID NO.: 27).

107. The peptide of claim 103, wherein the peptide comprises or consists of the sequence FYLL-OH (SEQ ID NO.: 28).

108. The peptide of claim 103, wherein the peptide comprises or consists of the sequence YQLL-OH (SEQ ID NO.: 29).

109. The peptide of claim 103, wherein the peptide comprises or consists of the sequence REAPRWDAPLRDPALRQLL-OH (SEQ ID NO.: 53).

110. The peptide of claim 103, wherein the peptide comprises or consists of the sequence REALRWDAPLRDPAPRQLL-OH (SEQ ID NO.: 54).

111. The peptide according to claim 103, wherein the peptide comprises or consists of the sequence REAPRWDAPLRDPALFQLL-OH (SEQ ID NO.: 58).

112. The peptide of claim 103, wherein the peptide comprises or consists of the sequence REAPRWDAPLRDPALRYLL-OH (SEQ ID NO.: 59).

113. The peptide of claim 103, wherein the peptide comprises or consists of the sequence REAPRWDAPLRDPALRQYL-OH (SEQ ID NO.: 60).

114. The peptide of claim 103, wherein the peptide comprises or consists of the sequence REAPRWDAPLRDPALRYYL (SEQ ID NO.: 61).

115. The peptide of claim 103, wherein the peptide comprises or consists of the sequence APLRDPAPRQLL-OH (SEQ ID NO.: 57).

116. The peptide of claim 103, wherein the peptide comprises or consists of the sequence PYILKRQLYENKPRRPYIL-OH (SEQ ID NO.: 55).

117. The peptide of claim 103, wherein the peptide comprises or consists of the sequence LYENKPRRPYIL-OH (SEQ ID NO.: 56).

118. A peptide according to any one of claims 103 to 104, wherein the peptide is capable of binding to sortilin with a dissociation constant of less than 50 μM, such as less than 2 μM, such as less than 0.5 μM, preferably less than 0.1 μM.

119. The bifunctional compound of claim 1, wherein S L For protein.

120. The bifunctional compound of claim 119, wherein S L It is an antibody, antibody fragment or nanobody.

121. The bifunctional compound of any one of claims 119 to 120, wherein the bifunctional compound is capable of binding to Sortilin.

122. A bifunctional compound according to any one of the preceding claims, wherein S L Derived from a protein, peptide or compound that can bind to sortilin with a dissociation constant of less than 50 μM, such as less than 40 μM, such as less than 30 μM, such as less than 20 μM, such as less than 10 μM, such as less than 5 μM, such as less than 4 μM, such as less than 3 μM, such as less than 2 μM, such as less than 1 μM, such as less than 0.8 μM, such as less than 0.6 μM, such as less than 0.5 μM, such as less than 0.4 μM, such as less than 0.3 μM, such as less than 0.2 μM, such as less than 0.1 μM.

123. A bifunctional compound according to any of the preceding claims, wherein the bifunctional compound is capable of binding to sortilin with a dissociation constant of less than 50 μM, such as less than 40 μM, such as less than 30 μM, such as less than 20 μM, such as less than 10 μM, such as less than 5 μM, such as less than 4 μM, such as less than 3 μM, such as less than 2 μM, such as less than 1 μM, such as less than 0.8 μM, such as less than 0.6 μM, such as less than 0.5 μM, such as less than 0.4 μM, such as less than 0.3 μM, such as less than 0.2 μM, such as less than 0.1 μM, such as less than 0.05 μM, such as less than 0.04 μM, such as less than 0.03 μM, such as less than 0.02 μM, such as less than 0.01 μM.

124. The bifunctional compound according to any one of the preceding claims, wherein the bifunctional compound can be used in a range of 50 μM to 0.001 μM, for example 50 μM to 0.001 μM, for example 50 μM to 40 μM, for example 40 μM to 30 μM, for example 30 μM to 20 μM, for example 20 μM to 10 μM, for example 10 μM to 5 μM, for example 5 μM to 4 μM, for example 4 μM to 3 μM, for example 3 μM to 2 μM, for example 2 μM to 1 μM, for example Such as 1 μM to 0.9 μM, for example 0.9 μM to 0.8 μM, for example 0.8 μM to 0.7 μM, for example 0.7 μM to 0.6 μM, for example 0.5 μM to 0.4 μM, for example 0.4 μM to 0.3 μM, for example 0.3 μM to 0.2 μM, for example 0.2 μM to 0.1 μM, for example 0.1 μM to 0.05 μM, for example 0.05 μM to 0.01 μM, for example 0.01 μM to 0.001 μM.

125. An isolated polynucleotide encoding a peptide according to any one of claims 61 to 118 or a protein according to any one of claims 119 to 120.

126. A vector comprising the polynucleotide according to claim 125.

127. The vector according to claim 126, wherein the vector is an expression vector, such as a bacterial vector or a viral vector.

128. A host cell comprising the polynucleotide of claim 127 and / or the vector of any one of claims 128 to 129.

129. The bifunctional compound of any one of claims 1 to 124, wherein the extracellular target molecule is a target protein.

130. The bifunctional compound of any one of claims 1 to 124, wherein the target molecule is TNFα.

131. The bifunctional compound of claim 129, wherein the target protein is selected from the group consisting of: TNF-α, ANGPTL-3, antibody light chain, IgG, IgE, IgAIL-1, IL-2, IL-6, IFN-γ, VEGF, TFG-β1, IL-21, IL-22, IL-5, IL-10, IL-8, cholinesterase, human CCL2, carboxypeptidase B-2, neutrophil elastase, factor Xa, factor XI, factor XIa, factor XII, factor XIII, prothrombin, factor VII, factor IX, Fibroblast growth factor 1, FGF-2, fibronectin 1, kallikrein-1, lipoprotein lipase, human matrix metallopeptidase 1, macrophage migration inhibitory factor, transforming growth factor-p (TGF-p), thrombospondin-1 (TSP-T), CD40 ligand, urokinase-type plasminogen activator, tissue-type plasminogen activator (TPA), plasminogen (PLG), plasminogen activator inhibitor-1, placental growth factor, phospholipase A2 group IB, phospholipase A2 group IIA, complement factor B, complement factor D, complement factor H, complement component 5, and complement C1s.

132. A bifunctional compound according to any one of claims 129 to 131, wherein the bifunctional compound is L Binds to target molecule or protein.

133. The bifunctional compound of claim 129, wherein T L Derived from a compound, peptide or protein capable of binding to a target protein with a dissociation constant of less than 100 μM, such as less than 2 μM, such as less than 0.5 μM, preferably less than 0.1 μM.

134. A bifunctional compound according to any one of claims 1 to 124, wherein T L is a substituent derived from biotin.

135. A bifunctional compound according to any one of claims 1 to 124, wherein T L According to formula (XII): Where R L Indicates that L I connection.

136. A bifunctional compound according to any one of claims 1 to 124, wherein T L is a substituent derived from dinitrophenyl.

137. A bifunctional compound according to any one of claims 1 to 124, wherein T L According to formula (XII): Where R L Indicates that L I connection.

