Bifunctional molecules that selectively induce degradation of extracellular targets within lysosomes

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to bifunctional molecules comprising a protein-binding moiety of interest linked via a linker group to a cellular receptor-binding moiety, preferably a moiety that binds to the receptor sortilin, encoded by the gene SORT1. Pharmaceutical compositions based on these bifunctional molecules represent a further aspect of the invention. These compounds and / or compositions can be used to treat disease states and conditions by removing secreted and / or transmembrane proteins through degradation inside the cells of a patient or subject in need thereof. Methods of treating disease states and / or conditions in which circulating proteins are associated with the disease state and / or condition are also described herein. [Background technology]

[0002] Protein degradation is a vital part of biomolecule turnover and recycling and is a natural process that occurs in all cells. Cytoplasmic proteins are generally degraded in the proteasome after ubiquitination, whereas extracellular biomolecules are degraded in the lysosomal compartment. Specific ubiquitination of cytoplasmic disease-associated proteins (DAPs) is promoted by bifunctional molecules called proteolysis targeting chimeras (PROTACs) [1]. PROTACs are bifunctional molecules composed of a DAP-binding warhead at one end, which is linked to an E3 ubiquitin ligase-binding small molecule at the other end. PROTACs thereby link E3 ligases to DAPs, leading to ubiquitination and subsequent degradation in the proteasome, effectively functioning as chemical DAP knockdown. However, because PROTACs rely on the proteasome for function, they are only applicable to intracellular proteins. Data have demonstrated that non-cytosolic proteins can be targeted for degradation in the lysosomal compartment by binding to the protein sorting mannose-6-phosphate receptor (M6P-R) using bifunctional molecules [2]. Banik et al. showed that antibodies tagged with M6P-R-binding sugar moieties promote lysosomal degradation of extracellular and membrane-bound targets. These types of molecules, termed lysosomal targeting chimeras (LYTACs), facilitate the lysosomal delivery and degradation of DAPs, providing the first preclinical proof-of-concept (PoC) that they have therapeutic potential. The M6P-R binding motif is a phosphorylated sugar polymer; therefore, developing an orally available modality will require considerable effort. Both PROTACs and LYTACs are designed to hitchhike to natural cellular mechanisms; therefore, the warhead does not need to provide functionality by itself. This is particularly advantageous compared to traditional drug development, as warhead development simply requires optimization of binding affinity and linker conjugation strategy. Furthermore, the absence of these requirements further enables engagement with targets considered undruggable due to current constraints.

[0003] The functionality of LYTACs depends on the successful recruitment of lysosomal trafficking receptors, exemplified by M6P-R [2]. Another lysosomal receptor protein is sortilin. Sortilin shares many common 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 ability is demonstrated by the plasma accumulation of its natural ligand, the frontotemporal dementia (FTD)-associated protein progranulin, which is increased 3.5-fold in the plasma of mice lacking 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 are being explored. The result is a large number of small molecule high affinity sortilin binders with different pharmacological profiles [8-10], including orally bioavailable compounds and compounds with CNS exposure [8].

[0004] Summary of the Invention

[0005] In one main aspect, the present invention provides a bifunctional compound having a structure according to formula (I): T L -L I -S L (I) (In the formula, S L is the moiety that binds to sortilin; L I is a linker or bond; T L is the moiety that binds to the extracellular target molecule) or a pharmaceutically acceptable salt thereof.

[0006] In another aspect, the present invention relates to a bifunctional compound for use in treating a disorder or condition in a subject. In one aspect, the disorder or condition is mediated by an extracellular protein.

[0007] In yet another aspect, the present invention provides a method for targeted lysosomal degradation of extracellular proteins, said method comprising administering an effective amount of a bifunctional compound of formula (I) as described herein.

[0008] In another aspect, the present invention provides a method for removing extracellular proteins from the plasma of a subject in need thereof, said method comprising administering to said subject an effective amount of a bifunctional compound of formula (I) as described herein.

[0009] Thus, in one aspect, the present disclosure provides a method for targeted lysosomal degradation of TNFα, comprising administering an effective amount of a bifunctional compound described herein.

[0010] In another aspect, the present disclosure provides a method for removing TNFα from the plasma of a patient or subject in need thereof, comprising administering a bifunctional compound described herein.

[0011] In a further aspect, the present disclosure relates to pharmaceutical compositions comprising the bifunctional compounds described herein.

[0012] In one aspect, the invention relates to novel small molecule sortilin binding agents described herein.

[0013] In another aspect, the invention relates to the novel sortilin-binding peptides described herein.

[0014] In another aspect, the invention relates to an isolated polynucleotide or bifunctional compound encoding a peptide described herein. In another aspect, the invention relates to a vector or bifunctional compound comprising an isolated polynucleotide encoding a peptide described herein. In another aspect, the invention relates to a host cell or bifunctional compound comprising an isolated polynucleotide or vector encoding a peptide described herein. In another aspect, the invention relates to novel TNFα-binding agents as described herein. [Brief explanation of the drawings]

[0015] [Figure 1] MST binding experiments using fluorescent sortilin-6his (100 nM) and compounds according to SEQ ID NO: 147 (A) and SEQ ID NO: 157 (B). [Figure 2] Proximity induced HTRF ratio as a function of compound concentration for compounds according to SEQ ID NO: 147 and SEQ ID NO: 157. [Figure 3A] 1 shows the cell-associated fluorescence intensity (FI) signal after 3 hours of incubation of HEK293 / Sortilin with a concentration series of SEQ ID NO: 147, SEQ ID NO: 157, or SEQ ID NO: 155 and a neutravidin fluorescent derivative (NA650). Data points are shown as mean + / - SEM. [Figure 3B] Figure 1 shows cell-associated fluorescence intensity (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 the small molecule sortilin binding agent AF38496 (Schroder et al) (0.1 nM to 100 μM), SEQ ID NO: 172 peptide without the biotin warhead (0.025 nM to 25 μM), or biotin alone (0.1 nM to 100 μM) (n=2). Data points are shown as mean ± SEM. [Figure 4A]Fluorescence microscopy imaging of lysosomal markers in HEK293 / Sortilin cells fixed and immunostained with anti-LAMP1 (Alexa-488) after 2 hours of incubation with the bifunctional molecules SEQ ID NO: 147 (300 nM) and NA650 (NA650) (100 nM). Nuclei were stained with Hoechst. Scale bar 20 μm. [Figure 4B] SDS-PAGE gel showing NA650 in HEK293 / Sortilin cell lysates incubated with peptide bifunctional compound (300 nM) and leupeptin (80 μM) (Sigma-Aldrich) for 24 hours before harvesting the cell lysates, and proteins separated on an SDS-PAGE gel. The bar graph shows quantification of the NA650 upper band (average of standard values). [Figure 5] NA650 fluorescence signal in HEK293 / Sortilin cell culture supernatants after incubation with SEQ ID NO: 147 (20 nM-10 μM) and NA650 (Invitrogen) (100 nM) for up to 24 h, 48 h, and 72 h. [Figure 6] NA650 fluorescence signal in HEK293 / Sortilin cell culture supernatants after incubation with NA650 (Invitrogen) (100 nM) and SEQ ID NO: 147 (20 nM to 5 μM) or SEQ ID NO: 155 (20 nM to 5 μM). [Figure 7A] SDS-PAGE analysis of the conjugation 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 + heat) conditions. [Figure 7B] SDS-PAGE analysis of the conjugation of NHS-linker or NHS-linker-RQLL to adalimumab under non-reducing or reducing (2 mM DTT + heat) conditions. [Figure 8] 1 shows the binding response of MST to sortilin as a function of antibody concentration for alirocumab-link-RQLL and alirocumab-link. [Figure 9A]1 shows complex formation between PCSK9-6HIS and alirocumab-linker-RQLL after purification by gel filtration. [Figure 9B] 1 shows complex formation between PCSK9-6HIS and the alirocumab linker after purification by gel filtration. [Figure 9C] SDS-PAGE analysis of peak fractions from protein complexes. [Figure 10] Baseline-corrected HTRF (665 / 620 nm) ratios normalized to the alirocumab-link plotted against a concentration gradient (290 nM to 3.5 fM) of alirocumab conjugated to a linker with or without RQLL after 2.5 hours of incubation. (Error bars = SD, n = 2) [Figure 10B] Corrected for the lowest global signal after 2.5 h of incubation, plotted against a concentration gradient (400 nM to 4.7 fM) of adalimumab conjugated to a linker with or without RQLL (error bars = SD, n = 3). [Figure 10C] Corrected for lowest global signal plotted against concentration gradient of adalimumab conjugated to sortilin-binding peptides SEQ ID NO:184-SEQ ID NO:187 (400 nM-4.7 fM) after 2.5 hours of incubation (Error bars = SD, n = 3). [Figure 11A] Cell-associated fluorescent signal after 3 hours of incubation of HEK293 / Sortilin with a concentration series of Alirocumab-link-RQLL (SEQ ID NO: 159) (1 nM to 0.5 μM) and Cy5-PCSK9 (200 nM). [Figure 11B] In control experiments, cells were incubated with alirocumab, a concentration series (250 pM to 250 nM) of a peptide lacking the C-terminal sortilin binding sequence alirocumab-link (SEQ ID NO: 160) and adalimumab-link-RQLL (SEQ ID NO: 161), and TNFα (100 nM). [Figure 11C]In control experiments, cells were incubated with a concentration series (250 pM to 250 nM) of adalimumab containing a peptide lacking the C-terminal sortilin-binding sequence adalimumab-link (SEQ ID NO: 162) and adalimumab conjugated to the sortilin-binding peptides of SEQ ID NO: 184 to SEQ ID NO: 187, and TNFα (100 nM). [Figure 12A]

[0023] Figure 1 shows the size exclusion chromatography elution profile and final sample of NB-Linker-RQLL (SEQ ID NO: 158). Pooled and concentrated fractions from size exclusion are shown in grey boxes. [Figure 12B] Figure 1 shows the size exclusion chromatography elution profile and final sample of NB-linker (SEQ ID NO: 71). Pooled and concentrated fractions from size exclusion are shown in grey boxes. [Figure 13A] Figure 1 shows that complex formation between the IgG target and NB-linker-RQLL (SEQ ID NO: 158) can be formed and purified by gel filtration. SDS-PAGE analysis of the main peak is shown on the right, with pooled and concentrated fractions from size exclusion indicated in grey boxes. [Figure 13B] Figure 1 shows that complex formation between the IgG target and the NB-linker (SEQ ID NO: 71) can be formed and purified by gel filtration. SDS-PAGE analysis of the main peak is shown on the right, and the pooled and concentrated fractions obtained from size exclusion are shown in grey boxes. [Figure 14A] FIG. 1 shows cell-associated FI signal after 3 hours of incubation of HEK293 / Sortilin with NB-Linker-RQLL, SEQ ID NO: 158 (8 nM to 8 μM) and a concentration series of Cy5-conjugated IgG (5, 50, or 500 nM). [Figure 14B] Figure 1 shows Cy5 FI in cell culture supernatants (top blot) and lysates (middle blot) of HEK293 / Sortilin cells after 72 hours of incubation with Cy5-conjugated IgG (κ-light chain) (50 nM) and NB-linker-RQLL, SEQ ID NO: 158, as indicated. The lower blot confirms sortilin expression in the cells by Western blotting. [Figure 14C] Cy5 FI (top blot) and Western blot (n=2) of IgG signal in lysates of HEK293 / Sortilin cells after 6 h 30 min incubation 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. The two lower blots confirm gel loading (anti-β-actin) and intracellular sortilin expression by Western blotting. Bar graphs show quantification of Ig HC as mean + / - SEM. [Figure 15] Detection of binding by titrated BF025, BF023, and BF020 (maximum concentration 12.5 μM) with constant NTA-labeled sortilin-6His (100 nM) in MST analysis. (Error bars = SD, n = 2) [Figure 16] HTRF (665 / 620 nm) signal, baseline corrected for the lowest local signal, plotted against bifunctional compound concentration (5 μM to 0.6 pM) after 2.5 h of incubation. [Figure 17] Figure 1 shows the cell-associated FI signal after 3 hours of incubation of HEK293 / Sortilin with a concentration series (2 nM to 2 µM) of compounds B025, B023, or B020 together with NA650 (100 nM). [Figure 18] NA650 FI signal in HEK293 / Sortilin cell culture supernatants after incubation of NA650 (100 nM) with bifunctional compounds BF025, BF023, or BF20 (20 nM–5 μM) as shown for 72 h. [Figure 19] FI signals in HEK293 / Sortilin lysates collected after 24 h of incubation with NA650 (100 nM), B025 (300 nM), AF38469 (10 μM), and leupeptin (80 μM) as indicated. [Figure 20]Cell-associated FI signals after 3 hours of incubation of HEK293 / Sortilin with a concentration series of compounds BF011, BF006, or BF005 (1.9 nM to 2 μM) and AlexaFluor488-anti-DNP antibody (100 nM) (n=2). [Figure 21A] FI signal in lysates of HEK293 / Sortilin cells incubated with AlexaFluor488-anti-DNP antibody (Thermo Fisher) (100 nM) and BF005 (30 nM) for 3 hours before lysing the cells as described above. The target and degrading agent were removed by replacing the medium with assay medium, and then incubated for 0 to 24 hours. Western blots of anti-β-actin and anti-Sortilin 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). [Figure 21B] FI in HEK293 / Sortilin lysates incubated with AlexaFluor488-anti-DNP antibody (100 nM), BF005 (30 nM), and leupeptin (80 μM) for 6 h 30 min as indicated. SB001 (30 nM) and AF38469 (10 μM) were included as controls. Bar graphs show quantification of FI signal of HC bands normalized to β-actin signal (mean ± SEM) (n = 2). [Figure 22] Concentration series (2 nM to 2 μM) of three different bifunctional compounds: SEQ ID NO: 155, NB-link-RQLL (SEQ ID NO: 158), or a small molecule bifunctional compound (BF005), as shown, and cell-associated FI signal after 3 hours of incubation with the corresponding targets and HEK293 / Sortilin. [Figure 23] 1 is a scheme representing a non-limiting example for the preparation of a bifunctional compound. [Figure 24A] Detection of binding of a constant NTA-labeled TNFα-6His (100 nM) by titrated BF080, BF081, and BF082 (maximum concentration 12.5 μM) in an MST assay (error bars = SD, n = 4-6). [Figure 24B]Mouse anti-TNFα (Invitrogen, MA5-23720) Western blot of cell lysates after 24-hour incubation of HEK393 / Sortilin with a concentration series of compounds BF040 and BF043 (100 nM to 10 μM) along with TNFα (100 nM). Control cells were incubated without compound and TNFα, or with TNFα alone. An anti-β-actin (Sigma, A5441) Western blot is shown as a control. [Figure 24C] Cell-associated FI signals after 24 h incubation of HEK293 / Sortilin with a concentration series of BF040, BF043, and BF042 (0.5 nM to 20 μM), and Cy5-TNFalpha (100 nM) in the presence of 80 μM leupeptin. [Figure 24D] Cell-associated FI signal after 24 h incubation of HEK293 / Sortilin cells with BF042 (0.5 nM-20 μM) and Cy5-TNFalpha with or without leupeptin (80 μM). [Figure 24E] Figure 1 shows TNFα levels in culture supernatants of HEK293 / Sortilin cells 72 hours after addition of 20 nM TNFα and the bifunctional molecules BF040 (, BF043) or BF042. TNFα is shown as a percentage normalized to the level measured in culture supernatants from cells without the addition of the bifunctional molecules. [Figure 24F] Anti-TNFα Western blotting of HEK293 / Sortilin cell lysates harvested at the indicated time points (0 h to 24 h) after 24 h of preincubation with BF042 (3 μM) and TNFα (100 nM). Preincubation medium was replaced at 0 h. Anti-β-actin Western blotting is shown as a control. [Figure 24G] Cell-associated FI signals after 24 h incubation of HEK293 / Sortilin with a concentration series (0.5 nM to 20 μM) of BF042 and Cy5-TNFα (100 nM) with the addition of sortilin binding agent SB013 (1.25, 5.0, and 10 μM) or DMSO. [Figure 24H]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 is shown. [Figure 25] 1 shows the efficacy of TNFα degraders in an in vivo model of LPS-induced acute systemic inflammation.

[0016] definition The term "alkyl," as used herein, refers to a straight-chain or branched-chain hydrocarbon moiety.

[0017] As used herein, the term "alkoxy" refers to a group of the formula -O-alkyl, where "alkyl" is as defined above. In particular, C1-C3-alkoxy is intended to indicate such a hydrocarbon group having 1, 2, or 3 carbon atoms. Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, and isopropoxy.

[0018] As used herein, the term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced with halogen atoms, e.g., one or more hydrogen atoms are replaced with either F, Cl, Br, or I.

[0019] As used herein, the term "cycloalkyl" or "carbocycle" refers to a monocyclic or polycyclic system. As used herein, the term "cycloalkyl" can also optionally include one or more unsaturated groups or substituents.

[0020] 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(s). As used herein, the term "heteroaromatic" or "heteroaryl," alone or in combination, refers to a saturated or unsaturated, aromatic ring containing 5 to 6 ring atoms, wherein at least one ring atom is a heteroatom(s). "Heteroatom" is intended to mean sulfur, oxygen, or nitrogen.

[0021] The terms "aromatic" or "aryl" refer to a cyclic or polycyclic moiety having a conjugated, unsaturated (4η+2) π-electron system, sometimes referred to as a delocalized π-electron system, where n is a positive integer.

[0022] The term "alkenyl" embraces radicals having at least one carbon-carbon double bond.

[0023] As used herein, the term "substituent" or "substituted," alone or in combination, refers to a group that can be used to replace hydrogen. A substituted molecule may itself be further substituted in some embodiments of the present invention. As referred to herein, a "substituent derived from" refers to a group of atoms derived from a particular molecule or formula at any position of said molecule or formula. In some embodiments, a substituent derived from a molecule is the corresponding molecule with a hydrogen atom removed. For example, a substituent derived from CH4 can be -CH3.

[0024] 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 affinity of the binding.

[0025] As described herein, ternary complex is a complex that comprises three different molecules that are bound together.As described herein, bifunctional compound can form a ternary complex between Sortilin and target molecule.This means that Sortilin is bound to the target protein and the bifunctional compound at the same time as the three-member complex.

[0026] An extracellular molecule or protein as described herein refers to a molecule or protein that is not completely enclosed within a cell, which means, for example, a protein that is completely outside the cell, but also a membrane-bound protein or a membrane-bound protein that has an extracellular domain.

[0027] As used herein, TNF-alpha may be referred to as TNFa, TNF-a, TNF-α, TNF-alpha or TNF-alpha. DETAILED DESCRIPTION OF THE INVENTION

[0028] In one main aspect, the present invention provides a bifunctional compound having a structure according to formula (I): T L -L I -S L (I) (In the formula, S L is the moiety that binds to sortilin; L I is a linker or bond; T L is the moiety that binds to the extracellular target molecule) or a pharmaceutically acceptable salt thereof.

[0029] The compounds of formula (I) function to bind extracellular target molecules or proteins of interest to Sortilin in a ternary complex, which then recruits the target molecules or proteins to the lysosomal pathway of the cell, leading to degradation of the target molecules or proteins. Thus, the present invention relates to a platform of chemical compounds that can effect targeted degradation of extracellular disease targets by binding to the Sortilin receptor.

[0030] Advantages of the present invention include, for example: Ability to induce targeted degradation of extracellular disease proteins.

[0031] Ability to target a broad disease space due to the wide range of body compartments where sortilin is expressed, including the bloodstream, CNS, PNS, CSF, cells of the immune system, and tumor subtypes.

[0032] The inventors have demonstrated the ability to build such a platform by generating bifunctional compounds according to formula (I) in multiple forms, from small molecules to large molecules and through medium-sized peptides.

[0033] Sortilin Binding: Sortilin (SEQ ID NO: 1) is a membrane protein expressed in most tissues that promotes the lysosomal degradation of several known ligands [4][5][6]. Structural studies by several independent investigators have shown that sortilin interacts with ligands inside the cavity of a large, 10-bladed β-propeller, releasing cargo after maturation and acidification of endosomal vesicles, leading to lysosomal degradation of the cargo and recycling of sortilin back to the plasma membrane

[12] . In summary, sortilin is a well-described lysosomal trafficking receptor with multiple distinct high-affinity small molecule binding partners. The present disclosure relates to a new platform of bifunctional compounds that can target Sortilin to recruit target molecules or proteins and internalize them to the lysosomal compartment, where they are degraded under lysosomal conditions. Thus, a large number and variety of different binders of Sortilin can be used.