138. A bifunctional compound according to any one of claims 1 to 124, wherein T L According to formula XVIIa, in, R XVIIa With formula Where R XVIId and R XVIIe is independently selected from C1-C5 alkyl, C1-C5 alkoxy, cyano, halogen and C1 haloalkyl, each of which is optionally independently substituted; X is an atom selected from N or CH, and * represents a connection with formula (XVIIa); R XVIIb and R XVIIb’ Each independently selected from H and C1-C3 alkyl; R XVIIc For R L or selected from Formula XVIIa-1 and Formula XVIIa-2: Where R XVIIf is selected from optionally substituted C1-C5 alkyl, wherein one or more methylene groups are replaced by one selected from carbonyl, ester, amide, -NH- or -O-, and * represents a connection to Formula XVIIa; and R L Indicates that L I connection.

139. The bifunctional compound of claim 138, wherein R XVIIb or R XVIIb ' is –CH3.

140. The bifunctional compound of claim 138, wherein R XVIIb and R XVIIb ' is –CH3.

141. A bifunctional compound according to any one of claims 137 to 140, wherein R XVIIa Selected from: in * Indicates linkage to Formula XVIIa.

142. A bifunctional compound according to any one of claims 1 to 124 or 137 to 141, wherein T L According to Formula XVIIa-3: Where R L Indicates that L I connection.

143. A bifunctional compound according to any one of claims 1 to 124 or 137 to 141, wherein T L According to Formula XVIIa-4: Where R L Indicates that L I connection.

144. A bifunctional compound according to any one of claims 1 to 124 or 137 to 141, wherein T L According to Formula XVIIa-5: Where R L Indicates that L I connection.

145. A bifunctional compound according to any one of claims 1 to 124, wherein T L According to Formula XVIIb Where R XVIIi is selected from a bond and an optionally substituted piperazine group; R XVIIj Selected from in * represents a connection to Formula XVIIb; and Where R L Indicates that L I connection.

146. The bifunctional compound of claim 145, wherein T L According to Formula XVIIb-1: Where R L Indicates that L I connection.

147. The bifunctional compound of claim 145, wherein T L According to Formula XVIIb-2: Where R L Indicates that L I connection.

148. A bifunctional compound according to any one of claims 1 to 124, wherein T L According to Formula XVIIc Where R XVIIg is a C1-C4 alkyl group, and R XVIIh is an aromatic ring or heteroaromatic ring optionally substituted by one or more groups selected from halogen, haloalkyl, cyano, hydroxyl, amino, hydroxyl, alkoxy, C3-C6 cycloalkyl and C3-C6 heterocycloalkyl; and Where R L Indicates that L I connection.

149. The bifunctional compound of claim 148, wherein R XVIIg is –CH3.

150. A bifunctional compound according to any one of claims 148 to 149, wherein T L According to Formula XVIIc-1 Formula (XVIIc-1), wherein R L Indicates that L I connection.

151. A bifunctional compound according to any one of claims 1 to 124, wherein T L According to Formula XVIIc-2 Formula (XVIIc-2), wherein R L Indicates that L I connection.

152. A bifunctional compound according to any one of claims 1 to 124, wherein T L According to Formula XVIId 153. A compound selected from any one of compounds TF001 to TF022 according to Table C in the "Compound List" section.

154. A bifunctional compound according to any one of claims 1 to 124, wherein T L Comprising or consisting of proteins, peptides or small molecules.

155. The bifunctional compound of claim 154, wherein T L These include antibodies, antibody fragments or nanobodies.

156. A bifunctional compound according to any one of claims 154 to 155, wherein T L Full length antibodies are included.

157. A bifunctional compound according to any one of claims 154 to 156, wherein T L Including antibody fragments.

158. The bifunctional compound of claim 157, wherein T L Includes antibody light chains.

159. The bifunctional compound of claim 157, wherein T L Includes antibody heavy chains.

160. The bifunctional compound of claim 157, wherein T L Including antibody heavy chain (V H )'s variable region.

161. The bifunctional compound of claim 157, wherein T L Including antibody light chain (V L )'s variable region.

162. A bifunctional compound according to any one of claims 1 or 154 to 155, wherein T L A fusion protein comprising two or more antibody fragments, such as 2 antibody fragments, such as 3 antibody fragments, such as 4 antibody fragments, such as 5 antibody fragments, such as 6 antibody fragments, such as 7 antibody fragments, such as 8 antibody fragments.

163. The bifunctional compound of claim 162, wherein T L A fusion protein comprising two antibody fragments.

164. A bifunctional compound according to any one of claims 154 to 163, wherein T L Includes single-chain antigen binding domains (scAb).

165. A bifunctional compound according to any one of claims 154 to 163, wherein T L Includes single chain variable fragments (scFv).

166. A bifunctional compound according to any one of claims 154 to 156, wherein T L Including nanobodies.

167. A bifunctional compound according to any one of claims 1 or 154 to 155, wherein T L is a protein, and the target protein is PCSK9, TNF-α or an antibody.

168. The bifunctional compound of any one of claims 1 to 167, wherein the target protein is PCSK9.

169. A bifunctional compound according to any one of claims 1 to 167, wherein T L Capable of binding antibodies.

170. The bifunctional compound of claim 169, wherein T L Capable of binding IgG.

171. A bifunctional compound according to any one of claims 169 to 170, wherein T L Capable of binding to the light chain of an antibody.

172. A bifunctional compound according to any one of claims 169 to 170, wherein T L Capable of binding to the heavy chain of an antibody.

173. A bifunctional compound according to any one of claims 169 to 170, wherein T L Capable of binding to the heavy chain of an antibody (C H ) constant region or light chain (C L ) constant region.

174. The bifunctional compound of any one of claims 1 to 167, wherein the target protein is TNF-α.

175. A bifunctional compound according to any one of claims 1 to 124, wherein T L An antibody having binding specificity for PCSK-9 is included, the antibody comprising: A light chain region comprising SEQ ID NO.: 67, or an amino acid sequence having at least 70% sequence identity, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO.: 67; and / or A heavy chain region comprising SEQ ID NO.: 66, or an amino acid sequence having at least 70% sequence identity, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO.: 66; or an antigen-binding fragment thereof.

176. A bifunctional compound according to any one of claims 1 to 124, wherein T L An antibody having binding specificity for PCSK-9 is included, the antibody comprising: A light chain region comprising SEQ ID NO.: 67 and / or A heavy chain region comprising SEQ ID NO.: 66; or an antigen-binding fragment thereof, Or it is composed of the antibody or its antigen-binding fragment.

177. A bifunctional compound according to any one of claims 1 to 124, wherein T L An antibody having binding specificity to TNF-α is included, the antibody comprising: A light chain region comprising SEQ ID NO.: 69, or an amino acid sequence having at least 70% sequence identity, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO.: 69; and / or A heavy chain region comprising SEQ ID NO.: 68, or an amino acid sequence having at least 70% sequence identity, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO.: 68; or an antigen-binding fragment thereof.

178. A bifunctional compound according to any one of claims 1 to 124, wherein T L An antibody having binding specificity to TNF-α is included, the antibody comprising: A light chain region comprising SEQ ID NO.: 69 and / or A heavy chain region comprising SEQ ID NO.: 68 or an antigen-binding fragment thereof, Or it is composed of the antibody or its antigen-binding fragment.

179. A bifunctional compound according to any one of claims 1 to 124, wherein T L The invention comprises a Nanobody with binding specificity for the constant region of a second antibody, the Nanobody comprising the following sequence: MGGTHHHHHHENLYFQGQVQLQESGGGLVQPGGSLRLSCAASGRT ISRYAMSWFRQAPGKEREFVAVARRSGDGAFYADSVQGRFTVSRDDAKN TVYLQMNSLKPEDTAVYYCAIDSDTFYSGSYDYWGQGTQVTVSSE(SEQ IDNO.:70) Or an amino acid sequence having at least 70% sequence identity to SEQ ID NO.: 70, such as at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity.

180. A bifunctional compound according to any one of claims 1 to 124, wherein T L Contains or consists of the following sequence: MGGTHHHHHHENLYFQGQVQLQESGGGLVQPGGSLRLSCAASGRTISRYAMSWFRQAPGKEREFVAVARRSGDGAFYADSVQGRFTVSRDDAKNTVYLQMNSLKPEDTAVYYCAIDSDTFYSGSYDYWGQGTQVTVSSE (SEQ ID NO.: 70).

181. The bifunctional compound according to any one of the preceding claims, wherein the bifunctional compound is capable of binding to the extracellular target protein with a dissociation constant of less than 100 μM, such as less than 2 μM, such as less than 0.5 μM, preferably less than 0.1 μM.

182. The bifunctional compound of any one of claims 1 to 16, wherein the bifunctional compound is according to any one of formulas XIVa, XIVb, XIVc and XIVd: in Q 3 is a bond or -CH2-; R 3a is selected from H, halogen, alkoxy, -CF3 and optionally substituted C 1-4 alkyl.

183. The bifunctional compound of any one of claims 1 to 16, 138 to 151 or 181, wherein the bifunctional compound is according to any one of formula XVIIIa, XVIIIb, XVIIIb-1, XVIIIc, XVIIId, XVIIId-1, XVIIIe, XVIIIf, XVIIIf-1, XVIIIg, XVIIIh and XVIIIh-1: in Q 3 is a bond or –CH2-; R 3a is selected from H, halogen, alkoxy, -CF3 and optionally substituted C 1-4 alkyl; and L I Indicates a connector.

184. A bifunctional compound according to any one of claims 182 to 183, wherein R 3a For H.

185. A bifunctional compound according to any one of claims 182 to 183, wherein R 3a It is a halogen.

186. A bifunctional compound according to any one of claims 182 to 183, wherein R 3a is –CF3.

187. A bifunctional compound according to any one of claims 182 to 183, wherein R 3a It is an alkoxy group.

188. A bifunctional compound according to any one of claims 182 to 183, wherein R 3a is an optionally substituted C 1-4 alkyl.

189. A bifunctional compound according to any one of claims 182 to 183, wherein Q 3 is the key.

190. A bifunctional compound according to any one of claims 182 to 183, wherein Q 3 is –CH2-.

191. The bifunctional compound of any one of claims 1 to 2, 17 to 19, 25, 138 to 151 or 181, wherein the bifunctional compound is according to any one of formula D-III, D-IV, DV and D-VI:

192. The bifunctional compound of any one of claims 1 to 16, 19 to 25, 138 to 151, wherein the bifunctional compound is according to any one of formula XVIIIm, XVIIIn, XVIIIn-1, XVIIIo, XVIIIp:

193. The bifunctional compound of any one of claims 28 to 47, wherein the bifunctional compound is according to any one of formulas XVa, XVb, XVc and XVd: in, R F1 and R F2 are independently selected from CF3 or H, provided that R F1 or R F2 At least one of them is CF3.

194. The bifunctional compound of claim 193, wherein R F1 is –CF3 and R F2 For H.

195. The bifunctional compound of claim 1, wherein the bifunctional compound is selected from: X T -X L -RQLL-OH(SEQ ID NO.:72), X T -X L -FQLL-OH(SEQ ID NO.:73), X T -X L -KQLL-OH(SEQ ID NO.:74), X T -X L -PQLL-OH(SEQ ID NO.:75), X T -X L -PYIL-OH(SEQ ID NO.:76), X T -X L -RYLL-OH(SEQ ID NO.:77), X T -X L -FYLL-OH(SEQ ID NO.:78), X T -X L -YQLL-OH(SEQ ID NO.:79), X T -X L -LLQL-OH(SEQ ID NO.:80), X T -X L -FYIL-OH(SEQ ID NO.:81), X T -X L -RYIL-OH(SEQ ID NO.:82), X T -X L -PYLL-OH(SEQ ID NO.:83), X T -X L -PYYL-OH(SEQ ID NO.:84), X T -X L -PYFL-OH(SEQ ID NO.:85), X T -X L -RYYL-OH(SEQ ID NO.:86), X T -X L -RYFL-OH(SEQ ID NO.:87), X T -X L -RQFL-OH(SEQ ID NO.:88), X T -X L -RQYL-OH(SEQ ID NO.:89), X T -X L -LQLL-OH(SEQ ID NO.:90), X T -X L -HQLL-OH(SEQ ID NO.:91), X T -X L -IQLL-OH(SEQ ID NO.:92), X T -X L -FYYL-OH(SEQ ID NO.:93), X T -X L -PYMKLAPGELTIIL-OH(SEQ ID NO.:94), X T -X L -NEKLSQLQTYMI-OH(SEQ ID NO.:95), X T -X L -KDADLYTSRVMLSSQVP-OH(SEQ ID NO.:96), X T -X L -ITVDPRLFKKRRLRSPRVLFSTQPPR-OH(SEQ ID NO.:99), X T -X L -WSGPIGVSWGLRAAAAGGAFP-OH(SEQ ID NO.:100), X T -X L -WSGPIGVSWGLRAAAAGGAFPRGGRWRR-OH(SEQ ID NO.:101), X T -X L -GVSWGLR-OH(SEQ ID NO.:102), X T -X L -REAPRWDAPLRDPALRQLL-OH(SEQ ID NO.:103), X T -X L -REALRWDAPLRDPAPRQLL-OH(SEQ ID NO.:104), X T -X L -PYILKRQLYENKPRRPYIL-OH(SEQ ID NO.:105), X T -X L -LYENKPRRPYIL-OH(SEQ ID NO.:106), X T -X L -APLRDPAPRQLL-OH(SEQ ID NO.:107), X T -X L -REAPRWDAPLRDPALFQLL-OH(SEQ ID NO.:108), X T -X L -REAPRWDAPLRDPALRYLL-OH(SEQ ID NO.:109), X T -X L -REAPRWDAPLRDPALRQYL-OH(SEQ ID NO.:110), X T -X L -REAPRWDAPLRDPALRYYL-OH(SEQ ID NO.:111), X T -X L -TGGFM-OH (SEQ ID NO.: 112), and X T -X L -YGGFL-OH(SEQ ID NO.:113); Where X L For L I And X T Selected from: a) a group comprising biotin; b) a group comprising dinitrophenol; c) an antibody having binding specificity for PCSK-9, comprising: A light chain region comprising SEQ ID NO.: 67 and / or A heavy chain region comprising SEQ ID NO.: 66; or an antigen-binding fragment thereof, Or it is composed of the antibody or its antigen-binding fragment. d) an antibody having binding specificity for TNF-α, comprising: A light chain region comprising SEQ ID NO.: 69 and / or A heavy chain region comprising SEQ ID NO.: 68 or an antigen-binding fragment thereof, Or it is composed of the antibody or its antigen-binding fragment.