[0034] Due to the potential for targeting sortilin inhibition as a therapeutic approach for conditions, there are numerous disclosures of sortilin binding agents in the art, based on small molecule scaffolds as well as peptides and proteins.

[0035] small molecule 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] ). Subsequently, several promising scaffolds for developing sortilin binders have been proposed, such as Formula IV in WO2014 / 114779, Formulas II and VI in Stachel et al. 2020

[10] , or Andersen 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), phenylamidic acid of benzyl-substituted glutaric acid (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 references regarding protein scaffolds are incorporated herein by reference. [ka] Thus, in one embodiment, a bifunctional compound according to the present invention can bind to sortilin via a substituent derived from the above structure or a derivative thereof.

[0036] In one embodiment, the sortilin binding moiety (S L ) has a structure according to formula (II): [ka] (In the formula, R 1 is heteroaryl, aryl, heterocycle, C3-C 10 Cycloalkyl, -(C1-C5 alkyl)-aryl, and C1-C 10 -alkylalkyl, -BO-aryl, each of which may be selected from the group consisting of one or more identical or different substituents R IIa B is an optionally substituted C1-C5 alkyl; R 2 is C1-C 10 Alkyl and C2-C 10 alkenyl, each of which may be selected from the group consisting of one or more identical or different substituents R IIb is optionally replaced by; R IIa is selected from the group consisting of -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 the group consisting of H, -O-CH2-C(=O)O-, -O-CH2-C(=O)NH-, halogen, alkoxy, -CF3, and optionally substituted C1-C4 alkyl; R IIb is C1-C 10 Alkoxy, C3-C 10 Cycloalkyl, C1-C 10 selected from the group consisting of alkyl, and -CH2-aryl; where S L teeth, 1 or R IIa via L I conjugated to

[0037] In one embodiment, R 1 are two identical R IIa In one embodiment, R 1 are two different R IIa In one embodiment, R 1 Only one of the above R IIa It has a substituent.

[0038] In one embodiment, R 1 is an optionally substituted phenyl group. In one embodiment, R 1 is a phenyl group having one or two substituents selected from the group consisting of halogen or alkoxy. 1 is a phenyl group substituted with two chlorine atoms. In a further embodiment, R 1 is a phenyl group substituted with two chlorine atoms located meta to the point of attachment to Formula II. 1 is a phenyl group having one alkoxy substituent. In a further embodiment, the alkoxy substituent is —OCH. In one embodiment, R 1 is according to formula B: [ka] (In the formula, R L L I and * represents a bond to formula II).

[0039] In one embodiment, R 2 is C1-C6 alkyl. In one embodiment, R 2 is a substituted C alkyl, wherein the C alkyl is substituted with an optionally substituted C-C cycloalkyl. In one embodiment, R 2 is a substituted C alkyl, wherein the C alkyl is substituted with a C cycloalkyl. In one embodiment, R 2 is tert-butyl. In one embodiment, R 2 is -CH2-C-(CH3)3.

[0040] In one embodiment, the sortilin binding moiety (S L ) is the formula [ka] (III) having the structure: (In the formula, Q 1 is a bond or -CH2-; R 3 is of formula (IIIa), formula (IIIb), or formula B; [ka] During the ceremony, R 3a is selected from the group consisting of H, halogen, alkoxy, —CF3, and optionally substituted C1-C4 alkyl; In the formula, * represents a bond to formula (III), and R L L I (represents a bond with

[0041] As described herein, R L L I and S L The connection point between, or T L and L I Represents the connection point between

[0042] In some embodiments, S L is according to any one selected from the group consisting of formulas IIIc, IIId, IIIe, IIIf, IIIg, and IIIh: [ka] (In the formula, R 3a is selected from the group consisting of H, halogen, alkoxy, —CF3, and optionally substituted C1-4 alkyl; R L L I Represents a bond with

[0043] In one embodiment, the bifunctional compound according to the present disclosure is S according to formula IIIc L In one embodiment, the bifunctional compound according to the present disclosure has the formula S L In one embodiment, the bifunctional compound according to the present disclosure has the formula S L In one embodiment, the bifunctional compound according to the present disclosure has the formula S L In one embodiment, the bifunctional compound according to the present disclosure has the formula IIIg: L In one embodiment, the bifunctional compound according to the present disclosure has the formula IIIh L In each of formulas IIIc, IIId, IIIe, IIIf, IIIg, and IIIh, R 3a is selected from the group consisting of H, halogen, alkoxy, —CF, and optionally substituted C alkyl; R L is L as described in formula (I) I Represents a bond to

[0044] In one embodiment, R 3a is H. In another embodiment, R 3a is halogen or -CF. In one embodiment, R 3a is alkoxy. In yet another embodiment, R 3a is an optionally substituted C1-4 alkyl.

[0045] In some embodiments, S L has a structure according to any one of formulas IIIi, IIIj, IIIk, IIIm, IIIn, and IIIo. [ka] R L L I Represents a bond with

[0046] In one embodiment, the bifunctional compound according to the present disclosure is S according to formula IIIi L In one embodiment, the bifunctional compound according to the present disclosure has the formula IIIjL In one embodiment, the bifunctional compound according to the present disclosure has the formula IIIk L In one embodiment, the bifunctional compound according to the present disclosure has the formula IIIm L In one embodiment, the bifunctional compound according to the present disclosure has the formula S L In one embodiment, the bifunctional compound according to the present disclosure has the formula S L In formulae IIIi, IIIj, IIIk, IIIm, IIIn, and IIIo, R L is L as described in formula (I) I Represents a bond to

[0047] In another embodiment, the sortilin binding moiety (S L ) has a structure according to formula (DI): [ka] (In the formula, R L L I represents a bond to

[0048] In another embodiment, the sortilin binding moiety (S L ) has a structure according to formula (D-II): [ka] (In the formula, R L L I represents a bond to

[0049] The structures according to Formula III and IIIa-IIIo described herein have a stereocenter at the α position of the carboxylic acid group, as indicated below by "α" in Formula III: [ka] The stereocenter can exist in one of two configurations: R or S. These formulae III and IIIa-IIIo exist in either the S-stereoisomeric configuration or the R-stereoisomeric configuration.

[0050] Thus, in one embodiment, S L is according to the S stereoisomer of any one of formulas III or IIIa-IIIo described herein.

[0051] In another embodiment, S L is according to the R stereoisomer of any one of formula III or IIIa-IIIo described herein.

[0052] In another embodiment, the sortilin binding moiety (S L ) has a structure according to formula (IV): [ka] (R 4 is H or F; R 4’ is H; R 5 is halogen, H, C1-C6 alkyl, C2-6 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 aromatic monocyclic ring having 1 or 2 heteroatom(s), wherein the aromatic heterocycle is optionally substituted with 1 or 2 substituents individually selected from the group consisting of: -CN; C-C alkyl; halogenated C-C alkyl; C-C alkoxy; halogen; -C(O)NH-C-C alkyl; aryl optionally substituted with -C(O)NH-C-C alkyl, and optionally substituted heteroaryl. or a pharmaceutically acceptable salt thereof (wherein, SL is R 7 via L I conjugated).

[0053] In one embodiment, R 5 is halogen, H, or C1-C6 haloalkyl; R 6 is halogen, H, C1-C6 alkyl, or C1-C6 haloalkyl.

[0054] In another embodiment, R 4 is H and R 5 is -CF3 or H; R 6 is -CF3 or H, and R 4 ' is H.

[0055] In one embodiment, Q 2 is a bond. 2 is -CH2-.

[0056] In one embodiment, R 7 is a 6-membered aromatic heterocycle having one heteroatom substituted with C alkyl, said aromatic heterocycle being optionally further substituted with an optionally substituted aryl; S L is R 7 via L I It is conjugated to

[0057] In one embodiment, Q 2 is a bond and R 4 is H and R 5 is -CF3 or H, and R 6 is -CF3 or H, and R 4 ' is H, Q 2 is a bond and R 7 is a 6-membered aromatic heterocycle having one heteroatom substituted with C alkyl, said aromatic heterocycle being optionally further substituted with an optionally substituted aryl; S L is R 7 via LI It is conjugated to

[0058] In one embodiment, R 5 or R 6 At least one of R is —CF. 5 or R 6 At least one of the groups is —CF3 and the other is hydrogen.

[0059] In one embodiment, R 7 has a structure according to Formula IVa or Formula IVb: [ka] (where * represents a bond to formula (IV), R L L I represents a bond to

[0060] In another embodiment, S L is selected from any one of the group consisting of formulas IVc, IVd, IVe, and IVf: [ka] (In the formula, R L L I represents a bond to

[0061] In one embodiment, the bifunctional compound according to the present disclosure is S according to formula IVc L In one embodiment, the bifunctional compound according to the present disclosure has the formula IVd: L In one embodiment, the bifunctional compound according to the present disclosure has the formula IVe: L In one embodiment, the bifunctional compound according to the present disclosure has the formula IVf: L In formulas IVc, IVd, IVe, and IVf, R L L I Represents a bond to

[0062] In another embodiment, the sortilin binding moiety (SL ) is a substituent derived from a compound of formula VI: [ka] (In the formula, R 8 is C1-C 10 is alkyl; R 9 is C1-C 10 selected from the group consisting of alkyl, aryl, and heteroaryl, each of which may be one or more identical or different substituents R VIa is optionally replaced by; R VIa is selected from the group consisting of aryl, heteroaryl, -O-aryl, -O-heteroaryl, and halogen, wherein each of the aryl, heteroaryl, -O-aryl, and -O-heteroaryl is optionally substituted with one or more halogen(s).

[0063] In one embodiment, R 8 is C1-C5 alkyl. In one embodiment, R 8 is tert-butyl. In another embodiment, R 8 is -CH2-C-(CH3)3.

[0064] In one embodiment, R 9 is optionally substituted aryl. In one embodiment, R 9 is optionally substituted benzyl. In one embodiment, R 9 is -C1-C3-aryl.

[0065] In one embodiment, R 9 teeth, [ka] is one selected from the group consisting of wherein * represents a bond to formula VI.

[0066] In one embodiment, R9 is one or two substituents R VIa In a further embodiment, one or two substituents R VIa is a halogen.

[0067] In one embodiment, Formula VI is R 9 via the linker moiety (L I )

[0068] In another embodiment, the sortilin binding moiety (S L ) is a substituent derived from a compound of formula VI: [ka] (In the formula, R 10 teeth, [ka] and; R 11 is an optionally substituted heteroaryl or an optionally substituted aryl).

[0069] In one embodiment, R 11 is a pyridyl group. In one embodiment, Formula VII is 11 via the linker moiety (L I )

[0070] In one embodiment, the sortilin binding moiety (S L ) is a substituent derived from the following compound: [ka]

[0071] In one embodiment, the sortilin binding moiety (S L ) is a substituent derived from the following compound: [ka]

[0072] In one embodiment, the sortilin binding moiety (S L ) is a substituent derived from the following compound: [ka]

[0073] In another embodiment, the sortilin binding moiety (S L ) is a substituent derived from a compound of formula VIII or formula IX: [ka] (In the formula, R VIIIa is a carboxylic acid or ester thereof, where any of the carbons of the norbornene ring is C 1-6 may be substituted with an alkyl group or the carboxylic acid may be C 1-5 It may also be condensed as an ester.

[0074] In the formula, R IXa is an optionally substituted divalent C1-C5 alkyl, in which one or more methylene groups are optionally replaced with -CO(NH)-; R IXb is a group selected from the group consisting of hydroxyl, alkoxy, amino, or aminoalkyl).

[0075] In one embodiment, the sortilin binding moiety (S 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.

[0076] In one embodiment, the sortilin binding moiety (S 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.

[0077] In one embodiment, the sortilin binding moiety (S 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.

[0078] In one embodiment, the sortilin binding moiety (S 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.

[0079] In another embodiment, the sortilin binding moiety (S L ) is a substituent derived from a compound of formula X: [ka] (In the formula, R Xa and R Xb independently, C 1- C5 alkyl, acyl, amino, sulfono, chloro, bromo, iodo, or fluoro; R Xc C 1- C5 alkyl or the acid is replaced with tetrazole).

[0080] In one embodiment, the sortilin binding moiety (S 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.

[0081] In another embodiment, the sortilin binding moiety (S L ) is a substituent derived from a compound of formula XI: [ka] (In the formula, R XIa is C 1-5 is alkyl, acyl, amino, sulfono, chloro, bromo, iodo, or fluoro; R XIb is a bond or C optionally substituted with C1-C5 alkyl 1- is a C3 alkyl, R XIc is a carboxylic acid, a C1-C5 ester of a carboxylic acid, or a tetrazole).

[0082] In one embodiment, the sortilin binding moiety (S L ) is a substituent derived from the compound 3-oxo-1,2,3,4-tetrahydro-2-quinoxalinyl)acetic acid.

[0083] Other sortilin binding agents are described in WO2023 / 031440, which is incorporated herein by reference, such as those described by formula (A): [ka] (In the ceremony A 1 , A 2 , and A 3 are each independently selected from the group consisting of halogen, H, C1-C4 alkyl, C1-C4 haloalkyl, C2-C5 alkenyl, and C2-C5 haloalkenyl; A 4 is selected from the group consisting of 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; A5 is C3-C 20 Aryl, C3-C 20 heteroaryl, and 3- to 12-membered heterocycle; wherein the aryl, heteroaryl, or heterocyclic ring is optionally substituted with one or more substituents independently selected from halogen, —OH, cyano, carbonyl, C1-C4 alkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, C3-C8 aryl, and C3-C8 heteroaryl; or A 4 and A 5 together form a 6- to 20-membered heterocyclic ring, The heterocycle is monocyclic, bicyclic, or tricyclic and is optionally substituted with one or more substituents independently selected from halo, —OH, cyano, carbonyl, C1-C4 alkyl, C1-C4 haloalkyl, acetyl, C1-C4 haloalkoxy, C1-C4 haloalkoxy).

[0084] For example, the following compound: [ka] Thus, in one embodiment, a bifunctional compound according to the present invention can bind to sortilin via a substituent derived from the above structure or a derivative thereof.

[0085] In one embodiment, the sortilin binding moiety (S L ) is a substituent derived from a compound according to formula (A) above.

[0086] In one embodiment, the sortilin binding moiety (S L ) according to any one of the formulae AI to A-III: [ka] or a derivative thereof, wherein R L L I Represents a bond to

[0087] In one embodiment, the sortilin binding moiety (S L ) is according to formula AI. In one embodiment, the sortilin binding moiety (S L ) is according to Formula A-II. In one embodiment, the sortilin binding moiety (S L ) is according to formula A-III.

[0088] Other sortilin binding agents are described in WO2023 / 101595, the contents of which are incorporated herein by reference, and are described, for example, by formula (E): [ka] (In the formula, Ring E A is a 5- or 6-membered aromatic, heteroaromatic, or heterocyclic ring having 0-2 heteroatom(s) selected from among N, O, and S, or an 8-10-membered bicyclic, heterocyclic, or heteroaromatic ring having 1 or 2 heteroatom(s) selected from among N, O, and S, wherein the aromatic, heteroaromatic, heterocyclic, or bicyclic ring A is optionally selected from C1-C4 alkyl, -N(E 1 )(E 2 ), -C(=O)C(E Z )3, -OE 4 substituted with one or more substituents independently selected from the group consisting of , halogen, and ═O; E Y is absent or -O-, -OCH2-, -CH2-, -NE 3 - or -CH(NH2)-; Ring E B is a 5- or 6-membered aromatic or heteroaromatic ring having no or 0-4 heteroatom(s) selected from N, O, and S, and the aromatic or heteroaromatic ring E B is C1-C4 alkyl, -N(E 1 )(E 2 ), -C(=O)C(EZ )3, -OE 4 optionally substituted with one or more substituents independently selected from the group consisting of: , halogen, and =O; E 1 , E 2 , E 3 , and E 4 are each independently selected from halogen or C1-C4 alkyl; E Z is a halogen; E 5 is hydroxyl or C1-C4 alkoxy.

[0089] In one embodiment, the sortilin binding moiety (S L ) is a substituent derived from a compound according to formula (E) above.

[0090] In one aspect, the present invention provides novel sortilin ligands that have high affinity for sortilin. Accordingly, one embodiment of the present disclosure provides a compound according to Formula III, or a pharmaceutically acceptable salt thereof: [ka] (In the formula, Q 1 is a bond or -CH2-; R 3 is of formula (IIIp), formula (IIIq), or formula B-II; [ka] (In the formula, R 3b is H, halogen, alkoxy, -CF3, and optionally substituted C 1-5 alkyl, C 1- One or more methylene groups of the C5 alkyl may optionally be individually -O-, -NH-, -C(O)-, ester, amide, carbamate, thiourea, sulfonamide, urea 、 , [ka] optionally substituted carbocycles; optionally substituted heterocycles, and [ka] wherein X is NH or O; and wherein * represents a bond to formula (III).

[0091] In one embodiment, R 3b is R 3 is of formula (IIIq), then it is not H.

[0092] In one embodiment, Q 1 is -CH2-. In one embodiment, Q 1 is a bond.

[0093] In one embodiment, R 3 is of formula IIIr: [ka] (In the formula, R 3b is H, halogen, alkoxy, -CF3, and optionally substituted C 1-5 alkyl, C 1-5 One or more methylene groups of the alkyl may optionally be independently selected from -O-, -NH-, -C(O)-, ester, amide, carbamate, thiourea, or [ka] and wherein * represents a bond to formula (III).

[0094] In one embodiment of the disclosure, the compound is selected from Formula IIIs, Formula IIIt, or Formula B-III: [ka] (In the formula, R 3bis H, halogen, alkoxy, -CF3, and optionally substituted C 1-5 alkyl, C 15 One or more methylene groups of the alkyl may optionally be individually replaced by -O-, -NH-, -C(O)-, ester, amide, carbamate, thiourea, sulfonamide, urea 、 , [ka] optionally substituted carbocycles; optionally substituted heterocycles, and [ka] wherein X is replaced by one or more of the group consisting of NH or O.

[0095] In one embodiment, R 3b is H.

[0096] In one embodiment, R 3b is selected from halogen or —CF 3 .

[0097] In one embodiment, R 3b is optionally replaced by C 1- C5 alkyl, C 1-5 One or more methylene groups of the alkyl may optionally be individually selected from -O-, -NH-, -C(O)-, esters, amides, carbamates, thioureas, and [ka] is replaced with one or more of the group consisting of:

[0098] In one embodiment, at least one of the methylene groups is selected from the group consisting of -O-, -NH-, -C(O)-, esters, amides, carbamates, thioureas, and [ka] is replaced with one or more of the group consisting of:

[0099] In one embodiment, R 3b is C 1- C5 alkyl, C 1-5 One or more methylene groups of the alkyl may optionally be individually selected from -O-, amide, and [ka] is replaced with one or more of the group consisting of:

[0100] In one embodiment, R 3b is C 1- C5 alkyl, C 1-5 One or more methylene group(s) of the alkyl are optionally individually replaced with -O-.

[0101] In one embodiment, R 3b is C 1- C5 alkyl, C 1-5 One or more methylene groups of the alkyl are optionally individually replaced with an amido.

[0102] In one embodiment, R 3b is C 1- C5 alkyl, C 1-5 One or more methylene groups of the alkyl may optionally be individually [ka] has been replaced with

[0103] In one embodiment, R 3b is C 1- C5 alkyl, C 1-5 One or more methylene groups of an alkyl may optionally be individually optionally substituted carbocyclic rings, e.g., [ka] wherein n is an integer selected from 0, 1, 2, or 3.

[0104] In one embodiment, R 3b is C 1- C5 alkyl, C 1-5 One or more methylene groups of an alkyl may optionally be individually optionally substituted heterocycles, e.g., [ka] wherein n is an integer selected from 0, 1, 2, or 3; or [ka] is replaced by a heterocycle according to

[0105] In one embodiment, R 3b is -O-CH2-C(=O)N(H)R IIIa where R IIIa is selected from H and C1-C6 alkyl.

[0106] In one embodiment, R 3b is -O-CH2-CO2R IIIa where R IIIa is selected from H and C1-C6 alkyl.

[0107] In one embodiment, R 3b is C 1- C5 alkyl, C 1-5 One or more methylene groups of the alkyl are optionally individually replaced with one of the groups shown in Table Z in the "Linker" section. In one embodiment, the compound is an S-stereoisomer based on the configuration of the carbon alpha to the carboxylic acid.