196. The bifunctional compound of claim 1, wherein the bifunctional compound is: Biotin-X L -X5-X1X2X3X4 (SEQ ID NO.: 116) in, X5 is an optional amino acid residue or a peptide comprising 2 to 30 amino acid residues or a bond, X1 is R, P, F, Y, L, K, G or H; X2 is Q, Y, L, E or G; X3 is Y, F, L, I, Q, E or N; X4 is M, K, L or a conservative substitution of L; Where X L For L I .

197. The bifunctional compound of claim 196, wherein X5 is a bond.

198. The bifunctional compound of claim 196, wherein X5 is APLRDPAP (SEQ ID NO.: 21).

199. The bifunctional compound of claim 196, wherein X5 is REAPRWDAPLRDPAL (SEQ ID NO.: 17).

200. The bifunctional compound of claim 196, wherein X5 is REALRWDAPLRDPAP (SEQ ID NO.: 18).

201. The bifunctional compound of claim 196, wherein X1 is selected from Y, F or R, X2 is selected from Q or Y, X3 is L, X4 is L.

202. The bifunctional compound of claim 196, wherein X1 is F or R.

203. The bifunctional compound of claim 196, wherein X2 is Q.

204. The bifunctional compound of claim 196, wherein X3 is Q.

205. The bifunctional compound of claim 196, wherein X4 is L.

206. The bifunctional compound of any one of claims 196 to 205, wherein the bifunctional compound is selected from any one of: Biotin-X L -RQLL-OH (SEQ ID NO.: 117) Biotin-X L -FQLL-OH (SEQ ID NO.: 118) Biotin-X L -RYLL-OH (SEQ ID NO.: 119) Biotin-X L -FYLL-OH (SEQ ID NO.: 120) Biotin-X L -YQLL-OH (SEQ ID NO.: 121) Biotin-X L -REAPRWDAPLRDPALRQLL-OH(SEQ ID NO.:122) Biotin-X L -REALRWDAPLRDPAPRQLL-OH(SEQ ID NO.:123) Biotin-X L -REAPRWDAPLRDPALFQLL-OH(SEQ ID NO.:124) Biotin-X L -REAPRWDAPLRDPALRYLL-OH(SEQ ID NO.:125) Biotin-X L -REAPRWDAPLRDPALRQYL-OH(SEQ ID NO.:126) Biotin-X L -REAPRWDAPLRDPALRYYL-OH(SEQ ID NO.:127) Biotin-X L -APLRDPAPRQLL-OH(SEQ ID NO.:128) Biotin-X L -PYILKRQLYENKPRRPYIL-OH (SEQ ID NO.:129); and Biotin-X L -LYENKPRRPYIL-OH(SEQ ID NO.:130) Where X L For L I .

207. The bifunctional compound of claim 1, wherein T L For at least 1 unit of –L I -S L Coupling, for example, with 1 unit of -L I -S L Coupling, for example, with 2 units of -L I -S L Coupling, for example, with 3 units of -L I -S L Coupling, for example with 4 units of –L I -S L Conjugated monoclonal antibodies.

208. The bifunctional compound of claim 207, wherein T L It is the monoclonal antibody amorobizumab (an antibody having a heavy chain of SEQ ID NO.: 66 and a light chain of SEQ ID NO.: 67).

209. The bifunctional compound of claim 207, wherein T L It is the monoclonal antibody adalimumab (an antibody having a heavy chain of SEQ ID NO.: 68 and a light chain of SEQ ID NO.: 69).

210. The bifunctional compound of any one of claims 207 to 209, wherein S L A peptide comprising at its C-terminus a sequence selected from the group consisting of: RQLL-OH (SEQ ID NO.: 22) FQLL-OH (SEQ ID NO.: 23) RYLL-OH (SEQ ID NO.: 27) FYYL-OH (SEQ ID NO.: 28) YQLL-OH (SEQ ID NO.: 29) and PYILKRQLYENKPRRPYIL-OH (SEQ ID NO.:55); Where L I Attached at the N-terminus.

211. A bifunctional compound according to any one of claims 207 to 209, wherein S L is a peptide comprising RQLL-OH (SEQ ID NO.: 22) at the C-terminus.

212. A bifunctional compound according to any one of claims 207 to 209, wherein S L is a peptide comprising FQLL-OH (SEQ ID NO.: 23) at the C-terminus.

213. A bifunctional compound according to any one of the preceding claims, wherein L I Has the following structure: in * Indicates that T L or S L The connection point; L1 and L2 are each independently selected from a bond, -C(H2)-, -O-, -N(H)-, a functional group selected from carbonyl, ester, amide, carbamate, thiourea, urea, sulfonamide and triazole; and a C1-C3 hydrocarbon chain, wherein one or more methylene groups are individually and optionally replaced by carbonyl, ester, amide, carbamate, thiourea, urea sulfonamide and triazole; Z is selected from divalent saturated or unsaturated straight or branched chain C 1- C 30 A hydrocarbon chain in which one or more methylene groups are individually and optionally replaced by one or more groups selected from: -O-, -N(H)-, -N(R L1 )-, -OC(=O)-, -C(=O)O-, -C(=O)-, -N(H)C(=O)-, -N(R L1 )C(=O)-, -C(=O)N(H)-, -NHC(O)NH-, -NHC(O)O--C(=O)N(R L1 )-, -S-, -S(=O)-, -S(=O)2-, -N(R L1 )S(=O)2-、-S(=O)2N(R L1 )-; optionally substituted aromatic group; optionally substituted carbocyclic ring; optionally substituted heterocyclic ring; optionally substituted aromatic heterocyclic ring; R L1 Selected from C 1-5 alkyl; n and w are each independently an integer from 1 to 9.

214. A bifunctional compound according to any one of the preceding claims, wherein L I Has the following structure: in * Indicates that T L or S L The connection point, L1 and L2 are each independently selected from -C(H2)-, -O-, -N(H)- and amide; Z is a divalent saturated or unsaturated straight or branched chain C 1- C 30 A hydrocarbon chain in which one or more methylene groups are individually and optionally replaced by one or more groups selected from: -O-, -N(H), -N(R L1 )-, -N(H)C(=O)-, -N(R)C(=O)-, -C(=O)N(H)-, -C(=O)N(R L1 )-、 R L1 Selected from C 1-5 alkyl; n and w are each independently an integer from 1 to 9.

215. A bifunctional compound according to any one of claims 213 to 214, wherein the C1-C 30 The hydrocarbon chain is C5-C 30 Hydrocarbon chains, such as C8-C 30 Hydrocarbon chains, such as C 10 -C 30 Hydrocarbon chains, such as C 12 -C 30 Hydrocarbon chain.

216. A bifunctional compound according to any one of claims 213 to 214, wherein the C1-C 30 The hydrocarbon chain is C 10 -C 25 Hydrocarbon chains, such as C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , C 24 or C 25 Hydrocarbon chain.

217. A bifunctional compound according to any one of claims 213 to 214, wherein the C1-C 30 The hydrocarbon chain is C 14 -C 20 Hydrocarbon chain.

218. The bifunctional compound according to any one of claims 213 to 214, wherein the C1-C 30 The hydrocarbon chain is C7-C 13 Hydrocarbon chain.

219. A bifunctional compound according to any one of claims 213 to 214, wherein Z is C 1- C 30 A hydrocarbon chain in which one or more methylene groups are individually and optionally replaced by one or more groups selected from: -O-, -N(H), -N(H)-C(=O)-, -C(=O)-N(H)-, triazole, an optionally substituted carbocycle; an optionally substituted heterocycle and -CH2-CH2-O-.

220. A bifunctional compound according to any one of claims 213 to 214, wherein Z comprises C1-C 30 Hydrocarbon chains, such as C1-C 20 , for example C1-C 15 , for example C1-C 10 wherein one or more methylene groups are individually and optionally replaced by one or more groups selected from the group consisting of: -O-, -N(H)-, -N(R L1 )-, -OC(=O)-, -C(=O)O-, -C(=O)-, -N(H)C(=O)-, -, -NHC(=O)O-, -N(R L1 )C(=O)-, -C(=O)N(H)-, -C(=O)N(R L1 )-, -S-, -S(=O)-, -S(=O)2-, -N(R L1 )S(=O)2-、-S(=O)2N(R L1 )-, -CH2-CH2-O-, optionally substituted carbocycle; optionally substituted heterocycle, and triazole; and R L1 C 1-5 alkyl.

221. A bifunctional compound according to any one of claims 213 to 220, wherein one or more methylene groups, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 methylene groups of the hydrocarbon chain in Z are individually and optionally replaced by one or more groups selected from the group consisting of: -O-, -N(H)-, -N(R L1 )-, -OC(=O)-, -C(=O)O-, -C(=O)-, -N(H)C(=O)-, -N(R L1 )C(=O)-, -C(=O)N(H)-, -C(=O)N(R L1 )-, -S-, -S(=O)-, -S(=O)2-, -N(R L1 )S(=O)2-、-S(=O)2N(R L1 )-, -CH2-CH2-O-, optionally substituted carbocycle; optionally substituted heterocycle and triazole; and R L1 C 1-5 alkyl.

222. A bifunctional compound according to any one of claims 213 to 220, wherein Z comprises one or more groups -NH-SO 2 -group.

223. The bifunctional compound of any one of claims 213 to 221, wherein Z comprises one or more triazole groups.

224. The bifunctional compound of any one of claims 213 to 223, wherein Z comprises one or more groups selected from the group consisting of optionally substituted carbocyclic groups and optionally substituted heterocyclic groups.

225. The bifunctional compound of any one of claims 213 to 224, wherein Z comprises two groups, each group being independently selected from: a triazole, an optionally substituted carbocyclic group, and an optionally substituted heterocyclic group.

226. The bifunctional compound of any one of claims 213 to 224, wherein Z comprises three groups, each group being independently selected from: a triazole, an optionally substituted carbocyclic group, and an optionally substituted heterocyclic group.

227. A bifunctional compound according to any one of claims 213 to 226, wherein the carbocyclic ring is according to wherein n is an integer selected from 0, 1, 2 or 3.

228. The bifunctional compound of any one of claims 213 to 227, wherein Z comprises one or more heterocyclic groups.

229. The bifunctional compound of any one of claims 213 to 228, wherein the heterocyclic group may be an optionally substituted 3 to 6 membered ring, wherein one or two carbon atoms of the ring have been replaced by N.

230. A bifunctional compound according to any one of claims 213 to 229, wherein the heterocyclic group is according to wherein n is an integer selected from 0, 1, 2 or 3.

231. A bifunctional compound according to any one of claims 213 to 230, wherein Z comprises one or more groups, each of which is individually selected from the groups shown in Table Z in the "Linker" section.

232. A bifunctional compound according to any one of claims 213 to 231, wherein Z comprises two groups, each of which is independently selected from the groups shown in Table Z in the "Linker" section.

233. The bifunctional compound according to any one of claims 213 to 232, wherein Z comprises three groups, each of which is individually selected from the groups shown in Table Z in the "Linker" section.

234. The bifunctional compound according to any one of claims 213 to 233, wherein Z comprises one or more groups, each of which is individually selected from the groups shown in Table ZI in the "Linker" part.

235. A bifunctional compound according to any one of claims 213 to 234, wherein the triazole group is 236. A bifunctional compound according to any one of claims 213 to 235, wherein Z comprises wherein n is an integer from 1 to 10.

237. A bifunctional compound according to any one of claims 213 to 236, wherein Z comprises wherein n is an integer from 1 to 10 and t or w is an integer from 1 to 20.

238. A bifunctional compound according to any one of claims 213 to 237, wherein Z comprises wherein n is an integer from 1 to 10 and each of t and t′ is independently an integer from 1 to 20.

239. A bifunctional compound according to any one of claims 213 to 238, wherein Z comprises wherein each of n and n′ is independently an integer from 1 to 10 and t is an integer from 1 to 20.

240. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 or L 2 It is an amide group.

241. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and L 2 It is an amide group.

242. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 or L 2 A carbonyl group.

243. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and L 2 A carbonyl group.

244. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 or L 2 It is an ester group.

245. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and L 2 It is an ester group.

246. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 or L 2 A carbamate group.

247. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and L 2 A carbamate group.

248. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and L 2 It is a urea group.

249. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 or L 2 It is a urea group.

250. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 or L 2 It is –NH-S(=O)2-.

251. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and L 2 It is –NH-S(=O)2-.

252. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 or L 2 A triazole group.

253. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and L 2 A triazole group.

254. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 or L 2 It is –O-.

255. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and L 2 It is –O-.

256. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 or L 2 It is –NH-.

257. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and L 2 It is –NH-.

258. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 or L 2 It is –S(=O)2-.

259. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and L 2 It is –S(=O)2-.

260. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 or L 2 for 261. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and L 2 for 262. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 or L 2 for 263. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and L 2 for 264. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and L 2 For different groups.

265. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and / or L 2 for 266. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and / or L 2 for 267. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and / or L 2 for wherein X is an atom selected from N or O.

268. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and / or L 2 for wherein X is an atom selected from N or O.

269. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and L 2 for the same.

270. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 is the key.

271. A bifunctional compound according to any one of claims 213 to 239, wherein L 2 is the key.

272. A bifunctional compound according to any one of claims 213 to 239, wherein L 1 and L 2 is the key.

273. A bifunctional compound according to any one of the preceding claims, wherein L I Selected from: n and w are each independently an integer from 1 to 9; and * Indicates that T L or S L connection.

274. A bifunctional compound according to any one of the preceding claims, wherein L I Any one of formula (XVIa) to formula (XVIae) in Table Z-II selected from the "Linker" section; wherein R L Indicates that S L The connection point and R T Indicates that T L connection point.

275. A bifunctional compound according to any one of the preceding claims, wherein L I Any one of formulas XVIaf to XVIbw in Table Z-III selected from the "Linker" section, wherein * represents a L or T L connection.

276. The bifunctional compound of claim 1, wherein L I For peptides.

277. The bifunctional compound of claim 276, wherein L I For peptides.

278. A bifunctional compound according to any one of claims 276 to 277, wherein L I A peptide with a length between 1 and 30 amino acids.

279. A bifunctional compound according to any one of claims 276 to 278, wherein L I It is a peptide with a length between 3 and 20 amino acids.