[0108] In one embodiment, the compound is an R-stereoisomer, based on the configuration of the carbon alpha to the carboxylic acid.

[0109] In one embodiment, the disclosure provides the following compound: [ka] In another embodiment, the present disclosure provides the following compound: [ka] or a pharmaceutically acceptable salt thereof.

[0110] In one embodiment, the present disclosure provides the following compound: [ka] or a pharmaceutically acceptable salt thereof.

[0111] In one aspect, the present disclosure provides a compound according to any one of the following: [ka] or a pharmaceutically acceptable salt thereof.

[0112] The present invention demonstrates several bifunctional compounds that can bind to sortilin. As shown in the examples, these compounds have affinity for sortilin. As will be appreciated by those skilled in the art, S L , and L I Fragments of the bifunctional compounds that encompass part or all of Sortilin also function as sortilin binding agents. Thus, in one embodiment, the present disclosure provides a bifunctional compound comprising S L -L I The present invention provides a sortilin binding agent, or fragment thereof, described by any combination of:

[0113] In one embodiment, the present invention provides a compound according to any one of formulas IIIt or B-III, wherein R 3b is a fragment of any one of the structures shown in Table Z-III in the "Linker" section.

[0114] Additionally, the present disclosure provides compounds capable of binding to sortilin, wherein said compounds are intermediates in the preparation of bifunctional compounds as described in the "Synthetic Protocols" section herein.

[0115] peptide Peptides have also been shown to bind to sortilin, such as 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 can also bind to sortilin; for example, the C-terminal sequence of the natural sortilin binder progranulin (SEQ ID NO: 3) has been shown to be essential for the binding of progranulin to sortilin.

[0116] Other examples of synthetic peptides have been used to target sortilin, such as the peptides described in WO2020 / 037434: IKLSGGVQAKAGVINMDKSESSM (SEQ ID NO: 7) IKLSGGVQAKAGVINMFKSESY (SEQ ID NO: 8) IKLSGGVQAKAGVINMFKSESYK (SEQ ID NO: 9) GVQAKAGVINMFKSESY (SEQ ID NO: 10) GAKAGVRNMFKSESYVR (SEQ ID NO: 11) GAKAGVRN(Nle)FKSESY (SEQ ID NO: 12) YKSLRRKAPRWDAPLRDPALRQLL (SEQ ID NO: 13) YKSLRRKAPRWDAYLRDPALRQLL (SEQ ID NO: 14) YKSLRRKAPRWDAYLRDPALRPLL (SEQ ID NO: 15) The sortilin binding group (S L ) can also be a peptide.

[0117] There are several proteins that have affinity for sortilin. Therefore, in one embodiment of the present disclosure, the sortilin binding group (S L ) includes peptide fragments of the amino acid sequences of such proteins.

[0118] 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: 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 having at least 60% sequence identity, such as at least 80%, for example at least 90%, for example at least 95% sequence identity to the corresponding original protein fragment of any one of a) to e).

[0119] In one embodiment, S L comprises or consists of a peptide fragment of the amino acid sequence of Progranulin (SEQ ID NO: 3), or a variant of said fragment having at least 60% sequence identity with the corresponding original fragment of SEQ ID NO: 3, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity.

[0120] In one embodiment, S L comprises or consists of a peptide fragment of the amino acid sequence of Progranulin (SEQ ID NO: 3) or a variant of said fragment having 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, for example at most 1 amino acid substitution compared to the corresponding original fragment of SEQ ID NO: 3.

[0121] In one embodiment, S Lcomprises or consists of a peptide fragment of the amino acid sequence of neurotensin (SEQ ID NO: 4), or a variant of said fragment having at least 60% sequence identity, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity compared to the corresponding original fragment of SEQ ID NO: 4.

[0122] In one embodiment, S L comprises or consists of a peptide fragment of the amino acid sequence of neurotensin (SEQ ID NO: 4) or a variant of said fragment having 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, for example at most 1 amino acid substitution compared to the corresponding original fragment of SEQ ID NO: 4.

[0123] In one embodiment, S L comprises or consists of a peptide fragment of the amino acid sequence of ApoB (SEQ ID NO: 176), or a variant of said fragment having at least 60% sequence identity, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity, compared to the corresponding original fragment of SEQ ID NO: 176.

[0124] In one embodiment, S L comprises or consists of a peptide fragment of the amino acid sequence of ApoB (SEQ ID NO: 176) or a variant of said fragment having 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, for example at most 1 amino acid substitution compared to the corresponding original fragment of SEQ ID NO: 176.

[0125] In one embodiment, S Lcomprises or consists of a peptide fragment of the amino acid sequence of BDNF (SEQ ID NO: 177), or a variant of said fragment having at least 60% sequence identity, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity, compared to the corresponding original fragment of SEQ ID NO: 177.

[0126] In one embodiment, S L comprises or consists of a peptide fragment of the amino acid sequence of BDNF (SEQ ID NO: 177) or a variant of said fragment having 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, for example up to 1 amino acid substitution compared to the corresponding original fragment of SEQ ID NO: 177.

[0127] In one embodiment, S L comprises or consists of a peptide fragment of the amino acid sequence of NGF (SEQ ID NO: 178), or a variant of said fragment having at least 60% sequence identity, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity, compared to the corresponding original fragment of SEQ ID NO: 178.

[0128] In one embodiment, S L comprises or consists of a peptide fragment of the amino acid sequence of NGF (SEQ ID NO: 178) or a variant of said fragment having 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, for example at most 1 amino acid substitution compared to the corresponding original fragment of SEQ ID NO: 178.

[0129] In one embodiment, S L is a peptide comprising a sequence selected from the group consisting of: H-PYMKLAPGELTIIL-OH (SEQ ID NO: 44), H-NEKLSQLQTYMI-OH (SEQ ID NO: 45), H-KDADLYTSRVMLSSQVP-OH (SEQ ID NO: 46) H-RLFKKRRLRSPRVLF-NH2 (SEQ ID NO: 47), H-ITVDPRLFKKRRLRSPRVLF-NH2 (SEQ ID NO: 48), H-ITVDPRLFKKRRLRSPRVLFSTQPPR-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) WSGPIGVSWGLRAAAAGFQLL-OH (SEQ ID NO: 179).

[0130] S L comprises or consists of a peptide, L I may be attached to any amino acid residue of the peptide. L If contains a peptide, L I is linked to any free amino group of the peptide.

[0131] In a preferred embodiment, S L comprises or consists of a peptide, L I is attached to the N-terminus of the peptide.

[0132] The present inventors have shown that the C-terminal tetrapeptide RQLL (SEQ ID NO: 1) of Progranulin is sufficient to bind to Sortilin and promote lysosomal degradation of Progranulin. Furthermore, the present inventors have shown that several peptide analogs with selected substitutions in the RQLL sequence are good binders of Sortilin.

[0133] Thus, in one embodiment, S L comprises or consists of a peptide comprising SEQ ID NO: 16 below.

[0134] X5-X1X2X3X4 (SEQ ID NO: 16) (In the formula, X5 is any amino acid residue or peptide containing 2 to 30 amino acid residues or bonds; 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 a conservative substitution for M, K, L, or L).

[0135] In one embodiment, X5 is any amino acid residue or peptide comprising at least 2 amino acid residues, such as at least 3, for example at least 4, such as at least 5, for example at least 6, such as at least 7, for example at least 8, such as at least 9, for example at least 10, such as at least 11, for example at least 12, such as at least 13, for example at least 14, such as at least 15, for example at least 20, such as at least 25 amino acid residues, for example at least 28 amino acid residues, such as at least 30 amino acid residues.

[0136] In one embodiment X5 is a peptide comprising 32 or less, such as 29 or less, for example 28 or less, such as 27 or less, for example 26 or less, such as 25 or less, for example 24 or less, such as 23 or less, for example 22 or less, such as 21 or less, for example 20 or less, such as 15 or less, for example 10 or less, for example 5 or less amino acid residues.

[0137] In one embodiment, X5 is a peptide fragment of the amino acid sequence of a protein selected from the group consisting of: 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 (SEQ ID NO: 178) or a variant of said fragment having at least 60% sequence identity, such as at least 80%, for example at least 90%, for example at least 95% sequence identity to the corresponding original protein fragment of any one of a) to e).

[0138] In one embodiment, 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 having at least 60% sequence identity, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity, compared to the corresponding original fragment of SEQ ID NO: 3.

[0139] In one embodiment, 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 having up to 5 amino acid substitutions compared to the corresponding original fragment of SEQ ID NO: 3, such as up to 4 amino acid substitutions, such as up to 3 amino acid substitutions, such as up to 2 amino acid substitutions, for example up to 1 amino acid substitution.

[0140] In one embodiment, 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 having at least 60% sequence identity, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity, compared to the corresponding original fragment of SEQ ID NO: 4.

[0141] In one embodiment, 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 having at most 5 amino acid substitutions compared to the corresponding original fragment of SEQ ID NO: 4, such as at most 4 amino acid substitutions, such as at most 3 amino acid substitutions, such as at most 2 amino acid substitutions, for example at most 1 amino acid substitution.

[0142] In one embodiment, X5 is selected from the group consisting of: 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 APLRDAP (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.

[0143] In one embodiment, X5 is a bond.

[0144] In one embodiment, X5 is APLRDAP (SEQ ID NO: 21). In one embodiment, X5 is REAPRWDAPLRDPAL (SEQ ID NO: 17). In one embodiment, X5 is REALRWDAPLRDPAP (SEQ ID NO: 18).

[0145] In one embodiment, X 5 is an amino acid residue selected from L, T, or Y. In one embodiment, X 5 is PYILKRQLYENKPRR (SEQ ID NO: 19). 5 is LYENKPR (SEQ ID NO: 20).

[0146] In one embodiment, X 1 is F or R. In one embodiment, X 2 is Q. In another embodiment, X 3 is L, and in one embodiment, X 4 is L.

[0147] In one embodiment, 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; X4 is L.

[0148] In one embodiment, 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; X4 is L.

[0149] In one embodiment, X1 is selected from Y, F, or R; X2 is selected from Q or Y; X3 is L, X4 is L.

[0150] In one embodiment, X1 is selected from F or R; X2 is Q, X3 is L, X4 is L.

[0151] In one embodiment, S L comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 22 to 65, 182 to 187, 196, and 197, and L I are connected at the N-terminus.

[0152] In one embodiment, S L is a peptide comprising any of the sortilin binding sequences described herein at its C-terminus.

[0153] In one embodiment, S L is a peptide consisting of RQLL-OH (SEQ ID NO: 22). L is a peptide containing RQLL-OH (SEQ ID NO: 22) at the C-terminus.

[0154] In one embodiment, S Lis a peptide consisting of FQLL-OH (SEQ ID NO: 23). L is a peptide containing FQLL-OH (SEQ ID NO: 23) at the C-terminus.

[0155] In one embodiment, S L is a peptide consisting of RYLL-OH (SEQ ID NO: 27). L is a peptide containing RYLL-OH (SEQ ID NO: 27) at the C-terminus.

[0156] In one embodiment, S L is a peptide consisting of FYLL-OH (SEQ ID NO: 28). L is a peptide containing FYYL-OH (SEQ ID NO: 28) at the C-terminus.

[0157] In one embodiment, S L is a peptide consisting of YQLL-OH (SEQ ID NO: 29). L is a peptide containing YQLL-OH (SEQ ID NO: 29) at the C-terminus.

[0158] In one embodiment, S L is a peptide consisting of REAPRWDAPLRDPALRQLL-OH (SEQ ID NO: 53). L is a peptide containing REAPRWDAPLRDPALRQLL-OH (SEQ ID NO: 53) at the C-terminus.

[0159] In one embodiment, S L is a peptide consisting of REALRWDAPLRDPAPRQLL-OH (SEQ ID NO: 54). L is a peptide containing REALRWDAPLRDPAPRQLL-OH (SEQ ID NO: 54) at the C-terminus.

[0160] In one embodiment, S L is a peptide consisting of REAPRWDAPLRDPALFQLL-OH (SEQ ID NO: 58). Lis a peptide containing REAPRWDAPLRDPALFQLL-OH (SEQ ID NO: 58) at the C-terminus.

[0161] In one embodiment, S L is a peptide consisting of REAPRWDAPLRDPALRYLL-OH (SEQ ID NO: 59). L is a peptide containing REAPRWDAPLRDPALRYLL-OH (SEQ ID NO: 59) at the C-terminus.

[0162] In one embodiment, S L is a peptide consisting of REAPRWDAPLRDPALRQYL-OH (SEQ ID NO: 60). L is a peptide containing REAPRWDAPLRDPALRQYL-OH (SEQ ID NO: 60) at the C-terminus.

[0163] In one embodiment, S L is a peptide consisting of REAPRWDAPLRDPALRYYL-OH (SEQ ID NO: 61). L is a peptide containing REAPRWDAPLRDPALRYYL-OH (SEQ ID NO: 61) at the C-terminus.

[0164] In one embodiment, S L is a peptide consisting of APLRDPAPRQLL-OH (SEQ ID NO: 57). L is a peptide containing APLRDPAPRQLL-OH (SEQ ID NO: 57) at the C-terminus.

[0165] In one embodiment, S L is a peptide consisting of PYILKRQLYENKPRRPYIL-OH (SEQ ID NO: 55). L is a peptide containing PYILKRQLYENKPRRPYIL-OH (SEQ ID NO: 55) at the C-terminus.

[0166] In one embodiment, S Lis a peptide consisting of LYENKPRRPYIL-OH (SEQ ID NO: 56). L is a peptide containing LYENKPRRPYIL-OH (SEQ ID NO: 56) at the C-terminus.

[0167] In one embodiment, S L is a peptide consisting of AARL-OH (SEQ ID NO: 196). L is a peptide containing AARL-OH (SEQ ID NO: 196) at the C-terminus.

[0168] In one embodiment, S L is a peptide consisting of PIPLV-OH (SEQ ID NO: 197). L is a peptide containing PIPLV-OH (SEQ ID NO: 197) at the C-terminus.

[0169] In one preferred embodiment, L I has S at the N-terminus L is connected to.

[0170] In one embodiment, S L 1 to 4 amino acid residues thereof are substituted with other amino acids, for example, 1 amino acid, for example, 2, for example, 3, for example, 4 amino acids are substituted.

[0171] In one embodiment, S L In one embodiment, one amino acid residue of S L Two amino acid residues have been substituted.

[0172] In one embodiment, the substitution is a conservative amino acid substitution. In another embodiment, the substitution is with a non-naturally occurring amino acid. Thus, in one embodiment, S L In another embodiment, S can have one substitution with one unnatural amino acid. L can have two substitutions with unnatural amino acids.

[0173] In one embodiment, S L1 to 4 amino acid residues are chemically modified, for example, 1 amino acid residue, for example 2, for example 3, for example 4 amino acids are modified.

[0174] In one embodiment, the chemical modification can be any chemical modification. In one embodiment, the chemical modification is selected from the group consisting of acylation, amidation, acetylation, esterification, and / or alkylation. Thus, S L In another embodiment, S may have one chemically modified amino acid. L may have two chemically modified amino acids.

[0175] In one embodiment, S L is a peptide comprising 50 or less amino acid residues, such as 45 or less, for example 40 or less, such as 35 or less, for example 32 or less, such as 30 or less, for example 28 or less, such as 26 or less, for example 24 or less, such as 22 or less, for example 20 or less, such as 19 or less, for example 18 or less, such as 17 or less, for example 16 or less, such as 15 or less, for example 14 or less, such as 13 or less, for example 12 or less, such as 11 or less, for example 10 or less, such as 9 or less, for example 8 or less, for example 7 or less, for example 6 or less, such as 5 or less, for example 4 or less amino acid residues.

[0176] In one embodiment, S L is a peptide containing 28 or fewer amino acid residues. L is a peptide containing four or fewer amino acid residues.

[0177] In one embodiment, S L comprises or consists of a peptide of at least 4 amino acid residues, such as at least 5, for example at least 6, such as at least 7, for example at least 8, such as at least 9, for example at least 10, such as at least 12, for example at least 14, such as at least 16, for example at least 18, such as at least 20, at least 22, for example at least 24, at least 26, for example at least 28.

[0178] In one embodiment, S L 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.

[0179] In one embodiment, S L comprises or consists of a peptide four amino acid residues in length. L comprises or consists of a peptide five amino acid residues in length. L comprises or consists of a peptide six amino acid residues in length. L comprises or consists of a peptide seven amino acid residues in length. L comprises or consists of a peptide eight amino acid residues in length. L comprises or consists of a peptide 9 amino acid residues in length. L comprises or consists of a peptide 10 amino acid residues in length. L comprises or consists of a peptide 11 amino acid residues in length. L comprises or consists of a peptide 12 amino acid residues in length.

[0180] In one embodiment the SL comprises or consists of a peptide which is capable of binding to sortilin with a dissociation constant of less than 50 μM, such as less than 40 μM, for example less than 30 μM, such as less than 20 μM, for example less than 10 μM, such as less than 5 μM, for example less than 4 μM, such as less than 3 μM, for example less than 2 μM, such as less than 1 μM, for example less than 0.8 μM, such as less than 0.6 μM, for example less than 0.5 μM, such as less than 0.4 μM, for example less than 0.3 μM, such as less than 0.2 μM, for example less than 0.1 μM, such as less than 0.05 μM, for example less than 0.04 μM, such as less than 0.03 μM, for example less than 0.02 μM, for example less than 0.01 μM.

[0181] It is an aspect of the present invention to provide sortilin-binding peptides. In one embodiment, such peptides are Sortilin-binding peptides as disclosed herein. L by.

[0182] It is an aspect of the present invention to provide a sortilin-binding peptide. In one embodiment, the sortilin-binding peptide comprises the following sequence: X5-X1X2X3X4 (SEQ ID NO: 16) (wherein X1, X2, X3, X4, and X5 are as described herein.

[0183] In one embodiment, the peptide is 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 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 in length.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] In one embodiment the peptide according to the present disclosure has a dissociation constant (K) of less than 50 μM, such as less than 40 μM, for example less than 30 μM, such as less than 20 μM, for example less than 10 μM, such as less than 5 μM, for example less than 4 μM, such as less than 3 μM, for example less than 2 μM, such as less than 1 μM, for example less than 0.8 μM, such as less than 0.6 μM, for example less than 0.5 μM, such as less than 0.4 μM, for example less than 0.3 μM, such as less than 0.2 μM, for example less than 0.1 μM, such as less than 0.05 μM, for example less than 0.04 μM, such as less than 0.03 μM, for example less than 0.02 μM, for example less than 0.01 μM. D ) can bind to sortilin.

[0199] In one aspect, the disclosure provides an isolated polynucleotide encoding a peptide or protein described herein.

[0200] In one aspect, the present disclosure provides a vector comprising a polynucleotide described herein. In one embodiment, the vector is an expression vector, such as a bacterial or viral vector.

[0201] In one aspect, the present disclosure provides a host cell comprising a polynucleotide and / or vector described herein.

[0202] protein Bifunctional compounds according to the present invention can be prepared using any antibody or antibody fragment capable of binding to Sortilin. L may be selected from an antibody or antibody fragment capable of binding to sortilin.

[0203] Antibodies or antibody fragments capable of binding to sortilin have been disclosed, for example, in WO2017 / 009327, WO2019 / 016247, WO2021 / 263279, WO2021 / 116290, WO2020 / 252066, WO2020 / 014617, US2017 / 0218058, or WO2016 / 164637, the contents of which are incorporated herein by reference.

[0204] Thus, in one embodiment, S L is selected from one of the antibodies or antibody fragments capable of binding to Sortilin described in the above disclosures. In one embodiment, S L is selected from the antibodies or antibody fragments described in the disclosure above, and L I and / or T L is selected as described herein.

[0205] In one embodiment, S L is an antibody, antibody fragment, or nanobody capable of binding to sortilin.

[0206] In one embodiment, S L 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, for example at least 0.1 μM.

[0207] As seen above, examples of sortilin binding agents suitable for preparing bifunctional compounds according to the present disclosure are numerous. Thus, in one embodiment, the S in the bifunctional compound L The group is selected from any one of the structures, peptides, and proteins described above.

[0208] In one embodiment, the bifunctional compound according to the present disclosure has a dissociation constant (K) of less than 50 μM, such as less than 40 μM, for example less than 30 μM, such as less than 20 μM, for example less than 10 μM, such as less than 5 μM, for example less than 4 μM, such as less than 3 μM, for example less than 2 μM, such as less than 1 μM, for example less than 0.8 μM, such as less than 0.6 μM, for example less than 0.5 μM, such as less than 0.4 μM, for example less than 0.3 μM, such as less than 0.2 μM, for example less than 0.1 μM, such as less than 0.05 μM, for example less than 0.04 μM, such as less than 0.03 μM, for example less than 0.02 μM, for example less than 0.01 μM. D ) can bind to sortilin.