280. A bifunctional compound according to any one of claims 276 to 279, wherein L I A peptide between 3 and 20 amino acids in length consisting of any combination of glycine, serine and cysteine.

281. A bifunctional compound according to any one of claims 276 to 280, wherein L I A peptide between 3 and 20 amino acids in length consisting of any combination of glycine and serine.

282. A bifunctional compound according to any one of claims 276 to 281, wherein L I is a peptide selected from any one of the following: GGSGGGGSGGGGSGG(SEQ ID NO.:131) GGSGGGG (SEQ ID NO.: 132) GGGGSGGGGSGGGGSGG(SEQ ID NO.:133) GGSGGGGSGGGGS (SEQ ID NO.: 134) GGSGGGGSGGG (SEQ ID NO.: 135) GGSGGGGSG (SEQ ID NO.: 136) GGSGGGG (SEQ ID NO.: 137) GGSGG (SEQ ID NO.: 138) GGS (SEQ ID NO.: 139) CGGSGGGGSGGGGSGG (SEQ ID NO.:140).

283. A bifunctional compound according to any one of claims 276 to 282, wherein L I By L I The C-terminus is connected to S L And L I By L I The N-terminus is connected to T L .

284. The bifunctional compound of claims 1 to 16, wherein S L According to formula IIIai: Wherein w is an integer from 1 to 9.

285. A bifunctional compound according to any one of claims 1 to 47, 138 to 151 or 213 to 275, wherein the bifunctional compound is selected from any one of compounds BF001 to BF028 shown in Table A in the "Compound Listing" section.

286. A bifunctional compound according to any one of claims 1 to 47, 138 to 151 or 213 to 275, wherein the bifunctional compound is selected from any one of compounds BF030 to BF149 shown in Table A in the "Compound Listing" section.

287. The bifunctional compound of any one of claims 1 to 47, 138 to 151, or 213 to 275, wherein the bifunctional compound has: aS L , which is selected from any one of Formulae IIIi, IIIj, IIIk, IIIm, IIIn and IIIo; or selected from Formula DI described above in the "Binding of Sortilin" section; and b. can be less than 50 μM, for example, less than 40 μM, for example, less than 30 μM, for example, less than 20 μM, for example, less than 10 μM, for example, less than 5 μM, for example, less than 4 μM, for example, less than 3 μM, for example, less than 2 μM, for example, less than 1 μM, for example, less than 0.8 μM, for example, less than 0.6 μM, for example, less than 0.5 μM, for example, less than 0.4 μM, for example, less than 0.3 μM, for example, less than 0.2 μM, for example, less than 0.1 μM. D ) binds to the target molecule.

288. The bifunctional compound of any one of claims 1 to 47, 59 to 60, 138 to 151, or 213 to 275, wherein the bifunctional compound has: aS L , which is selected from any one of formula IIIi, IIIj, IIIk, IIIm, IIIn and IIIo; or selected from formula DI described in the "Binding of Sortilin" section; and B L I , which is selected from any one of the formulas XVIaf to XVIbw in Table Z-III in the "Linkers" section above; and T L , which is selected from any one of Formulas XVIIa-3, XVIIa-4, XVIIc-1 and XVIIc-2 described in the "Targeted Warhead" section.

289. The bifunctional compound of any one of claims 1 or claims 61 to 118 or 276 to 282, wherein the bifunctional compound is selected from: Biotin-GGSGGGGSGRQLL-OH (SEQ ID NO.: 141) Biotin-GGSGGGGSGGGGSRQLL-OH (SEQ ID NO.: 142) Biotin-GGSGGGGFQLL-OH (SEQ ID NO.: 143) Biotin-GGSGGFQLL-OH (SEQ ID NO.: 144) Biotin-GGSGGGGSGFQLL-OH (SEQ ID NO.: 145) Biotin-GGSGGGGSGGGGSFQLL-OH (SEQ ID NO.:146) Biotin-GGSGGGGSGGGGSGGRQLL-OH (SEQ ID NO.:147) Biotin-GGSGGGGSGGGFQLL-OH (SEQ ID NO.:148) Biotin-GGSGGGGSGGGRQLL-OH (SEQ ID NO.: 149) Biotin-GGSGGGGRQLL-OH (SEQ ID NO.: 150) Biotin-GGSGGRQLL-OH (SEQ ID NO.: 151) Biotin-GGSRQLL-OH (SEQ ID NO.: 152) Biotin-GGRQLL-OH (SEQ ID NO.: 153) Biotin-RQLL-OH (SEQ ID NO.: 154) Biotin-GGSGGGGSGGGGSGGFQLL-OH (SEQ ID NO.: 155) Biotin-REAPRWDAPLRDPALFQLL-OH (SEQ ID NO.: 189), and Biotin-REAPRWDAPLRDPALRQLL-OH (SEQ ID NO.: 191).

290. A bifunctional compound according to any one of claims 1 to claims 61 to 129 or claims 276 to 282, wherein the bifunctional compound is: MGGTHHHHHHENLYFQGQVQLQESGGGLVQPGGSLRLSCAASGRTISRYAMSWFRQAPGKEREFVAVARRSGDGAFYADSVQGRFTVSRDDAKNTVYLQMNSLKPEDTAVYYCAIDSDTFYSGSYDYWGQGTQVTVSSEGGGGSGGGGSGGGGSGGRQLL (SEQ ID NO.: 158).

291. A bifunctional compound according to any one of claims 1 to 61 to 129 or claims 276 to 282, wherein the T L The monoclonal antibody amorobizumab (an antibody having a heavy chain of SEQ ID NO.: 66 and a light chain of SEQ ID NO.: 67) is combined with at least one unit -L I -S L Coupling, the –L I -S L Consists of CGGSGGGGSGGGGSGGRQLL (SEQ ID NO.: 175).

292. A bifunctional compound according to any one of claims 1 to 61 to 129 or claims 276 to 282, wherein the T L The monoclonal antibody adalimumab (an antibody having a heavy chain of SEQ ID NO.: 68 and a light chain of SEQ ID NO.: 69) is combined with at least one unit -L I -S L Coupling, the –L I -S L Selected from: CGGSGGGGSGGGGSGGRQLL(SEQ ID NO.:175) CREAPRWDAPLRDPALRQLL(SEQ ID NO.:184) CREAPRWDAPLRDPALFQLL-OH (SEQ ID NO: 186).

293. An isolated polynucleotide encoding the bifunctional compound of claim 290.

294. A vector comprising the polynucleotide according to claim 293.

295. The vector of claim 294, wherein the vector is an expression vector, such as a bacterial vector or a viral vector.

296. A host cell comprising a polynucleotide according to claim 293 and / or a vector according to any one of claims 294 to 295.

297. The bifunctional compound of any preceding claim, wherein the compound is capable of forming a ternary complex between sortilin and the target protein.

298. The bifunctional compound of any preceding claim, wherein the compound is capable of binding to sortilin and the target protein simultaneously.

299. A bifunctional compound according to any one of the preceding claims, wherein S L The dissociation constant for binding to sortilin is less than 50 μM, such as less than 2 μM, such as less than 0.5 μM, preferably less than 0.1 μM, and T L The dissociation constant for binding to its target is less than 100 μM, such as less than 0.5 μM, such as less than 0.1 μM.

300. The bifunctional compound of any preceding claim, wherein the compound is capable of binding to Sortilin at the surface of a cell.

301. A bifunctional compound according to any one of the preceding claims, wherein in S L Binds to sortilin on the cell surface and T L After binding to the target protein, the target protein is internalized into the cell.

302. The bifunctional compound of claim 301, wherein the target protein is degraded after internalization into the cell.

303. The bifunctional compound of any one of claims 1 to 124, 138 to 151, 177 to 178, 183 to 192, 286 and 292, wherein the compound is capable of forming a ternary complex between sortilin and TNFa.

304. The bifunctional compound of any one of claims 1 to 124, 138 to 151, 177 to 178, 183 to 191, 213 to 283, or 286 to 292 and 303, wherein the compound is capable of binding to sortilin and TNFα simultaneously.

305. A bifunctional compound according to any one of claims 297 to 304, wherein S L The dissociation constant for binding to sortilin is less than 50 μM, such as less than 2 μM, such as less than 0.5 μM, preferably less than 0.1 μM, and T L The dissociation constant for binding to TNFa is less than 100 μM, such as less than 0.5 μM, such as less than 0.1 μM.

306. The bifunctional compound of any one of claims 297 to 305, wherein the compound is capable of binding to Sortilin at the surface of a cell.

307. A bifunctional compound according to any one of claims 302 to 306, wherein in S L Binds to sortilin on the cell surface and T L After binding to TNFa, TNFa is internalized into the cell.

308. The bifunctional compound of any one of claims 307, wherein TNFa is degraded after internalization into the cell.

309. A pharmaceutical composition comprising a bifunctional compound according to any one of claims 1 to 308.

310. A bifunctional compound according to any one of claims 1 to 308 for use as a medicament.

311. The bifunctional compound of any one of claims 1 to 308 for use in treating a disorder or condition in a subject in need thereof.

312. The bifunctional compound for use according to claim 311, wherein the disorder or condition is mediated by an extracellular protein.

313. The bifunctional compound for use according to claim 312, wherein the extracellular protein is selected from the group consisting of: PCSK9, TNF-α, ANGPTL-3, antibody light chain, IgG, IgE, IgAIL-1, IL-2, IL-6, IFN-γ, VEGF, TFG-β1, IL-21, IL-22, IL-5, IL-10, IL-8, cholinesterase, human CCL2, carboxypeptidase B-2, neutrophil elastase, factor Xa, factor XI, factor XIa, factor XII, factor XIII, prothrombin, coagulation factor VII, coagulation factor Factor IX, fibroblast growth factor 1, FGF-2, fibronectin 1, kallikrein-1, lipoprotein lipase, human matrix metallopeptidase 1, macrophage migration inhibitory factor, transforming growth factor-p (TGF-p), thrombospondin-1 (TSP-T), CD40 ligand, urokinase-type plasminogen activator, tissue-type plasminogen activator (TPA), plasminogen (PLG), plasminogen activator inhibitor-1, placental growth factor, phospholipase A2 group IB, phospholipase A2 group IIA, complement factor B, complement factor D, complement factor H, complement component 5, and complement C1s.

314. A bifunctional compound for use according to claim 312, wherein the extracellular protein is PCSK9.

315. The bifunctional compound for use according to claim 314, wherein the disorder or condition is a disorder of lipoprotein metabolism.

316. The bifunctional compound for use according to claim 315, wherein the lipoprotein disorder is associated with abnormal PCSK9 levels.

317. The bifunctional compound for use according to claim 315 or 316, wherein the lipoprotein disorder is selected from dyslipidemia, hypercholesterolemia and coronary heart disease.

318. The bifunctional compound for use according to claim 312, wherein the extracellular protein is TNF-α.

319. A bifunctional compound for use according to claim 318, wherein the disorder or condition is an inflammatory disease.

320. The bifunctional compound for use according to claim 318, wherein the disorder or condition is an autoimmune disease.

321. A bifunctional compound for use according to claim 318, wherein the disorder or condition is cancer.

322. A bifunctional compound for use according to claim 311 or 318, wherein the disorder or condition is selected from rheumatoid arthritis, inflammatory bowel disease, graft-versus-host disease, ankylosing spondylitis, psoriasis, hidradenitis suppurativa, refractory asthma, systemic lupus erythematosus, diabetes and induction of cachexia.

323. The bifunctional compound for use according to claim 312, wherein the extracellular protein is an antibody light chain or IgG.

324. A bifunctional compound for use according to claim 323, wherein the disorder or condition is selected from type 1 autoimmune pancreatitis, interstitial nephritis, Riddle's thyroiditis, storiform fibrosis, Mikulicz's disease, Kuttner's tumor, inflammatory pseudotumor, mediastinal fibrosis, retroperitoneal fibrosis (Ormond's disease), aortitis, periaortitis, proximal bile duct stenosis, idiopathic hypocomplementemic tubulointerstitial nephritis, multifocal fibrosclerosis, pachymeningitis, pancreatic enlargement, tumor-like lesions, pericarditis, rheumatoid arthritis (RA), inflammatory bowel disease, multiple sclerosis, myasthenia gravis, thyroid eye disease, chronic inflammatory demyelinating polyneuropathy, warm autoimmune hemolytic anemia, ankylosing spondylitis, primary Sjögren's syndrome, psoriatic arthritis and systemic lupus erythematosus (SLE), sclerosing cholangitis and IgG monoclonal gammopathy, monoclonal gammopathy of undetermined significance (MGUS).

325. A bifunctional compound for use according to any one of claims 310 to 324, wherein the extracellular protein is present at abnormally high levels.

326. The bifunctional compound for use according to any one of claims 310 to 324, wherein the extracellular protein is mutated or misfolded compared to the corresponding endogenous protein.

327. A bifunctional compound for use according to any one of claims 310 to 326, wherein the subject is a mammal.

328. The bifunctional compound for use according to claim 327, wherein the mammal is a human.

329. A method for targeting lysosomal degradation of extracellular proteins, comprising administering an effective amount of a bifunctional compound according to any one of claims 1 to 308.

330. A method of removing an extracellular target protein from the plasma of a patient or subject in need thereof, comprising administering a bifunctional compound according to any one of claims 1 to 308.

331. Use of a bifunctional compound according to any one of claims 1 to 308 for the preparation of a medicament for the treatment of a disease or condition.

332. A method of treating a disease or condition comprising administering to a subject in need thereof a bifunctional compound according to any one of claims 1 to 312.

333. Use of a bifunctional compound according to any one of claims 1 to 312 for the preparation of a medicament for the treatment of a disorder or condition mediated by an extracellular protein.

334. A method of treating a disorder or condition mediated by an extracellular protein comprising administering to a subject in need thereof a bifunctional compound according to any one of claims 1 to 312.

335. A method of targeting lysosomal degradation of TNFa, comprising administering to a subject in need thereof an effective amount of a bifunctional compound according to any one of claims 1 to 124, 138 to 151, 177 to 178, 183 to 192, 286 to 292 and 303 to 308.

336. A method of removing TNFa from the plasma of a subject in need thereof, comprising administering a bifunctional compound according to any one of claims 1 to 124, 138 to 151, 177 to 178, 183 to 192, 286 to 292 and 303 to 308.

337. The method according to any one of claims 335 to 336, wherein the compound is selected from any one of compounds BF030 to BF149 in Table A of the "Compound Listing" section, or a pharmaceutically acceptable salt thereof.