[0209] In one embodiment, the bifunctional compound according to the present disclosure has a concentration of 50 μM to 0.001 μM, such as 50 μM to 40 μM, for example 40 μM to 30 μM, for example 30 μM to 20 μM, such as 20 μM to 10 μM, for example 10 μM to 5 μM, for example 5 μM to 4 μM, such as 4 μM to 3 μM, for example 3 μM to 2 μM, for example 2 μM to 1 μM, for example 1 μM to 0.9 μM, for example a dissociation constant (K D ) can bind to sortilin.

[0210] Targeted Warheads: Bifunctional compounds according to the present disclosure comprise a moiety capable of binding to an extracellular target molecule or protein, such as a growth factor, cytokine, hormone, lipoprotein, neurotransmitter, capsid, extracellular secreted protein, antibody, etc.

[0211] In one embodiment, the extracellular target molecule is a protein.The extracellular protein described herein refers to a protein that is not completely enclosed inside a cell.This means, for example, a protein that is completely outside a cell, but also a membrane-bound protein or a membrane-bound protein that has an extracellular domain.

[0212] The bifunctional compounds according to formula (I) bind to a targeting ligand (T L ) binds to an extracellular target molecule. In one embodiment, T L is a substituent of a small organic molecule (i.e., a non-biological substance) that binds sufficiently 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 binding fragment thereof that binds sufficiently to an extracellular target molecule.

[0213] T LThe moiety can be, for example, but not limited to, a substituent derived from an approved drug or a clinical stage drug, or a substituent derived from a compound being reviewed as a drug by a regulatory agency such as the FDA or EMA.

[0214] Extracellular target proteins exert beneficial therapeutic effects through their degradation. L The target protein may be any amino acid sequence to which a bifunctional compound comprising: can bind. In one embodiment, the target protein is a non-endogenous peptide, such as one derived from a pathogen or toxin. In another embodiment, the target protein may be an endogenous protein that mediates a disorder. The endogenous protein may be either a normal or abnormal form of the protein. For example, the target protein may be an extracellular mutant protein, or a protein in which, for example, a partial or complete gain or loss of function is encoded by a nucleotide polymorphism. In some embodiments, the bifunctional compound targets the abnormal form of the protein and not the normal form of the protein.

[0215] The targeting ligand (T) of the bifunctional compound according to the present disclosure L The 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 selected for lysosomal degradation, the target protein mediating disease in the host, as described in more detail below.

[0216] Several exemplary extracellular proteins of medical therapy interest described below have characteristic structural information in the well-known Protein Data Bank ("PDB"), a database of three-dimensional structural information for large biomolecules such as proteins and nucleic acids. The PDB contains X-ray crystallographic and other information contributed by scientists worldwide and is freely accessible. See, for example, www.rcsb.org, wwwrwwpdb.org, and www.uniprot.org in connection with the code provided below.

[0217] For example, one skilled in the art can visualize T using available visualization tools, including those available on the PDB website. L The skilled artisan can also determine where the T is docked to the extracellular protein. L The results can be imported into modeling software (including, for example, PyMOL, Glide, Maestro, RasMol, Visual Molecular Dynamics, Jrnol, and AutoDock) to determine which portion of the extracellular protein targeting ligand is bound to the extracellular protein. The T of the bifunctional compound can then be analyzed. L The linker (L) is suitable for binding to extracellular proteins without excessively adversely affecting the binding. I ) or sortilin binding group (S L ) is combined with

[0218] Non-limiting examples of extracellular proteins include: In one embodiment, the extracellular target protein is selected from the group consisting of PCSK9, TNF-α, ANCEPTL-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, and cytochrome P450. The phospholipase A2 is one selected from the group consisting of lipoprotein lipase, human matrix metallopeptidase 1, macrophage migration inhibitory factor, transforming 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, placenta 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.

[0219] Immunoglobulin G (IgG) In some embodiments, the target protein is human immunoglobulin G (IgG). IgG accounts for approximately 75% of human serum antibodies. IgG is the most common type of antibody found in the blood circulation. IgG antibodies are large globular proteins with a molecular weight of approximately 150 kDa, composed of four peptide chains. They contain two identical gamma (gamma) heavy chains of approximately 50 kDa and two identical light chains of approximately 25 kDa, i.e., a tetrameric quaternary structure. The two heavy chains are linked to each other and to the light chain by disulfide bonds. The resulting tetramer has two identical halves that together form a Y-like shape. Each end of the fork contains an identical antigen-binding site. The various regions and domains of a typical IgG are shown on the left of the figure. The Fc region of IgG contains a highly conserved N-glycosylation site at asparagine 297 in the constant region of the heavy chain. The N-glycans attached to this site are primarily complex, core-fucosylated, biantennary structures. Furthermore, a small proportion of these N-glycans also contain biantennary GlcNAc and α-2,6-linked sialic acid residues. The N-glycan composition in IgG has been linked to several autoimmune, infectious, and metabolic diseases. Furthermore, overexpression of IgG4 has been associated with IgG4-related diseases, which generally involve multiple organs, including type 1 autoimmune pancreatitis, interstitial nephritis, Riedel's thyroiditis, Mikulicz's disease, Kleiner's tumor, inflammatory pseudotumor (in various body sites), some cases of mediastinal and retroperitoneal fibrosis, aortitis, retroperitoneal fibrosis, proximal biliary stricture, tubulointerstitial nephritis, meningitis, pancreatic hypertrophy, and pericarditis.

[0220] The Protein Data Bank website has crystal structures of IgG searchable for 1H3X (Krapp, S., et al., J. Mol. Biol., 2003, 325: 979); and 5V43 (Lee, CH, et al., Nat. Immunol., 2017, 18: 889-898); as well as 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) provides crystal structures of IgG binding to various compounds. Furthermore, Kiyosi M., et al. provide insights into the structural basis for binding of human IgG1 to its high-affinity human receptor FcyRI (Kiyosi M., et al., Nat Commun., 2015, 6, 6866).

[0221] TNF-alpha In some embodiments, the target protein is human TNF-α (UniProtKB-PC) 1375 (TNFA_HUMAN)). TNF-α is a pro-inflammatory cytokine active in the body's immune response and serious inflammatory diseases. TNF-α has been implicated in several disorders, including, but not limited to, rheumatoid arthritis, inflammatory bowel disease, graft-versus-host disease, ankylosing spondylitis, psoriasis, psoriatic abscesses, refractory asthma, systemic erythematosus, diabetes, and the induction of cachexia. As used herein, TNF-alpha may be referred to as TNFa, TNF-a, TNF-α, TNF-α, or TNF-alpha.

[0222] The Protein Data Bank website contains the crystal structures of TNF-α, searchable for 6RMJ (Va!entinis, B., et al, Int. J. Mol. Sci., 2019, 20); 5UUI (Carrington et al., Biophys J., 2017, 113 371-380); 600Y, 600Z, and 60PO (O'Connell, J., et al., Nat. Commun., 2019, 10 5795-5795); and 5TSVV (Cha, SS, J Biol Che ., 1998, 273 2153-2160), 5YOY (Ono et al., Protein Sci., 2018, 27 1038-1046); 2AZ5 (He., M. VI. et al., Science, 2005, 310: 1022-1025); 5WUX (Lee, JU, Int J Mol Sci., 2017, 18); 5MU8 (Blevitt et al., J Med Chem., 2017, 60 3511-3517); 4Y60 (Feldman J. I,., 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, 288 13799-13807).

[0223] Proprotein convertase sphatidylinosin / Kexm type 9 (PCSK-9) In some embodiments, the target protein is human proprotein convertase subtilisin / kexin type 9 (PCSK-9) (UniProtKB-Q8NBP7(PCSK9_HUMAN)). PCSK-9 is a key player in regulating plasma cholesterol homeostasis. PCSK-9 binds to members of the low-density lipid receptor family, low-density lipoprotein receptor (LDLR), very-low-density lipoprotein receptor (VLDLR), apolipoprotein E receptor (LRP1 / APOER), and apolipoprotein receptor 2 (LRP8 / APOER2), promoting their degradation in intracellular acidic compartments. It may act via a non-proteolytic mechanism to promote the degradation of LDLR in the liver via the clathrin-LDLRAP1 / ARH-mediated pathway, preventing LDLR recycling from endosomes to the cell surface or directing it to lysosomes for degradation. PCSK-9 has been associated with the development of high blood cholesterol and cardiovascular disease.

[0224] The Protein Data Bank website contains the crystal structure of PCSK-9, searchable for 2P4E (Cunningham, D., et al., Nat Struct Mol Biol., 2007, 14 413-419); as well as 3BPS (Kwon, H. J., et al., Proc Natl Acad Sci USA, 2008, 105 1820-1825); 6U26, 6U2N, 6U2P, 6U36, 6U38, and 6U3X (Petrilli, W. L., et al., Ceil Chem Biol., 2019, 27 32-40. e3); 50CA (Gustafsen, C., et al., Nat Commun., 2017, 8 503-503); 4NE9 (Schroeder, C. L., et al., Chem Biol., 2014, 21 284-294); 40V6 (Mitchell, T., et. al., J Pharmacol Exp Ther., 2014, 350412-424); and 4NMX (Zhang, Y., et. al., J Biol Chem., 2014, 289942-955). Additionally, Piper et al. have provided insights into the crystal structure of PCSK9 (Piper, DE, et al., Structure, 2007, 15(5), 545-52).

[0225] TNF-alpha In one embodiment, the present disclosure provides a bifunctional compound described herein that can target TNFα. Thus, in one embodiment, the target molecule is TNFα.

[0226] In one embodiment, the targeting moiety (TL) is according to Formula XVIIa: [ka] (In the formula, R XVIIa is the expression [ka] where R XVIId and R XVIIe are independently C1-C5 alkyl, C1-C5 alkoxy, cyano, halogen, and C1 haloalkyl, each of which is optionally and independently substituted; X is an atom selected from N or CH; * represents a bond to formula (XVIIa); R XVIIb and R XVIIb’ are each independently selected from H and C1-C3 alkyl; R XVIIc is R L or selected from the group consisting of formula XVIIa-1 and formula XVIIa-2: [ka] where R XVIIf is selected from the group consisting of optionally substituted C1-C5 alkyl, wherein one or more of the methylene groups is replaced by a member selected from the group consisting of carbonyl, ester, amide, -NH-, or -O-, and * represents a bond to formula XVIIa; R L L I (represents a bond with

[0227] In one embodiment, R XVIIb and / or R XVIIb In one embodiment, R XVIIb and R XVIIb ' is -CH3.

[0228] In one embodiment, R XVIIa is selected from the group consisting of: [ka] (wherein * represents a bond to formula XVIIa).

[0229] In one embodiment, T L is according to formula XVIIa-3: [ka] (In the formula, R L L I (represents a bond with

[0230] In one embodiment, T L is according to formula XVIIa-4: [ka] (In the formula, R L L I (represents a bond with

[0231] In one embodiment, T L is according to formula XVIIa-5: [ka] (In the formula, R L L I (represents a bond with

[0232] In one embodiment, T L is according to formula XVIIb: [ka] (In the formula, R XVIIi is selected from a bond and an optionally substituted piperazine group; R XVIIj teeth, [ka] wherein * represents a bond to formula XVIIb; where R L L I (represents a bond with

[0233] In one embodiment, T L is according to formula XVIIb-1: [ka] (In the formula, R L L I (represents a bond with

[0234] In one embodiment, T L is according to formula XVIIb-2: [ka] (In the formula, R L L I (represents a bond with

[0235] In one embodiment, T L is according to formula XVIIc: [ka] (In the formula, R XVIIg is C1-C4 alkyl, and R XVIIh is optionally substituted by one or more groups selected from halogen, haloalkyl, cyano, hydroxyl, amino, hydroxyl, alkoxy, C3-C6 cycloalkyl, and C3-C6 heterocycloalkyl; R L L I (represents a bond with

[0236] In one embodiment, R XVIIg is -CH3.

[0237] In one embodiment, T L is according to formula XVIIc-1: [ka] (In the formula, R L L I (represents a bond with In one embodiment, T L is according to formula XVIIc-2: [ka] (In the formula, RL L I (represents a bond with

[0238] In one embodiment, T L is according to formula XVIId: [ka] In certain embodiments, the present disclosure provides a compound selected from any one of compounds TF001-TF022 shown in Table C in the "List of Compounds" section.

[0239] TL biotin and DNP In one embodiment, T L In another embodiment, the T L The moiety has the following structure: [ka] (In the formula, R L L I (represents a bond with

[0240] Some 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 the group consisting of avidin, streptavidin, or neutravidin or their derivatives.In some cases, the biotin-binding protein is labeled with a tag moiety such as a fluorophore, a chromophore, or a radioactive labeling compound.

[0241] In one embodiment, T L The moiety is a substituent derived from dinitrophenol (DNP). L The moiety has the following structure: [ka] (In the formula, R L L I (represents a bond with

[0242] In one embodiment, extracellular target protein is DNP binding protein.For example, extracellular target protein is anti-DNP antibody, the fragment of anti-DNP antibody that can be bound to DNP, or its derivative.In some cases, anti-DNP antibody or its fragment is labeled with tag moiety such as fluorophore, chromophore or radiolabeled compound.

[0243] TL is a protein In some embodiments, T L is an antibody or antigen-binding fragment of an antibody or a nanobody capable of binding to an extracellular target molecule. L comprises an antibody or an antigen-binding fragment of an antibody or a nanobody capable of binding to an extracellular target protein.

[0244] According to the present disclosure, T L may comprise or consist of a full-length antibody or an antibody fragment. L The antibody light chain (LC), antibody heavy chain (HC), antigen binding region (Fab), and variable region of the light chain (V L ) or the variable region of the heavy chain (V H ) or consisting of an antibody fragment such as

[0245] In one embodiment, T L comprises or consists of an antibody heavy chain and / or an antibody light chain.

[0246] In one embodiment, T L is the variable region of the antibody light chain (V L In another embodiment, the compound comprises or consists of T L is the variable region of the antibody heavy chain (V H ) or consisting of it.

[0247] In one embodiment, T L In one embodiment, T comprises or consists of a full-length antibody. L comprises or consists of a monoclonal antibody.

[0248] In one embodiment, T L comprises or consists of a fusion protein of two or more antibody fragments, such as two antibody fragments, for example three antibody fragments, for example four antibody fragments, such as five antibody fragments, for example six antibody fragments, for example seven antibody fragments, for example eight antibody fragments.

[0249] For example, T L comprises or consists of a single chain antigen binding region (scAb) or a single chain variable fragment (scFv). L The scFv fragments contain the variable region of the heavy chain of a single antibody chain (V H ) and the light chain variable region (V L ) fusion protein.

[0250] In one embodiment, T L comprises or consists of a single-chain antigen-binding region (scAb). scAb consists of a light chain (V L ) and heavy chain (V H ) to the variable region of the antibody heavy chain (C H ) or light chain (C L ) is a fusion protein to which one or more regions of the constant region of either

[0251] In one embodiment, T L Nanobodies (also known as single domain antibodies) contain a single monomeric variable heavy chain (VH) that selectively binds to a specific antigen. H H) domain.

[0252] In one embodiment, T L consists of an antibody or antibody fragment or a fusion protein of antibody fragments as described herein.

[0253] Alirocumab is a monoclonal antibody that binds to PCSK-9 and is approved for the treatment of hypercholesterolemia. The heavy chain sequence of the alirocumab antibody is SEQ ID NO: 66, and the light chain is SEQ ID NO: 67.

[0254] In one embodiment, T L comprises an antibody or antigen-binding fragment thereof having binding specificity for PCSK-9: a light chain region comprising SEQ ID NO: 67 or an amino acid sequence having at least 70%, e.g., at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 67; and / or SEQ ID NO: 66 or a heavy chain region comprising an amino acid sequence having at least 70%, for example at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO:66.

[0255] In one embodiment, T L comprises or consists of an antibody or antigen-binding fragment thereof having binding specificity for PCSK-9: a light chain region comprising SEQ ID NO: 67; and / or , comprising or consisting of a heavy chain region comprising SEQ ID NO:66.

[0256] In one embodiment, T L comprises or consists of the full-length monoclonal antibody alirocumab (an antibody having a light chain according to SEQ ID NO: 67 and a heavy chain according to SEQ ID NO: 66).

[0257] Adalimumab is a monoclonal antibody that binds to TNF-α. The heavy chain sequence of Adalimumab is that of SEQ ID NO: 68, and the light chain sequence is that of SEQ ID NO: 69.

[0258] In one embodiment, T L comprises an antibody or antigen-binding fragment thereof having binding specificity for TNF-α: a light chain region comprising SEQ ID NO: 69, or an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 69, e.g., at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity; and / or SEQ ID NO: 68, Alternatively, it comprises a heavy chain region comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:68, for example at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

[0259] In one embodiment, T L comprises or consists of an antibody or antigen-binding fragment thereof having binding specificity for TNF-α: a light chain region comprising SEQ ID NO: 69; and / or It comprises or consists of a heavy chain region comprising SEQ ID NO:68.

[0260] In one embodiment, T L comprises or consists of the full-length monoclonal antibody adalimumab.

[0261] In one embodiment, T L comprises a Nanobody that has binding specificity for the κ-light chain of a secondary antibody comprising the following sequence: MGGTHHHHHHENLYFQGQVQLQESGGGLVQPGGSLRLSCAASGRTISRYAMSWFRQAPGKEREFVAVARRSGDGAFYADSVQGRFTVSRDDAKNTVYLQMNSLKPEDTAVYYCAIDSDTFYSGSYDYWGQGTQVTVSSE (SEQ ID NO: 70) or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:70, for example at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity.

[0262] In one embodiment, T Lhas the following sequence:MGGTHHHHHHENLYFQGQVQLQESGGGLVQPGGSLRLSCAASGRTISRYAMSWFRQAPGKEEFVAVARRSGDGAFYADSVQGRFTVSRDAKNTVYLQMNSLKPEDTAVYYCAIDSDTFYSGSYDYWGQGTQVTVSSE (SEQ ID NO: 70).

[0263] In one embodiment, the bifunctional compound according to the present disclosure has a dissociation constant (K) of less than 50 μM, such as less than 40 μM, for example less than 30 μM, such as less than 20 μM, for example less than 10 μM, such as less than 5 μM, for example less than 4 μM, such as less than 3 μM, for example less than 2 μM, such as less than 1 μM, for example less than 0.8 μM, such as less than 0.6 μM, for example less than 0.5 μM, such as less than 0.4 μM, for example less than 0.3 μM, such as less than 0.2 μM, for example less than 0.1 μM. D ) can bind to the target molecule.

[0264] Binding to a target protein can be measured via different methods, as known to those skilled in the art, for example, microscale thermophoresis (MST).

[0265] Identified SL and TL In one embodiment, the bifunctional compound is according to any one of formulas XVIIIa, XVIIIb, XVIIIb-1, XVIIIc, XVIIId, XVIIId-1, XVIIIe, XVIIIf, XVIIIf-1, XVIIIg, XVIIIh, and XVIIIh-1: [ka] TIFF2025530891000066.tif220159TIFF2025530891000067.tif86159 (in the formula, Q 3 is a bond or -CH2-; R 3ais selected from the group consisting of H, halogen, alkoxy, —CF3, and optionally substituted C1-4 alkyl; L I indicates a linker).

[0266] In one embodiment, R 3a is H.

[0267] In one embodiment, R 3a is a halogen or -CF3.

[0268] In one embodiment, R 3a is an alkoxy.

[0269] In one embodiment, R 3a is an optionally substituted C1-4 alkyl.

[0270] In one embodiment, Q 3 is a bond.

[0271] In one embodiment, Q 3 is -CH2-.

[0272] In one embodiment, the bifunctional compound is according to any one of formulas D-III, D-IV, DV, and D-VI: [ka]

[0273] In one embodiment, the bifunctional compound is according to any one of formulas XVIIIm, XVIIIn, XVIIn-1, XVIIIo, XVIIIp: [ka] In the formula, L I indicates a linker.

[0274] In one embodiment, the bifunctional compound is according to any one of formulas XIVa, XIVb, XIVc, and XIVd: [ka] (In the formula, Q 3 is a bond or -CH2-; R 3a is selected from the group consisting of H, halogen, alkoxy, —CF 3 , and optionally substituted C 1-4 alkyl).