338. Use of a bifunctional compound according to any one of claims 1 to 308 for the preparation of a medicament for treating a disease or condition.

339. A method of treating a disease or condition comprising administering to a subject in need thereof a bifunctional compound according to any one of claims 1 to 308.

340. Use of a bifunctional compound according to any one of claims 1 to 124, 138 to 151, 177 to 178, 183 to 192, 286 to 292 and 303 to 308 for the preparation of a medicament for treating a disorder or condition mediated by TNFa.

341. A method of treating a disorder or condition mediated by TNFa comprising administering to a subject in need thereof a bifunctional compound according to any one of claims 1 to 124, 138 to 151, 177 to 178, 183 to 192, 286 to 292 and 303 to 308.

342. A compound having a structure according to formula (III): or a pharmaceutically acceptable salt thereof, Where Q 1 is a bond or -CH2-; and R 3 Having formula (IIIp), or formula (IIIq) or formula B-II: Where R 3b is selected from H, halogen, alkoxy, -CF3 and optionally substituted C 1-5 Alkyl, wherein the C 1-5 One or more methylene groups of the alkyl group are optionally replaced individually by one or more of the following groups: -O-, -NH-, -C(O)-, ester, amide, carbamate, thiourea, sulfonamide, urea, optionally substituted carbocyclic ring; optionally substituted heterocyclic ring and wherein X is an atom selected from N and O; The condition is that when R 3 When it is formula (IIIq), R 3b is not H; and in * It indicates the connection with formula (III).

343. The compound according to claim 342, wherein Q 1 It is -CH2-.

344. The compound according to claim 342, wherein Q 1 is the key.

345. according to the compound described in any one of claims 342 to 344, wherein R 3 Having formula (IIIr): Where R 3b is selected from H, halogen, alkoxy, -CF3 and optionally substituted C 1-5 Alkyl, wherein the C 1-5 One or more methylene groups of the alkyl group are optionally replaced individually by one or more of the following groups: -O-, -NH-, -C(O)-, ester, amide, carbamate, thiourea, and and in * It indicates the connection with formula (III).

346. The compound of claims 342 to 345, wherein the compound has formula (IIIs), formula (IIIt) or formula B-III: Where R 3b is selected from H, halogen, alkoxy, -CF3 and optionally substituted C 1-5 Alkyl, wherein the C 1-5 One or more methylene groups of the alkyl group are optionally replaced individually by one or more of the following groups: -O-, -NH-, -C(O)-, ester, amide, carbamate, thiourea, sulfonamide, urea, optionally substituted carbocyclic ring; optionally substituted heterocyclic ring and Where X is N(H) or O; provided that when R 3 When it is formula (IIIt), R 3b Not H.

347. according to the compound described in any one of claims 342 to 346, wherein R 3b For H.

348. according to the compound described in any one of claims 342 to 346, wherein R 3b is halogen or –CF3.

349. according to the compound described in any one of claims 342 to 346, wherein R 3b C 1- C5 alkyl, where C 1-5 One or more methylene groups of the alkyl group are individually optionally replaced by one or more of the following groups: -O-, -NH-, -C(O)-, ester, amide, carbamate, thiourea, and 350. according to the compound described in any one of claim 342 to 346 or 349, wherein R 3b C 1- C5 alkyl, wherein the C 1-5 One or more methylene groups of the alkyl group are optionally replaced individually by one or more of the following groups: -O-, amide and 351. according to the compound described in any one of claim 342 to 346 or 349 to 350, wherein R 3b C 1- C5 alkyl, wherein the C 1-5 One or more methylene groups of the alkyl group are individually optionally replaced by -O-.

352. according to any one of claims 342 to 346 or 349 to 351, wherein R 3b C 1- C5 alkyl, wherein the C 1-5 One or more methylene groups of the alkyl group are individually optionally replaced by an amide.

353. according to the compound described in any one of claim 342 to 346 or 349 to 352, wherein R 3b C 1- C5 alkyl, wherein the C 1-5 One or more methylene groups of the alkyl group are optionally replace.

354. according to the compound described in any one of claims 342 to 346, wherein R 3b It is -C(=O)-(NH)-CH3.

355. according to the compound described in any one of claims 342 to 346, wherein R 3b It is -CH2-CH2-O-CH3.

356. according to the compound described in any one of claims 342 to 346, wherein R 3b It is -O-CH2-CH2-O-CH3.

357. according to the compound described in any one of claims 342 to 346, wherein R 3b It is -CH2-NH-C(=O)-CH3.

358. A compound according to any one of claims 342 to 346, wherein R 3b It is -O-CH2-CH2-NH-C(=O)-CH3.

359. according to the compound described in any one of claims 342 to 346, wherein R 3b It is tetrazole-CH2-CH2-O-CH3.

360. A compound according to any one of claims 342 to 346, wherein R 3b C 1- C5 alkyl, wherein the C 1-5 The one or more methylene groups of the alkyl group are optionally substituted with an optionally substituted carbocyclic ring, for example according to wherein n is an integer selected from 0, 1, 2 or 3.

361. A compound according to any one of claims 59 to 346, wherein R 3b C 1- C5 alkyl, wherein the C 1-5 One or more methylene groups of the alkyl group are optionally substituted with an optionally substituted heterocycle, for example according to wherein n is an integer selected from 0, 1, 2 or 3; or Heterocyclic replacement.

362. A compound according to any one of claims 342 to 346, wherein R 3b -O-CH2-C(=O)N(H)R IIIa , where R IIIa Selected from H and C1-C6 alkyl.

363. according to the compound described in any one of claims 342 to 346, wherein R 3b -O-CH2-CO2R IIIa , where R IIIa Selected from H and C1-C6 alkyl.

364. according to the compound described in any one of claims 342 to 346, wherein R 3b C 1- C5 alkyl, wherein the C 1-5 One or more methylene groups of the alkyl group are individually optionally replaced by one of the groups shown in Table Z in the "Linker" section.

365. The compound of any preceding claim, wherein the compound is an S-stereoisomer.

366. The compound of any preceding claim, wherein the compound is the R-stereoisomer.

367. The compound of any one of claims 342 to 347, wherein the compound is:

368. The compound of any one of claims 342 to 346, wherein the compound is:

369. A compound according to any one of claims 342 to 346 or 349 to 352, wherein the compound is selected from any one of compounds SB001, SB003, SB006, SB007, SB008, SB009, SB010, SB011 and SB012 according to Table B in the "Compound Listing" section, or a pharmaceutically acceptable salt thereof.

370. A compound selected from or a pharmaceutically acceptable salt thereof.

371. A compound having a structure selected from any of the following: or a pharmaceutically acceptable salt thereof.

372. A compound according to any one of claims 342 to 372, wherein the compound is capable of binding to sortilin with a dissociation constant of at least less than 50 μM, such as less than 40 μM, such as less than 30 μM, such as less than 20 μM, such as less than 10 μM, such as less than 5 μM, such as less than 4 μM, such as less than 3 μM, such as less than 2 μM, such as less than 1 μM, such as less than 0.8 μM, such as less than 0.6 μM, such as less than 0.5 μM, such as less than 0.4 μM, such as less than 0.3 μM, such as less than 0.2 μM, such as less than 0.1 μM.

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