[0275] In one embodiment, in any one of formulas XIVa, XIVb, XIVc, or XIVd, R 3a is H.

[0276] In one embodiment, in any one of formulas XIVa, XIVb, XIVc, or XIVd, R 3a is a halogen or -CF3.

[0277] In one embodiment, in any one of formulas XIVa, XIVb, XIVc, or XIVd, R 3a is an alkoxy.

[0278] In one embodiment, in any one of formulas XIVa, XIVb, XIVc, or XIVd, R 3a is an optionally substituted C1-4 alkyl.

[0279] In one embodiment, in any one of formulas XIVa, XIVb, XIVc, or XIVd, Q 3 is a bond.

[0280] In one embodiment, in any one of formulas XIVa, XIVb, XIVc, or XIVd, Q 3 is -CH2-.

[0281] In one embodiment, the bifunctional compound is according to any one of formulas XVa, XVb, XVc, and XVd: [ka] (In the formula, R F1 and R F2 are independently selected from CF or H, with the proviso that R F1 or R F2 At least one of the groups is CF3.

[0282] In one embodiment, in any one of formulas XIVa, XIVb, XIVc, or XIVd, R F1 is -CF3, and R F2 is H.

[0283] In one embodiment, the bifunctional compound is according to one selected from the group consisting of: 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), XT -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); (In the formula, X L L I and X Tis selected from the group consisting of: a) a group containing biotin; b) a group containing dinitrophenol; c) has binding specificity for PCSK-9: SEQ ID NO: 67 and / or a light chain region comprising SEQ ID NO: 66 An antibody or antigen-binding fragment thereof comprising or consisting of a heavy chain region comprising:

[0284] d) having binding specificity for TNF-α: a light chain region comprising SEQ ID NO: 69; and / or comprising or consisting of a heavy chain region comprising SEQ ID NO: 68 An antibody or antigen-binding fragment thereof.

[0285] In one embodiment, the bifunctional compound is according to: Biotin-X L -X5-X1X2X3X4 (SEQ ID NO: 116) (In the formula, X5 is any amino acid residue or peptide containing 2 to 30 amino acid residues or bonds; 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 a conservative substitution of M, K, L, or L; where X L L I (It is).

[0286] In one embodiment, X5 is a bond.

[0287] In one embodiment, X5 is APLRDAP (SEQ ID NO: 21).

[0288] In one embodiment, X5 is REAPRWDAPLRDPAL (SEQ ID NO: 17).

[0289] In one embodiment, X5 is REALRWDAPLRDPAP (SEQ ID NO: 18).

[0290] In one embodiment, X1 is selected from Y, F, or R; X2 is selected from Q or Y; X3 is L, X4 is L.

[0291] In one embodiment, X1 is F or R.

[0292] In one embodiment, X2 is Q.

[0293] In one embodiment, X3 is Q.

[0294] In one embodiment, X4 is L.

[0295] In one embodiment, the bifunctional compound is according to any one of the group consisting 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) Biotin-X L -LYENKPRPYIL-OH (SEQ ID NO: 130) (In the formula, X L L I (It is).

[0296] antibody conjugates In one embodiment, the bifunctional compound comprises a monoclonal antibody and -L I -S L It is prepared by forming a conjugate between a reactive precursor of the -L group. I -S L The group can be conjugated to a free amino group within the antibody sequence, such as the side chain of a lysine amino acid or the N-terminal group. Several methods and conditions for conjugating groups to free amino groups of antibodies are well known in the art. For example, L I -S L An N-hydroxysuccinimide (NHS) derivative of may be prepared and reacted with an antibody.

[0297] Thus, in one embodiment, the bifunctional compound is -L I -S L It is prepared by reacting an NHS derivative of the formula (I) with a monoclonal antibody.

[0298] As will be appreciated by those skilled in the art, --L I -S L The reaction of the NHS derivative with the monoclonal antibody is I -SL yields a product in which multiple units of the formula (I) can be conjugated to an antibody.

[0299] Thus, in one embodiment, the bifunctional compound comprises at least one -L I -S L Units, e.g., two -L I -S L Units, e.g., 3 -L I -S L Units, e.g. 4 -L I -S L Units, e.g. 5 -L I -S L Units, e.g. 6 -L I -S L Units, e.g. 7 -L I -S L Units, e.g. 8 -L I -S L Units, e.g. 9 -L I -S L T consisting of a monoclonal antibody conjugated to a L It has a group.

[0300] In one embodiment, the monoclonal antibody is alirocumab (an antibody having a heavy chain SEQ ID NO: 66 and a light chain SEQ ID NO: 67), and S L and / or L I is as described herein.

[0301] In one embodiment, the monoclonal antibody is alirocumab (an antibody having a heavy chain SEQ ID NO: 66 and a light chain SEQ ID NO: 67), and S L is a peptide comprising a C-terminal 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) FYLL-OH (SEQ ID NO: 28) YQLL-OH (SEQ ID NO: 29) and PYILKRQLYENKPRRPYIL-OH (SEQ ID NO: 55); (In the formula, L Iis attached to the N-terminus).

[0302] In one embodiment, the monoclonal antibody is alirocumab (an antibody having a heavy chain SEQ ID NO: 66 and a light chain SEQ ID NO: 67), and S L is a peptide containing the following sequence at the C-terminus: RQLL-OH (SEQ ID NO: 22).

[0303] In one embodiment, the monoclonal antibody is alirocumab (an antibody having a heavy chain SEQ ID NO: 66 and a light chain SEQ ID NO: 67), and S L is a peptide containing the following sequence at the C-terminus: FQLL-OH (SEQ ID NO: 23).

[0304] In one embodiment, the monoclonal antibody is adalimumab (an antibody having a heavy chain SEQ ID NO: 68 and a light chain SEQ ID NO: 69), and S L is as described herein.

[0305] In one embodiment, the monoclonal antibody is adalimumab (an antibody having a heavy chain SEQ ID NO: 68 and a light chain SEQ ID NO: 69), and S L is 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); (wherein LI is attached to the N-terminus).

[0306] In one embodiment, the monoclonal antibody is adalimumab (an antibody having a heavy chain SEQ ID NO: 68 and a light chain SEQ ID NO: 69), and S L is a peptide containing the following sequence at the C-terminus: RQLL-OH (SEQ ID NO: 22).

[0307] In one embodiment, the monoclonal antibody is adalimumab (an antibody having a heavy chain SEQ ID NO: 68 and a light chain SEQ ID NO: 69), and S Lis a peptide containing the following sequence at the C-terminus: FQLL-OH (SEQ ID NO: 23).

[0308] Linker: The bifunctional compounds according to the present disclosure comprise a sortilin binding moiety (S L ) and targeting moiety (T L ) covalently bonded to the linker. L Group and T L Covalent conjugation of groups to form bifunctional compounds can be achieved through several chemical strategies as are known in the art.

[0309] In one embodiment, the bifunctional compound according to the present disclosure is L according to the structure I Having: [ka] (In the formula, * indicates T L or S L represents a point of attachment to either of the following: L1 and L2 are each independently selected from the group consisting of a bond, -C(H2)-, -O-, -N(H)-; a functional group selected from carbonyl, ester, amide, carbamate; or a C1-C3 hydrocarbon chain in which one or more methylene groups are individually and optionally replaced with carbonyl, ester, amide, carbamate, thiourea, urea sulfonamide, and triazole; Z is a divalent, saturated or unsaturated, straight or branched chain, C 1- C 30 hydrocarbon chains, wherein one or more methylene groups are individually and optionally 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)-, -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 divalent aromatic groups, optionally substituted carbocycles, optionally substituted heterocycles, optionally substituted divalent aromatic heterocycles; [ka] and is replaced by one or more of the group consisting of: [ka] L 1 or L 2 represents a bond to; R L1 is C 1-5 selected from the group consisting of alkyl; n and w are each independently an integer from 1 to 8.

[0310] In one embodiment, the bifunctional compound according to the present disclosure comprises L I Having: [ka] (In the formula, * indicates T L or S L represents a point of attachment to either L1 and L2 are each independently selected from the group consisting of -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, wherein one or more methylene groups are individually and optionally -O-, -N(H), -N(R L1 )-, -N(H)C(=O)-, -N(R)C(=O)-, -C(=O)N(H)-, -C(=O)N(R L1 )-, [ka] and is substituted by one or more groups selected from: [ka] L 1 or L 2 represents a bond to; R L1 is C 1-5 selected from the group consisting of alkyl; n and w are each independently an integer from 1 to 8.

[0311] In one embodiment, Z is a divalent C-C 30 Hydrocarbon chains, e.g., C5-C 30 Hydrocarbon chains, e.g., C8-C 30 Hydrocarbon chains, e.g., C 10 -C 30 Hydrocarbon chains, e.g., 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.

[0312] In one embodiment, Z is a divalent C 10 -C 25 Hydrocarbon chains, e.g., 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 in which one or more methylene groups are replaced as described in Formula XVI or Formula XVI-2.

[0313] In one embodiment, Z is a divalent C 14 -C 20 In one embodiment, Z is a divalent C-C hydrocarbon chain in which one or more methylene groups are replaced as described in Formula XVI or Formula XVI-2.13 A hydrocarbon chain in which one or more methylene groups are replaced as described in Formula XVI or Formula XVI-2.

[0314] In one embodiment, L 1 is a bond. In one embodiment, L 2 is a bond. In one embodiment, T 1 and L 2 is a bond.

[0315] In one embodiment, Z is C 1- C 30 A hydrocarbon chain in which one or more methylene groups are individually and optionally replaced with 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-CH-O-.

[0316] In one embodiment, Z is C-C 30 Hydrocarbon chains, e.g., C1-C 20 , e.g. C1-C 15 , e.g. C1-C 10 a hydrocarbon chain, wherein one or more methylene groups may be individually and optionally selected from -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)2N(R L1 )-, -CH2-CH2-O-, an optionally substituted carbocycle, an optionally substituted heterocycle, and triazole; R L1 is C 1-5 alkyl.

[0317] In one embodiment, 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 may be individually and optionally replaced by —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-, an optionally substituted carbocycle, an optionally substituted heterocycle, and triazole; R L1 is C 1-5 alkyl.

[0318] In one embodiment, Z is one or more groups -NH-SO 2 - group.

[0319] In one embodiment, Z comprises one or more triazole groups. In one embodiment, the triazole is [ka] Refers to...

[0320] In one embodiment, Z comprises one or more groups selected from optionally substituted carbocyclic group(s) and optionally substituted heterocyclic group(s). In one embodiment, Z comprises two groups each independently selected from triazole, optionally substituted carbocyclic group(s), and optionally substituted heterocyclic group(s). In one embodiment, Z comprises three groups each independently selected from triazole, optionally substituted carbocyclic group(s), and optionally substituted heterocyclic group(s).

[0321] Non-limiting examples of carbocyclic and heterocyclic rings are described herein below. In one embodiment, one or more carbocyclic or heterocyclic rings are as described herein below.

[0322] In one embodiment, Z is [ka] wherein n is an integer selected from 0, 1, 2 or 3.

[0323] In one embodiment, Z comprises one or more heterocyclic groups. For example, the heterocyclic group can be an optionally substituted 3- to 6-membered ring in which one or two carbon atoms of the ring are replaced with N. In one embodiment, Z is [ka] wherein n is an integer selected from 0, 1, 2, or 3.

[0324] In one embodiment, Z comprises one or more groups, each independently: Selected from the group consisting of those shown in Table Z: [Table 1]

[0325] In one embodiment, Z comprises one or more groups each individually selected from the groups set forth in Table ZI: [Table 2]

[0326] In one embodiment, Z is [ka] where n is an integer from 1 to 10.

[0327] In one embodiment, Z is [ka] In the formula, n is an integer of 1 to 10, and t or w is an integer of 1 to 20.

[0328] In one embodiment, Z is [ka] wherein n is an integer from 1 to 10, and t and t' are each independently an integer from 1 to 20.

[0329] In one embodiment, Z is [ka] wherein n and n' are each independently an integer from 1 to 10, and t is an integer from 1 to 20.

[0330] In one embodiment, L 1 or L 2 is an amide group. 1 and L 2 is an amide group.

[0331] In one embodiment, L 1 or L 2 is a carbonyl group. 1 and L 2 is a carbonyl group.

[0332] In one embodiment, L 1 or L 2 is an ester group. In one embodiment, L 1 and L 2 is an ester group.

[0333] In one embodiment, L 1 or L 2 is a carbamate group. In one embodiment, L 1 and L 2 is a carbamate group.

[0334] In one embodiment, L 1 or L 2 is a urea group. 1 and L 2 is a urea group.

[0335] In one embodiment, L 1 or L 2 is -NH-S(=O)-. In one embodiment, L 1 and L 2 is -NH-S(=O)2-.

[0336] In one embodiment, L 1 or L 2 is a triazole group. 1 and L 2 is a triazole group. Triazole groups can be prepared via the reaction of an azide group with an alkyne group, optionally in the presence of a catalyst, for example using copper ions as a catalyst.

[0337] In one embodiment, L 1 or L 2 is —O—. In one embodiment, L 1 and L 2 is -O-.

[0338] In one embodiment, L 1 or L 2 is —NH—. In one embodiment, L 1 and L 2 is -NH-.

[0339] In one embodiment, L 1 or L 2 is -S(=O)2-. In one embodiment, L 1 and L 2 is -S(=O)2-.

[0340] In one embodiment, L 1 or L 2 teeth, [ka] In one embodiment, L 1 and L 2 teeth, [ka] is.

[0341] In one embodiment, L 1 or L 2 teeth, [ka] In one embodiment, L 1 and L 2 teeth, [ka] is.

[0342] In one embodiment, L 1 or L 2 teeth, [ka] wherein X is NH or O. In one embodiment, L 1 and L 2 teeth, [ka] wherein X is NH or O. In one embodiment, L 1 or L 2 teeth, [ka] In one embodiment, L 1 and L 2 teeth, [ka] is.

[0343] In one embodiment, L 1or L 2 teeth, [ka] wherein X is NH or O. In one embodiment, L 1 and L 2 teeth, [ka] wherein X is NH or O. In one embodiment, L 1 or L 2 teeth, [ka] In one embodiment, L 1 and L 2 teeth, [ka] is.

[0344] In one embodiment, L 1 and L 2 are different groups. In one embodiment, L 1 and L 2 are identical.

[0345] In one embodiment, the bifunctional compound according to the present disclosure comprises L according to one selected from the group consisting of: I Having: [ka] n and q are each independently an integer from 1 to 9; * indicates T L or S L represents a bond to either

[0346] In one embodiment, L I is due to: [ka] (wherein w is an integer of 1 to 9, and * is T L or S L (represents a bond to either

[0347] In one embodiment, L I is due to: [ka] (wherein n is an integer of 1 to 8, and * is T L or S L (represents a bond to either

[0348] In one embodiment, L I is due to: [ka] (wherein n and w are integers of 1 to 8, and * is T L or S L (represents a bond to either

[0349] In one embodiment, L I is due to: [ka] (wherein n is an integer of 1 to 9, and * is T L or S L (represents a bond to either

[0350] In one embodiment, L I is selected from one of the group consisting of formulas (XVIa) to (XVIae) in Table Z-II: [Table 3] TIFF2025530891000105.tif242155TIFF2025530891000106.tif60159

[0351] In one embodiment, L Iis selected from one of the group consisting of formulas XVIaf to XVIbw in Table Z-III: [Table 4] TIFF2025530891000108.tif238159TIFF2025530891000109.tif236159TIFF2025530891000110.tif242157TIFF2025530891000111.tif60159

[0352] In one embodiment, the linker L I is 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, may contain S at any position marked with *. L and T L For example, in one embodiment, the linker shown in Table Z-III can be connected to T through the position marked with an * that appears to the left of the formula shown in Table Z-III. L and S through the position marked with * on the right side of the formula shown in Table Z-III. L is connected to.

[0353] In some embodiments, the linker is a peptide. Thus, the linker can be produced by methods known in the art for preparing peptides, such as peptide linkers produced by solid phase synthesis using protected amino acids or by expression of a suitable vector in an organism of choice.

[0354] In one embodiment, L I is a peptide having a length of 1 to 30 amino acids, for example, 3 to 20 amino acids.

[0355] In one embodiment, L I is a peptide of 3 to 20 amino acids in length consisting of any combination of glycine, serine, and cysteine.

[0356] In one embodiment, L Iis a peptide of 3 to 20 amino acids in length consisting of any combination of glycine and serine.

[0357] In one embodiment, L I is a peptide selected from the group consisting of: 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)

[0358] In one embodiment, L I is GGSGGGGSGGGGSGG (SEQ ID NO: 131).

[0359] In one embodiment, L I is GGSGGGG (SEQ ID NO: 132).

[0360] In one embodiment, L I is GGGGSGGGGSGGGGSGG (SEQ ID NO: 133).

[0361] In one embodiment, L I is GGSGGGGSGGGGS (SEQ ID NO: 134).

[0362] In one embodiment, L I is GGSGGGGSGGG (SEQ ID NO: 135).

[0363] In one embodiment, L I is GGSGGGGSG (SEQ ID NO: 136).

[0364] In one embodiment, L I is GGSGGGG (SEQ ID NO: 137).

[0365] In one embodiment, L I is GGSGG (SEQ ID NO: 138).

[0366] In one embodiment, L I is GGS (SEQ ID NO: 139).

[0367] In one embodiment, L I is CGGSGGGGSGGGGSGG (SEQ ID NO: 140).

[0368] In one embodiment, L I L I via the C-terminus of S L connected to L I L I T via the N-terminus of L is connected to.

[0369] Bifunctional compounds: compound In one embodiment, the bifunctional compound is selected from any one of compounds BF030-BF149 shown in Table A in the "List of Compounds" section, or a pharmaceutically acceptable salt thereof.

[0370] In one embodiment, the bifunctional compound according to the present disclosure comprises: a) S selected from the group consisting of any one of formulas IIIi, IIIj, IIIk, IIIm, IIIn, and IIIo, or selected from formula DI described in the "Binding of Sortilin" section above. L and b) a dissociation constant (K) of less than 50 μM, such as less than 40 μM, for example less than 30 μM, such as less than 20 μM, for example less than 10 μM, such as less than 5 μM, for example less than 4 μM, such as less than 3 μM, for example less than 2 μM, such as less than 1 μM, for example less than 0.8 μM, such as less than 0.6 μM, for example less than 0.5 μM, such as less than 0.4 μM, for example less than 0.3 μM, such as less than 0.2 μM, for example less than 0.1 μM D ) can bind to the target molecule.

[0371] In one embodiment, the bifunctional compound according to the present disclosure comprises: a) S selected from the group consisting of any one of formulas IIIi, IIIj, IIIk, IIIm, IIIn, and IIIo, or selected from formula DI described in the "Binding of Sortilin" section above. L and b) L selected from any one of the formulas XVIaf to XVIbw in Table Z-III of the "Linker" section above. I and c) Having a TL according to any one of Formulas XVIIa-3, XVIIa-4, XVIIc-1, and XVIIc-2 described in the "Targeted Warhead" section above.

[0372] In one embodiment, the bifunctional compound according to the present disclosure is selected from any one of compounds BF001-BF028 shown in Table A in the "List of Compounds" section.

[0373] peptide In one embodiment, the bifunctional compound is selected from any one of the group consisting of: 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-NH2 (SEQ ID NO: 188) Biotin-REAPRWDAPLRDPALFQLL-OH (SEQ ID NO: 189) Biotin-REAPRWDAPLRDPALRQLL-NH2 (SEQ ID NO: 190), and Biotin-REAPRWDAPLRDPALRQLL-OH (SEQ ID NO: 191).

[0374] protein In one embodiment, the bifunctional compound is: MGGTHHHHHHENLYFQGQVQLQESGGGLVQPGGSLRLSCAASGRTISRYAMSWFQAPGKEREFVAVARRSGDGAFYADSVQGRFTVSRDDAKNTVYLQMNSLKPEDTAVYCAIDSDTFYSGSYDYWGQGTQVTVSSEGGGGSGGGGSGGGGSGGRQLL (sequence number 158).

[0375] In one embodiment, the bifunctional compound comprises at least one unit -L consisting of CGGSGGGSGGGGSGGRQLL (SEQ ID NO: 175). I -S L T consisting of the monoclonal antibody alirocumab conjugated to L and SEQ ID NO: 175 is conjugated via its N-terminus. This conjugate of alirocumab is referred to herein as: It is written as alirocumab-CGGSGGGGSGGGGSGGRQLL (SEQ ID NO: 159) or alirocumab-link-RQLL or alirocumab-linker-RQLL.

[0376] Also disclosed are variant conjugates that do not have the sequence RQLL, and are described herein as Alirocumab-CGGSGGGGSGGGGSGG (SEQ ID NO: 160) or referred to as alirocumab-link or alirocumab-linker.

[0377] The product set forth in SEQ ID NO: 159 may have multiple units of (SEQ ID NO: 175) conjugated to the antibody, such as two or more units, such as three or more units, such as four or more units.

[0378] In one embodiment, the bifunctional compound comprises at least one unit -L consisting of: I -S L T consisting of the monoclonal antibody adalimumab conjugated to L Having: CGGSGGGGSGGGGSGGRQLL (SEQ ID NO: 175), where SEQ ID NO: 175 is conjugated via its N-terminus. This conjugate of adalimumab is referred to herein as: Referred to as adalimumab-CGGSGGGGSGGGGSGGRQLL (SEQ ID NO: 161) or adalimumab-link-RQLL or adalimumab-linker-RQLL.

[0379] In one embodiment, the bifunctional compound comprises at least one unit -L consisting of the group consisting of: I -S L T consisting of the monoclonal antibody adalimumab conjugated to L Having: CGGSGGGGSGGGGSGGRQLL (SEQ ID NO: 175), CREAPRWDAPLRDPALRQLL (SEQ ID NO: 184) CREAPRWDAPLRDPALRQLL-NH2 (SEQ ID NO: 185) CREAPRWDAPLRDPALFQLL-OH (SEQ ID NO: 186) and CREAPRWDAPLRDPALFQLL-NH2 (SEQ ID NO: 187) (wherein SEQ ID NO: 175 or SEQ ID NOs: 184-187 are conjugated via their N-terminus.) These adalimumab conjugates are referred to herein as: Adalimumab - CGGSGGGGSGGGGSGGRQLL (SEQ ID NO: 161) Adalimumab-CREAPRWDAPLRDPALRQLL (SEQ ID NO: 192) Adalimumab-CREAPRWDAPLRDPALRQLL-NH2 (SEQ ID NO: 193) Adalimumab-CREAPRWDAPLRDPALFQLL-OH (SEQ ID NO: 194) Adalimumab-CREAPRWDAPLRDPALFQLL-NH2 (SEQ ID NO: 195).

[0380] Also disclosed are variant conjugates that do not have the sequence RQLL, and are described herein as It is referred to as Adalimumab-CGGSGGGGSGGGGSGG (SEQ ID NO: 162) or Adalimumab-link or Adalimumab-linker.

[0381] A product set forth in SEQ ID NO: 161 may have multiple units of (SEQ ID NO: 175) conjugated to an antibody, such as two or more units, for example three or more units, for example four or more units. Similarly, a product described as a conjugate by SEQ ID NOs: 192 to 195 may have multiple units of SEQ ID NOs: 184 to 187, such as two or more units, for example three or more units, for example four or more units.

[0382] Conjugated -L per antibody I -S L is the average number per antibody: N a In one embodiment, N a is an integer or decimal between 1 and 10. In one embodiment, the product set forth in SEQ ID NO: 161 is a Some -L of SEQ ID NO: 175, where L is 1 to 10 I -S L In one embodiment, the product set forth in any one of SEQ ID NOs: 192-195 may have N a is between 1 and 10, I -S L It may have units.

[0383] Functional Features The bifunctional compounds according to the present invention comprise a sortilin binding moiety (S L ) and can bind to sortilin via a protein targeting moiety (T L ) can bind to the target protein.

[0384] Thus, in one embodiment, the bifunctional compound of the present invention is capable of binding to Sortilin and a target protein simultaneously. This means that the bifunctional compound according to the present disclosure forms a ternary complex with Sortilin and a target protein. In the ternary complex, Sortilin and the target protein are simultaneously bound to the bifunctional compound.

[0385] The formation of ternary complex can be measured by different methods known in the art.For example, the formation of ternary complex can be measured by Förster resonance energy transfer (FRET) assay or time-resolved Förster resonance energy transfer (TR-FRET) assay.When measured by these assays, the increase in the homogeneous time-resolved fluorescence (HTRF) ratio indicates the formation of ternary complex.

[0386] As demonstrated in the Examples, bifunctional compounds according to the present disclosure are capable of binding to both sortilin and a target protein, forming a ternary complex.

[0387] In one embodiment, a bifunctional compound according to the present disclosure is capable of binding to sortilin at the cell surface, hi another embodiment, the bifunctional compound is capable of forming a ternary complex with sortilin and a target molecule at the cell surface.

[0388] In one embodiment, S L binds to sortilin located on the cell surface, and T L When the target protein binds 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.

[0389] In one embodiment, the bifunctional compound according to the present disclosure is present at less than 50 μM, such as less than 40 μM, for example less than 30 μM, such as less than 20 μM, for example less than 10 μM, such as less than 5 μM, for example less than 4 μM, such as less than 3 μM, for example less than 2 μM, such as less than 1 μM, for example less than 0.8 μM, such as less than 0.6 μM, for example less than 0.5 μM, such as less than 0.4 μM, for example less than 0.3 μM, such as less than 0.2 μM, for example less than 0.1 μM, such as less than 0.05 μM, for example less than 0. and has a binding dissociation constant to sortilin of less than 50 μM, such as less than 40 μM, for example less than 30 μM, such as less than 20 μM, for example less than 10 μM, such as less than 5 μM, for example less than 4 μM, such as less than 3 μM, for example less than 2 μM, such as less than 1 μM, for example less than 0.8 μM, such as less than 0.6 μM, for example less than 0.5 μM, such as less than 0.4 μM, for example less than 0.3 μM, such as less than 0.2 μM, for example less than 0.1 μM.

[0390] In one embodiment, the target protein is TNFα. Thus, in one embodiment, the bifunctional compound according to the present disclosure can form a ternary complex between sortilin and TNFα. In one embodiment, the bifunctional compound can simultaneously bind to sortilin and TNFα.

[0391] In one embodiment, the bifunctional compounds of the present disclosure have an S of less than 50 μM, such as less than 2 μM, for example less than 0.5 μM, preferably less than 0.1 μM. L to sortilin and a T of less than 100 μM, for example less than 0.5 μM, for example less than 0.1 μM. L and a binding dissociation constant for TNFα of 0.05.

[0392] In one embodiment, the bifunctional compounds of the present disclosure have a binding dissociation constant 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 T of less than 100 μM, such as less than 0.5 μM, such as less than 0.1 μM. L It has a dissociation constant for TNFα of 0.05%.

[0393] In one embodiment, the bifunctional compound binds to sortilin located on the cell surface. L binds, and T L When binds to TNFα, it causes TNFα to be internalized into cells.To evaluate whether target protein is internalized, methods such as detecting target protein in cell culture supernatant can be used.Any suitable method can be used, such as ELISA, detecting the fluorescence of conjugate, HPLC, gel electrophoresis combined with staining such as SDS-PAGE or Western blotting, or other well-known techniques in the art.The presence of target protein or its fragment in cells can also be evaluated by the same method after cell lysis.

[0394] This example demonstrates that bifunctional compounds according to the present disclosure can promote the internalization of a target protein into a cell via sortilin-mediated binding. The example also demonstrates that this internalization results in the degradation of the target protein.

[0395] In one embodiment, TNFα is degraded after internalization into the cell.

[0396] Preparation of bifunctional compounds The following abbreviations refer to their respective definitions herein: 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'-binaphthalene, BOC (tert-butoxycarbonyl), CBZ (carbobenzoxy), CDCl3 (deuterated chloroform), CD3OD (deuterated methanol), CH3CN (acetonitrile), c-Hex (cyclohexane), DCC (dicyclohexyl)carbodiimide), DCM (dichloromethane), DHP (O-(2,4-dinitrophenyl)-hydroxylamine), dppf (1,1'-bis(diphenylphosphine) pheno)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 (diethyl ether), EtOH (ethanol), FMOC (fluorenylmethyloxycarbonyl), 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-chloroperbenzoic acid), MD Autoprep (Mass Directed Autoprep), MeOH (methanol), MgSO4 (magnesium sulfate), MS (mass spectrometry), MSH (O-mesitylenesulfonylhydroxylamine), 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), PyBOP® (Benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium 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).

[0397] In general, compounds according to Formula (I) and related formulae of the present invention can be prepared from readily available starting materials. If such starting materials are not commercially available, they can be prepared by standard synthetic techniques. In general, the synthetic route for any individual compound of Formula (I) and related formulae will depend on the specific substituents of each molecule, and such factors will be recognized by those skilled in the art. Compounds of Formula (I) and related formulae may be prepared using the following general methods and procedures described below in the Examples. Reaction conditions, e.g., temperatures, solvents, or co-reagents, illustrated in the following schemes are given by way of example only and are not limiting. It will be understood that although typical or preferred process conditions (i.e., reaction temperatures, times, moles of reagents, solvents, etc.) are given, other process conditions can also be used unless otherwise specified. Optimum reaction conditions may vary with the particular reactants or solvents used; such conditions can be determined by one skilled in the art by routine optimization. For complete protection and deprotection methods, see Philip J. Kocienski, in "Protecting Groups", Georg Thieme Verlag Stuttgart, New York, 1994 and Theodora W. Greene and Peter G. M. Wuts, in "Protective Groups in Organic Synthesis", Wiley Interscience, 3rd Edition, 1999.

[0398] T L , L I , and S L Depending on the nature of T, different synthetic strategies can be chosen for the synthesis of compounds of formula (I). In the process shown in the scheme below, L , L I , and S L is as defined above in this description unless otherwise stated.

[0399] A compound of formula (I) L , L I , and SL (as defined above) can be prepared from alternative compounds of formula (I) using suitable interconversion procedures such as those described below in the Examples or conventional interconversion procedures well known by those skilled in the art.

[0400] In Figure 23, R represents H or methyl, and Q 1 represents -CH2- or a bond.

[0401] PG is a suitable protecting group that is compatible with the chemistries described in Schemes 1 to 19. Preferred groups PG are: carbobenzyloxy (Cbz), p-methoxybenzylcarbonyl (Moz or MeOZ), tert-butyloxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (FMOC), alkanoyl groups such as acetyl (Ac), benzoyl (Bz), benzyl (Bn), carbamate, p-methoxybenzyl (PMB), 4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), arylsulfonyl groups such as tosyl (Ts) or benzolsulfonyl.

[0402] Compounds of formula (I) (wherein TL, LI, and SL are defined as above) can be prepared by those skilled in the art from the reaction of a compound comprising a TL-LI moiety with SL, or from the reaction of a compound comprising a LI-SL moiety with TL, using methods and reactions known to those skilled in the art. Such reactions can 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 below in the Examples (Scheme 1).

[0403] Scheme 1 T L -L I +S L →T L -L I -S L T L +L I -S L →TL -L I -S L Alternatively, T L , L I , or S L may be used to synthesize compounds of formula (I) by reacting the precursors of formula (I) with other moieties that constitute compounds of formula (I). Further steps using methods and reactions known to those skilled in the art provide compounds of formula (I).

[0404] For example, but not limited to, S L corresponds to (IIIa), then T L -L I or L I The moiety can react with a phenol derivative (Aa) to give intermediates (Ba) or (Baa), respectively, where R La corresponds to the functional group that reacts with the LI moiety, and L I R as a bond with L (Figure 23). Reaction of intermediates (Ba) or (Baa) with ester (Ca) via aromatic substitution gives esters (Da) or (Daa), respectively. After saponification conditions, acids (Ea) or (Eaa), respectively, are obtained. Alternatively, aromatic substitution with the corresponding acid (Ca, R = H) will directly give acids (Ea) and (Eaa), respectively. Amide coupling with ester (Fa) gives intermediates (Ga) and (Gaa), respectively, which, after saponification, give compounds of formula (Ia) or intermediate (Haa). Under conditions described in the examples below or known by those skilled in the art, (Haa) and T L Further reaction with a moiety will provide a compound of formula (Ia).

[0405] Medical Uses: The present invention focuses on the degradation of circulating extracellular proteins that mediate diseases such as immune, inflammatory, hematopoietic / blood disorders (including those caused by or exacerbated by angiogenesis) and diseases involving abnormal cell proliferation, such as tumors and cancer.

[0406] The bifunctional compounds of the present invention can be administered in any manner that allows the bifunctional compound to bind to extracellular proteins, typically in the bloodstream, and deliver them to sortilin-bearing cells for endocytosis and degradation. Thus, examples of methods for delivering the degradative agents of the present invention include, but are not limited to, oral, intravenous, buccal, sublingual, subcutaneous, and nasal.

[0407] In one aspect, the present invention provides bifunctional compounds for use in targeted sortilin-mediated lysosomal degradation of an extracellular target molecule. In one embodiment, the target molecule is a protein associated with a disease or disorder.

[0408] In another aspect, the present disclosure relates to the use of the bifunctional compounds described herein for use as a pharmaceutical. The present disclosure relates to pharmaceutical compositions comprising the bifunctional compounds described herein.

[0409] In yet another aspect, the present disclosure relates to a method for targeted lysosomal degradation of an extracellular target protein, comprising administering to a subject in need thereof a bifunctional compound described herein.

[0410] 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.

[0411] In one aspect, the present disclosure provides a bifunctional compound as described herein for use in treating a disorder or condition mediated by an extracellular protein in a subject in need thereof.

[0412] In one aspect, the present disclosure relates to a bifunctional compound as described herein for use in removing an extracellular target molecule from the plasma of a subject.

[0413] In one embodiment, the disorder or disease is associated with abnormal levels of an extracellular protein. In one embodiment, the disorder or disease is associated with abnormally high levels of an extracellular target protein.

[0414] In one embodiment, the disorder or disease is associated with a mutant or misfolded extracellular protein.

[0415] In some embodiments, the extracellular target protein is selected from the group consisting of: PCSK9, TNF-α, ANCEPTL-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, Recreain-1, lipoprotein lipase, human matrix metallopeptidase 1, macrophage migration inhibitory factor, transforming 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.

[0416] In one embodiment, the extracellular protein is PCSK9. In a further embodiment, the extracellular target protein is PCSK9 and the disorder or condition is a disorder of lipoprotein metabolism. In a further embodiment, the disorder of lipoprotein metabolism is associated with abnormal PCSK9 levels. In another embodiment, the disorder is selected from dyslipidemia, hypercholesterolemia, and coronary heart disease.

[0417] In one embodiment, the extracellular protein is TNF-α. In a further embodiment, the extracellular target protein is TNF-α and the disorder or condition is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, graft-versus-host disease, ankylosing spondylitis, psoriasis, suppurative dacryoadenitis, refractory asthma, systemic erythrocytosis, diabetes, and induction of cachexia. In a further 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.

[0418] Thus, in one aspect, the present disclosure provides a method for targeted lysosomal degradation of TNFα, comprising administering an effective amount of a bifunctional compound described herein.

[0419] In another aspect, the present disclosure provides a method for removing TNFα from the plasma of a patient or subject in need thereof, comprising administering a bifunctional compound described herein.

[0420] In another aspect, the disclosure provides the use of a bifunctional compound described herein for the manufacture of a medicament for the treatment of a disease or condition.

[0421] In another aspect, the present disclosure provides the use of a bifunctional compound described herein for the manufacture of a medicament for the treatment of a TNFα-mediated disorder or condition.

[0422] 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.

[0423] 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.

[0424] In the Examples, it is demonstrated that bifunctional compounds according to the present disclosure that target TNFα promote the removal of TNFα from the plasma of animals.

[0425] In one embodiment, the extracellular protein is an antibody light chain or IgG. In further embodiments, the extracellular target protein is IgG and the disorder or condition is selected from the group consisting of type 1 autoimmune pancreatitis, interstitial nephritis, Riedel's disease, thyroid fibrosis, Mikulicz's disease, Kujetner's tumor, inflammatory pseudotumor, mediastinal fibrosis, retroperitoneal fibrosis (Ormond's disease), aortitis, periaortitis, proximal biliary stricture, idiopathic hypocomplemente interstitial nephritis, multifocal fibrosclerosis, pachymeningitis, pancreatic hyperplasia, neoplastic lesions, pericarditis, rheumatoid arthritis (RA), inflammatory bowel disease, multiple sclerosis, myalgia, goiter eye disease, chronic inflammatory demyelinating polyneuropathy, warm autoimmune hemolytic anemia, ankylosing spondylitis, primary Sjogren's syndrome, psoriatic arthritis, and systemic lupus erythematosus (SLE), sclerosing cholangitis, and IgG monoclonal gammopathy, monoclonal gammopathy of undetermined significance (MGUS).

[0426] In one embodiment, the subject is a mammal, hi one embodiment, the mammal is a human. [Example]

[0427] Example 1: Peptide-derived S L A bifunctional compound with α- and β-actin directly interacts with the ectodomain of sortilin the purpose In this example, S L is the equilibrium dissociation constant (K ) that describes the binary binding event between the bifunctional compound, which is derived from a peptide and the soluble ectodomain of sortilin-6his. D ) is measured by microscale thermophoresis (MST).

[0428] Materials and Methods In these experiments, a 12-point titration series of peptide solutions was prepared in a total volume of 1.2 μL in a TTP LVDS 384-well plate (SPT Labtech) using a Mosquito pipetting robot (SPT Labtech) in a buffer containing 16.7% DMSO (v / v) and 0.05% Tween 20 (v / v). The plate was spun down, after which 8.8 μL 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, and 0.05% Tween 20 (v / v) was added to each well. At this point, the plate was sealed with adhesive strips, placed in an Eppendorf thermomixer, and shaken at 600 rpm for 3 minutes, then spun on a Fisherbrand plate spinner for 15 seconds and further incubated at 19°C for 1 hour. MST was measured on a Monolith NT automated (Nanotemper) with standard treated capillaries at 5% LED power and high MST power. Dose-responses were extracted from raw thermograms at 20 seconds of hot time, and dissociation constants were estimated by sigmoidal curve fitting in the MO affinity analysis program (Nanotemper). All experiments were performed in duplicate.

[0429] FIG. 1A shows an MST binding experiment using fluorescent sortilin-6his (100 nM) and the compound according to SEQ ID NO: 147, and the measured dissociation constant (K D ) was 703 nM (N=2). FIG. 1B shows the same experimental setup as FIG. A1, but titrating the compound according to SEQ ID NO: 157, which is unable to bind fluorescent sortilin-6his due to specific amidation at the C-terminus.

[0430] conclusion Compounds of the invention can bind to sortilin [Table 5]

[0431] Example 2: Peptide-derived SL Bifunctional compounds comprising: the purpose This example addresses S L The ability of a peptide-derived bifunctional compound to induce the formation of a ternary complex composed of streptavidin, the bifunctional compound, and sortilin-6his. In this example, the bifunctional molecule consists of a biotin molecule at one end, followed by peptide linkers of various lengths and compositions, and finally a sortilin-binding peptide sequence. The induction of the tertiary complex is measured by a proximity-based TR-FRET assay.

[0432] Materials and Methods In these experiments, compounds of interest were titrated 24 times in a 384-well TTP LVDS plate (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.

[0433] This titration series (4 uL) was transferred to a black 384-well plate (Corning) and the following was added: 4 μL of 10 nM MAb anti-6HIS in 50 mM Bis-Tris propane (pH 8), 50 mM NaCl, 0.05% Tween 20. Tb (Cisbio), 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. The reaction was allowed to incubate at room temperature for 2.5 hours, at which point the HTRF signal ratio at 665 / 620 nm was determined using a plate reader (ClarioStar, BMG Labtech). All reactions were performed in triplicate, and data were expressed as the HTRF ratio as a function of bifunctional compound concentration.

[0434] Figure 2A shows the proximity-induced HTRF ratio as a function of compound concentration for two peptide compounds. For the compound according to SEQ ID NO: 147, a bell-shaped dose response was observed, which means that the formation of ternary complexes at both low and high compound concentrations is relatively low, which is a common pharmacological profile observed in bifunctional molecules and is commonly referred to as the hook effect. The compound according to SEQ ID NO: 157 is identical to the compound according to SEQ ID NO: 147 in all respects except for the addition of C-terminal amidation, and as a result, this peptide did not induce a dose-dependent increase in the HTRF ratio. This indicates that this peptide cannot mediate the formation of ternary complexes between streptavidin and sortilin.

[0435] [Table 6] conclusion The compounds of the present invention are capable of mediating the formation of a ternary complex between sortilin and a target protein.

[0436] Example 3: Internalization of target proteins This example addresses S L is the ability of sortilin to internalize the extracellular target of a peptide-derived bifunctional compound. In this example, the peptidic degrader consists of a sortilin binder motif linked to a biotin warhead that is thought to target the fluorescent NeutrAvidin-650 (NA650).

[0437] The cellular uptake of NA650 was investigated in HEK293 cells expressing the full-length sortilin receptor (Petersen et al.

[13] ). Cells were plated in poly-L-lysine-coated 96-well plates (Perkin Elmer) (40K / well) in 50 μL of medium (DMEM (Lonza) + 10% FBS (Sigma-Aldrich) + 1% penicillin-streptomycin (Sigma-Aldrich) + 1% GlutaMAX (Gibco) + 100 μg / mL Zeocin (Invitrogen)) in a cell incubator (37°C, humidified, 5% CO2) overnight.

[0438] The medium was replaced with assay medium (DMEM (Lonza) + 10% fetal bovine serum (Sigma-Aldrich) + 1% penicillin-streptomycin (Sigma-Aldrich) + 1% GlutaMAX (Gibco)) containing NA650 (Invitrogen) (100 nM) (Thermo Fischer) and peptidic bifunctional compounds (9.8 nM to 10 μM). After incubating the cells for 3 h in a cell incubator, they were washed with dPBS (Bionordica). The DyLight650 signal of the cell layer was detected using a fluorescent plate reader (BMG Labtech Clariostar).

[0439] Figure 3A shows the cell-associated FI signal after 3 hours of incubation of HEK293 / Sortilin with a concentration series 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) (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 tertiary complex formation. Data points are shown as mean ± SEM.

[0440] Figure 3B shows the cell-associated FI signal after 3 hours of incubation of HEK293 / Sortilin with SEQ ID NO:147 (300 nM) and NA650 (Invitrogen) (100 nM) in the presence of the small molecule sortilin binding agent AF38496 (Schroder et al. [8]) (0.1 nM to 100 μM), SEQ ID NO:172, a peptide without a biotin warhead (0.025 nM to 25 μM), or biotin alone (0.1 nM to 100 μM) (n=2). The data show that degrader-induced internalization of NA650 is inhibited by competitive binding to sortilin by AF38499 (IC50 of 589 nM) or the RQLL peptide (IC50 of 149 nM) and by competitive binding to the target by biotin (IC50 of 56 nM). Data points are shown as mean ± SEM.

[0441] conclusion This example demonstrates that a bifunctional compound consisting of a sortilin-binding motif linked to a biotin warhead mediates the uptake of extracellular NA650. Furthermore, the data provided demonstrate that target internalization is dependent on binding of the bifunctional compound to the sortilin receptor and the target.

[0442] Example 4: Sorting of targeted molecules to lysosomes for degradation the purpose This example addresses S LThe ability of peptide-derived bifunctional compounds to mediate the transport of extracellular targets through the endolysosomal pathway for degradation within lysosomes was investigated by fluorescence microscopy of the intracellular localization of internalized targets and by on-blot assessment of intracellular target levels after inhibition of lysosomal function.

[0443] Materials and Methods The cellular uptake of target NA650 was analyzed by fluorescence microscopy. For this assay, coverslips in 4-well plates were coated with poly-L-lysine, and HEK293 / Sortilin cells (25K cells / coverslip) were then seeded in culture medium (as described in Example 3) and incubated overnight in a cell incubator (37°C, humidified, 5% CO2). The culture medium was replaced with assay medium (as described in Example 3) containing NA650 (Invitrogen) (500 nM) (Thermo Fischer), the compound according to SEQ ID NO: 147 (2 μM), and AF38469 (100 μM, MedChemExpress). After 4 hours of incubation in the cell incubator, the cells were washed with dPBS (Bionordica) and subsequently fixed with dPBS + 4% PFA (Sigma Aldrich) for 15 minutes. Cells were permeabilized with TBS + 0.1% Triton X-100 (Sigma-Aldrich) for 15 minutes, washed with dPBS, and incubated with primary antibody (LAMP-1 mAb H4A3, Abcam Ab25630) overnight at 4°C. Incubation with secondary antibody (goat anti-mouse Alexa-Fluor 488 (ThermoFisher)) was performed at room temperature for 2 hours, followed by washing with dPBS and incubation in Hoechst (1:10,000, Sigma-Aldrich) for 10 minutes. Coverslips were briefly immersed in 70% ethanol (VWR) and mounted on glass slides using fluorescent mounting medium (Agilent Dako). Fluorescence signals were analyzed using a laser scanning confocal microscope (LSM-800).

[0444] Figure 4A provides fluorescence microscopy imaging data showing that target NA650 (DyLight650) co-localizes with a lysosomal marker in HEK293 / Sortilin cells fixed and immunostained with anti-LAMP1 (Alexa-488) after 2 hours of incubation with bifunctional molecules SEQ ID NO: 147 (300 nM) and NA650 (NA650) (100 nM). Nuclei were stained with Hoechst. Scale bar is 20 uM. The intracellular levels of NA650 after inhibition of lysosomal function were investigated in HEK293 / Sortilin cells. Cells were seeded in 250 μL of medium (as described in Example 3) on poly-L-lysine-coated 4-well plates (Thermo Fisher) (250K / well) and incubated overnight in a cell incubator.

[0445] The culture medium was replaced with assay medium (DMEM (Lonza), 10% fetal bovine serum (Sigma-Aldrich), 1% penicillin-streptomycin (Sigma-Aldrich), 1% GlutaMAX (Gibco)) containing or without NA650 (Invitrogen) (100 nM) (Thermo Fischer), peptidic bifunctional compound (300 nM), and leupeptin (80 μM) (Sigma-Aldrich). After 24 h of incubation in a cell incubator, the cells were washed with dPBS (Bionordica) and lysed in lysis buffer (STE buffer (Sigma-Aldrich) + 1% Nonidet™ P40 replacement (Sigma-Aldrich) + cOmplete (Roche)). The lysates were centrifuged, and the supernatants were mixed with LDS sample buffer (Thermo Fisher) and DTT (Sigma-Aldrich). After boiling for 5 min, proteins were separated on a 4-12% Bis-Tris gel (Thermo Fisher). Proteins were blotted onto nitrocellulose membranes (Thermo Fisher), and DyLight 650 signals were detected using an iBright 1500 (Thermo Fisher).

[0446] The membrane was blocked for 1 hour in blocking buffer (TBS (Fisher Scientific) containing 1% Tween 20 (Sigma-Aldrich) and 5% milk powder) 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 wash 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. After three washes in wash buffer, the blot was developed and analyzed using ECL reagent (Cytiva) and iBright 1500 (Thermo Fisher Scientific).

[0447] Figure 4B provides data further demonstrating intracellular accumulation of the target after inhibition of lysosomal cysteine ​​protease activity by the addition of leupeptin (80 μM). In the example shown, cells were incubated with 100 nM NA650 (Invitrogen), a peptidic bifunctional compound (300 nM), and leupeptin (80 μM) (Sigma-Aldrich) for 24 hours, followed by harvesting of cell lysates and separation of proteins on SDS-PAGE gels. The bar graph shows quantification of the upper NA650 band (average of the standard).

[0448] conclusion This example demonstrates that the target of the bifunctional compound is sorted from the extracellular space to the lysosomal compartment for subsequent degradation.

[0449] Example 5: Depletion of target molecules from the extracellular medium the purpose In this example, S L We investigate whether the peptide-derived bifunctional compounds are able to deplete targets from the extracellular space, as addressed by evaluation of fluorescence intensity (FI) in cell culture supernatants after incubation of cells with the bifunctional compounds and NA650.

[0450] Materials and Methods 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 selective antibiotic 100 μg / mL Zeocin (Invitrogen). 35k cells / well were seeded into poly-L-lysine-coated 96-well plates (Perkin Elmer) and incubated for 16–24 h in a cell incubator (37°C, humidified, 5% CO). 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). This medium was discarded and replaced with assay medium or assay medium containing 100 nM NA650 (Invitrogen). In wells assessing bifunctional compounds, the assay medium also contained concentrations ranging from 20 nM to 5 μM. After an incubation period of up to 72 h in a cell incubator, the medium was collected and the presence of NA650 was assessed by measuring the fluorescence intensity (FI) of DyLight650 using a fluorescent plate reader (BMG Labtech Clariostar).

[0451] Figure 5A shows the NA650 signal in HEK293 / Sortilin cell culture supernatants after incubation with SEQ ID NO:147 (20 nM to 10 μM) and NA650 (Invitrogen) (100 nM) for up to 72 hours. A single addition of the bifunctional compound results in a significant decrease in signal after 24 hours (16%), 48 hours (46%), and 72 hours (61%) of incubation. At all time points, the highest target uptake is observed with 312 nM of degrader.

[0452] Figure 6 shows the NA650 FI signal in HEK293 / Sortilin cell culture supernatants after incubation with NA650 (Invitrogen) (100 nM) and SEQ ID NO: 147 (20 nM-5 μM) or SEQ ID NO: 155 (20 nM-5 μM). The bifunctional compound SEQ ID NO: 155, with improved affinity for Sortilin, results in further reduction of target in the cell culture supernatant compared to SEQ ID NO: 147 after 48 and 72 hours.

[0453] conclusion This example demonstrates that the target concentration in the medium decreases over time, indicating target depletion by the bifunctional compound and suggesting a more sustainable mechanism of action.

[0454] Example 6: Peptide conjugation. the purpose In this example, the equilibrium dissociation constant (KD), representing the binary binding event between a peptide and the soluble ectodomain of sortilin-6his, is measured by microscale thermophoresis (MST).

[0455] Materials and Methods MST was performed as described in Example 1.

[0456] [Table 7] TIFF2025530891000115.tif54159 Conclusion In this example, we assess whether a functional peptide binds directly to the soluble ectodomain of sortilin and quantify the binding event by measuring the dissociation constant at equilibrium.

[0457] Example 7: Conjugation of sortilin-binding peptides to antibodies the purpose In this example, the monoclonal antibody alirocumab (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 conjugated to a peptide configured at one end with an NHS group linked to the C-terminal sortilin-binding peptide sequence RQLL, thereby generating the bifunctional alirocumab-link-RQLL (SEQ ID NO:159) or adalimumab-link-RQLL (SEQ ID NO:161). A negative control sample is made by conjugating alirocumab to the same peptide as above in all respects except lacking the C-terminal peptide sequence RQLL, generating the sample alirocumab-link (SEQ ID NO:160) or adalimumab-link (SEQ ID NO:162) linkage.

[0458] Materials and Methods The NHS-containing peptides NHS-PEG4-Mal-CGGSGGGGSGGGGSGGRQLL (SEQ ID NO: 168) and NHS-PEG4-Mal-CGGSGGGGSGGGGSGG (SEQ ID NO: 169) were solubilized to 5 mM in DMSO and added in 25 molar excess to a 4 mg / ml antibody solution in PBS. The reaction was incubated at room temperature for 24 hours, at which point unreacted NHS-containing peptide was removed by applying the sample to a 2 mL Zeba spin desalting column. Two separate samples, Antibody-Link-RQLL and Antibody-Link, were prepared following this protocol. The reaction was followed by SDS-PAGE analysis, as shown in Figures 7A and 7B.

[0459] Figure 7A shows SDS-PAGE analysis of the conjugation of NHS-linker (SEQ ID NO: 169) or NHS-linker-RQLL (SEQ ID NO: 168) to alirocumab under non-reducing or reducing conditions (2 mM DTT + heat). Successful conjugation of NHS-linker and / or NHS-linker-RQLL to alirocumab was observed, as seen by the mass shift compared to the unconjugated antibody.

[0460] Figure 7B shows SDS-PAGE analysis of the conjugation of NHS-linker or NHS-linker-RQLL to adalimumab under non-reducing or reducing (2 mM DTT + heat) conditions. Successful conjugation of NHS-linker (SEQ ID NO: 169) and / or NHS-linker-RQLL (SEQ ID NO: 168) to adalimumab was observed, as seen by the mass shift compared to the unconjugated antibody.

[0461] conclusion The compounds of the invention can be covalently bound to an antibody.

[0462] Example 8: Bifunctional compounds derived from antibody conjugates bind to sortilin with nM affinity the purpose In this example, the equilibrium dissociation constant (KD) representing the binary binding event between a monoclonal antibody conjugated to a sortilin binding agent (SEQ ID NO: 159) and the soluble ectodomain of sortilin-6his is measured by microscale thermophoresis (MST).

[0463] Materials and Methods MST binding experiments of alirocumab-link-RQLL (SEQ ID NO: 159) or alirocumab-link (SEQ ID NO: 160) solutions were performed using a protocol adapted from Example 1.

[0464] Figure 8 shows the MST binding response as a function of antibody concentration. From this experiment, a clear sigmoidal dose response was observed for alirocumab conjugated to a peptide containing the sortilin binding sequence (SEQ ID NO: 159 alirocumab-link-RQLL), resulting in a dissociation constant of 540 nM, whereas when alirocumab was conjugated to a peptide lacking the C-terminal sortilin binding sequence (alirocumab-link, SEQ ID NO: 160), it did not produce a sigmoidal binding curve. This means that this antibody does not bind to sortilin with a measurable dissociation constant.

[0465] conclusion Covalent coupling of the compounds of the invention to the antibody results in sortilin binding.

[0466] Example 9: Antibody-derived bifunctional compounds retain binding to antigen the purpose In this example, we further evaluated whether alirocumab-linker-RQLL and alirocumab-link retained their ability to bind to PCSK9-6HIS by gel filtration.

[0467] Materials and Methods Alirocumab-link-RQLL (SEQ ID NO: 159) or Alirocumab-link (SEQ ID NO: 160) was mixed with PCSK9-6HIS (SEQ ID NO: 171) at a molar ratio of 1:2.5 (1.67 μM Alirocumab and 4.175 μM PCSK9-6HIS) in a final volume of 200 μL and incubated at 4°C for 24 hours. The following day, samples were centrifuged at 13,400 rpm for 15 minutes at 4°C to remove potential aggregates, and the samples were loaded onto a Superdex200 increase 10 / 300 (Cytiva) column connected to an Akta Pure HPLC system (Cytiva). For each experiment, 0.5 mL fractions were collected and analyzed by SDS-PAGE (reducing and non-reducing). A negative control (PCSK9-6HIS alone) was prepared according to the same procedure as above.

[0468] Figure 9 shows that a complex between PCSK9-6HIS and alirocumab-linker-RQLL (Figure 9A) or alirocumab-linker (Figure 9B) can be formed and purified by gel filtration, as evidenced by the SDS-PAGE analysis in Figure 9C of peak fractions from the protein complex, which shows both a decrease in the peak size of PCSK-6HIS and its coelution with alirocumab-linker-RQLL.

[0469] conclusion Complex formation between the target and a) alirocumab-linker-RQLL (SEQ ID NO: 159) or b) alirocumab-linker (SEQ ID NO: 160) was possible and could be purified by gel filtration, as evidenced by c) SDS-PAGE analysis of peak fractions from the protein complex, which showed both a decrease in the peak size of PCSK-6HIS and co-elution with alirocumab-linker-RQLL.

[0470] Example 10: Tertiary complex formation with antibody-derived bifunctional compounds. the purpose Addressed in this example is the ability of monoclonal antibodies conjugated to sortilin-binding peptides to mediate the formation of ternary protein complexes with target proteins and sortilin.

[0471] Materials and Methods In these experiments, a Mosquito LV pipetting robot (SPT Labtech) was used to titrate the bifunctional compounds alirocumab-link-RQLL (SEQ ID NO: 159) and adalimumab-link-RQLL (SEQ ID NO: 161) into a 384-well TTP LVDS plate (SPT Labtech) in a total volume of 4 μL over 24 points to a final buffer composition of 50 mM Bis-Tris propane (pH 8), 50 mM NaCl, 0.05% Tween 20. The same study was performed with antibody conjugates lacking the binding motif (alirocumab-link SEQ ID NO: 160 and adalimumab-link SEQ ID NO: 162).

[0472] PCSK9 target: To a black 384-well plate (Corning) was added the following: 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, 50 mM Bis-Tris propane (pH 8), 50 mM NaCl, 0.05% Tween 20, 5 μL of 200 nM sortilin-6His in ... 4 μL of 200 nM biotin-avi-PCSK9 in bis-Tris propane (pH 8), 50 mM NaCl, 0.05% Tween 20, and 4 μL of alirocumab-link-RQLL (SEQ ID NO: 159) or alirocumab-link (SEQ ID NO: 160) in 50 mM Bis-Tris propane (pH 8), 50 mM NaCl, 0.05% Tween 20.

[0473] TNF-α target: To a black 384-well plate (Corning) was added the following: 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 Bis-Tris propane (pH 8), 50 mM NaCl, 0.05% Tween 20, 5 ... 4 μL of 200 nM TNF-α-biotin in Bis-Tris propane (pH 8), 50 mM NaCl, 0.05% Tween 20, and 4 μL of adalimumab-link-RQLL (SEQ ID NO: 161) or adalimumab-link (SEQ ID NO: 162) in 50 mM Bis-Tris propane (pH 8), 50 mM NaCl, 0.05% Tween 20.

[0474] After 2.5 hours of incubation at room temperature, the HTRF signal ratio 665 / 620 nm was determined on a ClarioStar plate reader (BMG Labtech). All reactions were performed in duplicate, and data were expressed as the HTRF ratio as a function of bifunctional compound concentration.

[0475] Figure 10A shows the HTRF (665 / 620 nm) ratio normalized to the alirocumab-link plotted against a concentration gradient (290 nM to 3.5 fM) of alirocumab conjugated to a linker with or without RQLL after 2.5 hours of incubation. This indicates that the formation of the ternary complex, visualized by the bell-shaped curve, is dependent on the RQLL motif. (Error bars = SD, n = 2) Figure 10B shows the resulting HTRF (665 / 620 nm) signal, baseline corrected to the lowest global signal, plotted against a concentration gradient (400 nM-4.7 fM) of adalimumab conjugated to a linker with or without RQLL after 2.5 hours of incubation. This indicates that the formation of the ternary complex, visualized by the bell-shaped curve, is dependent on the RQLL motif. (Error bars = SD, n = 3) Figure 10C shows the resulting HTRF (665 / 620 nm) signal, baseline corrected to the lowest global signal, plotted against a concentration gradient (400 nM-4.7 fM) of adalimumab conjugated to sortilin-binding peptides SEQ ID NO: 184-SEQ ID NO: 187 after 2.5 hours of incubation, indicating the formation of a ternary complex visualized by a bell-shaped curve (error bars = SD, n = 3).

[0476] conclusion The compounds of the present invention mediate the formation of a ternary complex between sortilin and a target.

[0477] Example 11: Antibody-derived bifunctional compounds mediate uptake of extracellular targets the purpose Addressed in this example is the ability of sortilin to promote the internalization of the extracellular target of a monoclonal antibody conjugated to a sortilin-binding peptide.

[0478] Materials and Methods In this example, a bifunctional antibody conjugate targeting PCSK9 alirocumab-link-RQLL (SEQ ID NO: 159) and TNFα adalimumab-link-RQLL (SEQ ID NO: 161) is presented.

[0479] Cellular uptake was measured as in Example 3 using Cy5 (Thermo Fisher) labeled targets (100-200 nM).

[0480] Figure 11A shows the cell-associated FI signal after a 3-hour incubation of HEK293 / Sortilin with a concentration series (1 nM to 0.5 μM) of alirocumab-RQLL (SEQ ID NO: 159) and Cy5-PCSK9 (200 nM). In a control experiment, cells were incubated with alirocumab and a peptide lacking the C-terminal sortilin-binding sequence alirocumab-link (SEQ ID NO: 160).

[0481] Figure 11B shows the cell-associated FI signal after a 3-hour incubation of HEK293 / Sortilin with a concentration series (250 pM to 250 nM) of adalimumab-link-RQLL (SEQ ID NO: 161) and TNFα (100 nM). In a control experiment, cells were incubated with adalimumab and a peptide lacking the C-terminal sortilin binding sequence adalimumab-link (SEQ ID NO: 162).

[0482] FIG. 11C shows the cell-associated FI signal after a 3-hour incubation of HEK293 / Sortilin with a concentration series of adalimumab (250 pM to 250 nM) conjugated to sortilin-binding peptides of SEQ ID NOs: 184 to 187 and TNFα (100 nM).

[0483] conclusion This example demonstrates that monoclonal antibodies conjugated to sortilin-binding peptides can mediate the uptake of extracellular disease proteins, such as PCSK9 or TNFα.

[0484] Example 12: Production of anti-LC VHH products: the purpose In this example, a bifunctional nanobody conjugated to a sortilin-binding peptide is generated that can bind both a kappa light chain antibody and sortilin (NB-linker-RQLL, SEQ ID NO: 158). A control without the RQLL motif is also prepared (NB-linker, SEQ ID NO: 71).

[0485] Materials and Methods 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 being plated on plates containing 100 micrograms / ml ampicillin. Cultures were grown overnight in 100 ml of LB medium (Luria-Bertani) at 30°C, 100 micrograms / ml ampicillin, and 175 RPM. Cell pellets from overnight cultures were resuspended in 1 liter of 2xYT medium (tryptone 16 g / l, yeast extract 10 g / l, NaCl 5 g / l, 100 micrograms / ml ampicillin) and incubated at 30°C and 200 RPM. At an OD 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.

[0486] After harvesting, cells were resuspended in 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 20 mM imidazole, and 1 mM PMSF and lysed by sonication. The lysate was clarified by centrifugation, and the supernatant was loaded onto a 5 mL HisTrap FF Crude column (Cytiva) equilibrated in 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 20 mM imidazole. The loaded column was washed to baseline, and elution was performed with a linear gradient of 20–300 mM imidazole. The protein was dialyzed overnight against 20 mM sodium acetate (pH 5.5), 50 mM NaCl (MWCO: 6–8 kDa) at 4°C and subsequently concentrated using a centrifugal concentrator (MWCO: 5 kDa). 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.

[0487] Figure 12 shows the size exclusion chromatography elution profiles and final sample of A) NB-Linker-RQLL (SEQ ID NO: 158) and NB-Linker (SEQ ID NO: 71). The pooled and concentrated fractions from size exclusion are shown in grey boxes.

[0488] Example 13: Nanobody-derived bifunctional compounds bind to target molecules the purpose 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 an antibody containing a kappa light chain. Alirocumab is used as a tool antibody with a kappa light chain in the example.

[0489] Materials and Methods NB-Linker-RQLL, SEQ ID NO: 158, and NB-Linker, SEQ ID NO: 71, were mixed with the tool antibody at a molar ratio of 2.2:1 in a final volume of 250 μL and incubated at room temperature for 1 hour. The sample was centrifuged at 13,400 rpm at 4°C for 15 minutes to remove potential aggregates, and the sample was loaded onto a Superdex75 Increase 10 / 300 (Cytiva) column connected to an Äkta Pure HPLC system (Cytiva). For each experiment, 0.5 mL fractions were collected and analyzed by SDS-PAGE. A negative control (NB-Linker alone) was prepared according to the same procedure as above.

[0490] Figure 13 shows that complexes formed between the IgG target and (Figure 13A) the NB-linker RQLL, SEQ ID NO: 158 or (Figure 13B) the NB-linker, SEQ ID NO: 71, can be formed and purified by gel filtration. SDS-PAGE analysis of the main peak confirms the presence of the complex with the target protein. The pooled and concentrated fractions from size exclusion are shown in grey boxes.

[0491] conclusion NB-Linker-RQLL, SEQ ID NO: 158, is capable of binding to and forming a complex with the target IgG. The complex can be separated by size exclusion chromatography and confirmed by analysis on SDS-PAGE, which shows co-elution of the target protein and the Nanobody bifunctional compound.

[0492] Example 14: Lysosomal degradation of extracellular targeted IgG the purpose This example addresses cellular uptake and degradation of an extracellular target by a nanobody with the sortilin-binding peptide sequence RQLL and a human IgG kappa light chain binding sequence (NB-linker-RQLL, SEQ ID NO: 158).

[0493] Materials and Methods The cellular uptake of Cy5-conjugated IgG was investigated in HEK293 cells expressing the sortilin receptor using methodology similar to that described in Example 3.

[0494] FIG. 14A shows the cell-associated FI signal after a 3-hour incubation of HEK293 / Sortilin with a concentration series of NB-Linker-RQLL, SEQ ID NO: 158 (8 nM to 8 μM), and Cy5-conjugated IgG (5, 50, or 500 nM).

[0495] In separate experiments, 35K or 250K cells were seeded in 4-well or 96-well plates (Thermo Fisher) in 100 or 250 μL of medium (as described in Example 3) and incubated overnight in a cell incubator (37°C, humidified, 5% CO2).

[0496] The medium was replaced with assay medium described in Example 3 containing SEQ ID NO: 158 or control SEQ ID NO: 71 (0-2 μM) and Cy5-conjugated IgG (50 nM). Additionally, leupeptin (80 μM) was added to a subset of samples to inhibit lysosomal proteases.

[0497] The cells were incubated in a cell incubator (for up to 72 hours), after which the cell supernatant was collected and the cells were harvested and lysed (as described in Example 4). The lysates and medium were subjected to SDS-PAGE (as described in Example 4). Proteins were blotted onto nitrocellulose membranes (Thermo Fisher), and Cy5 signals were detected using an iBright 1500 (Thermo Fisher). Alternatively, IgG was detected by Western blotting (as described in Example 4) using an anti-human IgG antibody (1:3500, Abcam) or an HRP-conjugated anti-rabbit IgG (1:3500, Abcam).

[0498] After anti-human IgG Western blotting, the membrane was probed with mouse anti-human β-actin (1:3500, Sigma-Aldrich) and HRP-conjugated anti-mouse IgG (1:3500, Abcam). β-actin blots were used as loading controls.

[0499] The membranes were then stripped in Restore stripping buffer (Thermo Scientific) for 15 min at 37°C and reprobed with anti-human NTR3 (sortilin) ​​Ab (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).

[0500] Figure 14B shows Cy5 FI in cell culture supernatants (top blots) and lysates (middle blots) of HEK293 / Sortilin cells after 72 hours of incubation with Cy5-conjugated IgG (κ-light chain) (50 nM) and NB-linker-RQLL, SEQ ID NO: 158, as indicated. The intensity of the bands corresponding to the IgG heavy chain (Ig HC) and light chain (Ig LC) decreases in the medium when the target is co-incubated with increasing concentrations of nanobody. No IgG bands are detectable in the culture supernatant from cells incubated with the highest nanobody concentration, indicating complete depletion of the target from the medium. In the corresponding lysate samples, a weak Ig LC signal and lower weight degradation products are detected. No Ig HC signal is observed in the cell lysates, indicating that the internalized IgG target is rapidly degraded after internalization. The lower blot confirms intracellular sortilin expression by Western blotting.

[0501] Figure 14C shows Cy5 FI (upper blot) and IgG Western blot signals in HEK293 / Sortilin cell lysates after 6 h 30 min of incubation with Cy5-conjugated IgG (50 nM), NB-linker-RQLL, SEQ ID NO: 158, or control nanobody (NB-linker, SEQ ID NO: 71) (250 nM), as indicated, and the lysosomal protease inhibitor leupeptin (80 μM) (n=2). Upon incubation with leupeptin, an increase in the intensity of the bands corresponding to Ig HC and Ig LC is observed. Furthermore, no degradation products are observed. Taken together, this indicates that IgG is internalized and subsequently degraded in lysosomes. In the lower two blots, Western blotting confirms gel loading (anti-β-actin) and intracellular sortilin expression. The bar graph shows quantification of Ig HC as mean + / - SEM.

[0502] conclusion Anti-LC nanobody-derived bifunctional compounds mediate cellular uptake and proteolysis of extracellular IgG via lysosomal degradation.

[0503] Example 15: Binding of small molecule bifunctional compounds to sortilin the purpose In this example, the equilibrium dissociation constants (KD) representing the binary binding events between different compounds and the soluble ectodomain of sortilin-6his are measured by microscale thermophoresis (MST).

[0504] Materials and Methods MST was performed as described in Example 1.

[0505] Figure 15 shows the binding of BF025, BF023, and BF020 (top concentration 12.5 μM) titrated with constant NTA Sortilin-6His (100 nM) in an MST assay. (Error bars = SD, n = 2) [Table 8] TIFF2025530891000117.tif42159

[0506] conclusion The small molecule compounds of the present invention bind to sortilin.

[0507] Example 16: S L Ternary complex formation promoted by bifunctional compounds derived from small molecules the purpose The ability of the bifunctional compounds to form ternary complexes with sortilin and target molecules is demonstrated.

[0508] Materials and Methods In these experiments, bifunctional compounds were titrated into 384-well TTP LVDS plates (SPT Labtech) using a Mosquito LV pipetting robot (SPT Labtech) in a total volume of 5 μL over 24 points to a final buffer composition of 50 mM Bis-Tris propane (pH 8), 50 mM NaCl, 0.05% Tween 20, and 8% DMSO.

[0509] The plates were transferred to a black 384-well plate (Corning) and the following was added: 5 μL of 8 nM MAb anti-6HIS in 50 mM Bis-Tris propane (pH 8), 50 mM NaCl, 0.05% Tween 20. Tb (Cisbio), 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, 5 μL of bifunctional compound in 50 mM Bis-Tris propane (pH 8), 50 mM NaCl, 0.05% Tween 20 with or without 8% DMSO.

[0510] After 2.5 h of incubation at room temperature, the HTRF signal ratio 665 / 620 nm was determined on a ClarioStar plate reader (BMG Labtech). All reactions were performed in triplicate.

[0511] Figure 16 shows the resulting HTRF (665 / 620 nm) signal, baseline corrected for the lowest local signal, plotted against bifunctional compound concentration (5 μM to 0.6 pM) after 2.5 hours of incubation. The dual interaction with sortilin by the bifunctional compound was demonstrated as visualized by the bell-shaped curve.

[0512] [Table 9]

[0513] conclusion The compounds of the present invention mediate the formation of a ternary complex between sortilin and a warhead binding target.

[0514] Example 17: Internalization of target extracellular proteins in cells mediated by bifunctional compounds the purpose This example addresses: L is the ability of sortilin to promote the internalization of extracellular targets by small molecule-derived bifunctional compounds.

[0515] Materials and Methods In this example, the small molecule degrader consists of a sortilin binding agent linked to a warhead biotin (BF017-BF028) to target the fluorescent neutravidin-650 (NA650).

[0516] The cellular uptake of NA650 was examined in HEK293 cells expressing the full-length sortilin receptor using methodology similar to that in Example 3.

[0517] FIG. 17 shows the cell-associated FI signal after a 3-hour incubation of HEK293 / Sortilin with a concentration series of compounds B025, B023, or B020 (2 nM to 2 μM) along with NA650 (100 nM).

[0518] [Table 10]

[0519] conclusion The compounds of the present invention mediate cellular internalization of the target.

[0520] Example 18: Bifunctional compounds mediate target depletion from extracellular cell culture medium the purpose Addressed in this example is the ability of small molecule bifunctional compounds to deplete targets from the extracellular space as investigated by assessment of fluorescence intensity (FI) in cell culture supernatants after co-incubation of cells with the bifunctional compounds and NA650.

[0521] Materials and Methods HEK293 / Sortilin cells (35k cells / 96 well) were seeded in medium (as described in Example 3) and incubated for 24 hours in a cell incubator. The medium was discarded, and the cells were washed with assay medium containing FluoroBright DMEM (Gibco) and supplements as described in Example 3. They were then incubated in assay medium containing 100 nM NA650 and small molecule bifunctional compounds (20 nM to 5 μM). After an incubation period of up to 72 hours, the medium was collected, and the presence of NA650 was assessed by measuring the FI of DyLight650 using a fluorescent plate reader (BMG Labtech Clariostar).

[0522] FIG. 18 shows the NA650 FI signal in HEK293 / Sortilin cell culture supernatants after incubation of NA650 (100 nM) with bifunctional compounds BF025, BF023, or BF020 (20 nM-5 μM) as shown for 72 hours.

[0523] conclusion This example demonstrates that small molecule bifunctional compounds can induce target depletion from cell culture supernatants.

[0524] Example 19: Targeting bifunctional compounds for sorting from the extracellular space to lysosomes for degradation. the purpose This example investigates the ability of small molecule bifunctional compounds to mediate target degradation in lysosomes, which was studied by assessing fluorescent target levels in blotted cell lysates after inhibition of lysosomal function.

[0525] Materials and Methods The 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) in 250 μL of medium as described in Example 3 and incubated overnight.

[0526] The medium was replaced with assay medium (as described in Example 3) containing or without NA650 (100 nM) (Thermo Fisher), B025 (300 nM), sortilin binding agent AF38469 (10 μM), and leupeptin (80 μM) (Sigma-Aldrich). After 24 h of incubation in a cell incubator, the cells were washed and lysed in lysis buffer (as described in Example 4). The lysates were centrifuged, and the supernatants were mixed with LDS sample buffer (Thermo Fisher) and DTT (Sigma-Aldrich). After boiling for 5 min, proteins were separated on a 4-12% Bis-Tris gel (Thermo Fisher). Proteins were blotted onto nitrocellulose membranes (Thermo Fisher), and DyLight650 signals were detected using an iBright 1500 (Thermo Fisher). After FI evaluation, the membranes were blocked for 1 h in blocking buffer (TBS (Fisher Scientific) containing 1% Tween 20 (Sigma-Aldrich) and 5% milk powder) and then incubated with primary anti-β-actin antibody (1:3500) (Sigma-Aldrich) for 2 h at room temperature. The blots were washed three times in wash 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 h at room temperature. After three washes in wash buffer, the blots were developed and analyzed using ECL reagent (Cytiva) and iBright 1500 (Thermo Fisher Scientific).

[0527] Figure 19 shows the FI signal in HEK293 / Sortilin lysates collected after 24 hours of incubation with NA650 (100 nM), B025 (300 nM), AF38469 (10 μM), and leupeptin (80 μM) as indicated. A band corresponding to NA650 is visible in cells incubated with B025 alone but is not seen when cells are co-incubated with the competing sortilin-binding agent AF38469. Furthermore, the band increases in cells incubated with leupeptin. The low molecular weight NA650 degradation products are most intense in cells in which lysosomal activity is not inhibited by leupeptin. An anti-β-actin Western blot is shown as a control.

[0528] conclusion This example provides data showing intracellular accumulation of targets after inhibition of lysosomal cysteine ​​protease activity by the addition of leupeptin. This indicates that small molecule bifunctional compound-mediated internalization leads to lysosomal degradation of the targets. Furthermore, the data show that small molecule bifunctional compound-mediated internalization is potently inhibited by the addition of a competitive sortilin-binding agent, thereby promoting sortilin.

[0529] Example 20: Internalization of extracellular targets the purpose Addressed in this example is the ability of sortilin to mediate the internalization of extracellular targets by bifunctional small molecules.

[0530] Materials and Methods In this example, the small molecule degraders consist of sortilin binding agents linked to a warhead DNP to target anti-DNP antibodies (BF001-BF016).

[0531] The cellular uptake of AlexaFluor488-anti-DNP antibody (Thermo Fisher) was investigated in HEK293 cells expressing the full-length sortilin receptor using a methodology similar to that described in Example 3.

[0532] Figure 20 shows the cell-associated FI signal after 3 hours of incubation of HEK293 / Sortilin with a concentration series of compounds BF011, BF006, or BF005 (1.9 nM to 2 μM) and AlexaFluor488-anti-DNP antibody (100 nM) (n=2). This bell-shaped response to increasing concentrations of the bifunctional compound is consistent with the hook effect observed in systems involving the formation of a ternary complex.

[0533] [Table 11]

[0534] conclusion The compounds of the present invention mediate targeted cellular uptake.

[0535] Example 21: Targeting bifunctional compounds for sorting from the extracellular space to lysosomes for degradation.

[0536] the purpose Addressed in this example is the ability of small molecule bifunctional compounds to mediate degradation of targets within lysosomes, which was investigated by assessing fluorescent target levels in blotted cell lysates after inhibition of lysosomal function.

[0537] Materials and Methods The intracellular levels of AlexaFluor488-anti-DNP antibody (Thermo Fisher) after internalization were investigated in HEK293 / Sortilin cells. Cells (250kJ) were seeded in 4-well plates in 250 μL of medium as described in Example 3 and incubated overnight.

[0538] The medium was replaced with assay medium (as described in Example 3) containing AlexaFluor 488-anti-DNP antibody (Thermo Fisher) (100 nM) and BF005 (30 nM). Selected samples of the sortilin inhibitor, AF38469 (10 μM), or the lysosomal protease inhibitor, leupeptin (80 μM) (Sigma-Aldrich) were added. After incubation for 3–6 h 30 min in a cell incubator, the cells were washed with dPBS (Bionordica), and the medium was replaced with assay medium without BF005 and AlexaFluor 488-anti-DNP antibody. The cells were then incubated for up to 24 h in a cell incubator or lysed. After incubation, the cells were lysed in lysis buffer, and the lysates were subjected to SDS-PAGE, followed by protein transfer to a nitrocellulose membrane as described in Example 4. The AlexaFluor 488 FI signal was detected using an iBright 1500 (Thermo Fisher). After evaluation of the FI signal, the membrane was blocked and incubated with a primary anti-β-actin antibody (1:3500) (Sigma-Aldrich) and a secondary anti-mouse HRP antibody (1:3500) (Abcam), and developed and analyzed using ECL reagents (Cytiva) and iBright 1500 (Thermo Fisher Scientific) as described in Example 4. The membrane was then stripped in Restore stripping buffer and probed with an anti-human NTR3 (sortilin) ​​Ab (1:3500, BD Bioscience) and an HRP-conjugated anti-mouse IgG (1:3500, Abcam).

[0539] Figure 21A shows FI in lysates of HEK293 / Sortilin cells incubated with AlexaFluor488-anti-DNP antibody (100 nM) and BF005 (30 nM) for 3 hours before cell lysis as described above. The target and degradant were removed by replacing the medium with assay medium, and the cells were further incubated for 0 to 24 hours. A band corresponding to Ig heavy chain (HC) is visible at 0 hours, and the band intensity decreases with increasing incubation time. 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).

[0540] In another experiment, HEK293 / Sortilin cells were incubated with or without AlexaFluor488-anti-DNP antibody (100 nM), BF005 (30 nM), the sortilin binding agent AF38469 (10 μM), and leupeptin (80 μM). After incubation for 6 h 30 min in a cell incubator, the cells were washed with dPBS and lysed for assessment of intracellular target accumulation as described above.

[0541] Figure 21B shows the FI in lysates of HEK293 / Sortilin cells incubated with AlexaFluor488-anti-DNP antibody (100 nM), BF005 (30 nM), and leupeptin (80 μM) for 6 hours and 30 minutes, as indicated. SB001 (30 nM) and AF38469 (10 μM) were included as controls. A band corresponding to the Ig heavy chain (HC) is visible in lysates from cells co-incubated with the target and the BF005 degrader. The band intensity significantly increases upon inhibition of lysosomal degradation by leupeptin and is absent in cells treated with a competitive sortilin binder (AF38469) or a control degrader without the DNP warhead (SB001). The bar graph shows quantification of the FI signal of the HC band normalized to the β-actin signal (mean ± SEM) (n = 2).

[0542] conclusion This example provides data demonstrating rapid degradation of internalized targets. Furthermore, the data show intracellular accumulation of targets after inhibition of lysosomal cysteine ​​protease activity by the addition of leupeptin, indicating lysosomal degradation of the desired targets. Furthermore, the data show that small molecule bifunctional compound-mediated internalization is potently inhibited by competitive sortilin binding.

[0543] Example 22: Compounds of the present invention mediate cellular uptake of target proteins via sortilin receptors. the purpose This example investigates the role of sortilin in the cellular internalization of extracellular targets mediated by bifunctional compounds of the present invention.

[0544] Materials and Methods Cellular uptake of the target was investigated in untransfected HEK239 cells and HEK293 cells expressing the sortilin receptor using methodology similar to that described in Example 3.

[0545] Figure 22 shows the cell-associated FI signal after a 3-hour incubation of HEK293 or HEK293 / Sortilin with a concentration series (2 nM to 2 μM) of three different bifunctional compounds: SEQ ID NO: 155, NB-Link-RQLL (SEQ ID NO: 158), or a small molecule bifunctional compound (BF005) and the corresponding target, as indicated. The data show a bell-shaped FI response to increasing degrader concentrations in Sortilin-expressing cells for all bifunctional compound modalities. No response is observed in HEK293 cells.

[0546] conclusion This example demonstrates that bifunctional compound-mediated target uptake requires cellular expression of sortilin.

[0547] Example 23: Binding to TNFα the purpose Compounds according to the present disclosure are assessed for binding to TNFα.

[0548] Materials and Methods MST was measured in the same manner as in Example 1 using TNFα-6his.

[0549] result Figure 24A: Detection of binding of titrated BF080, BF081, and BF082 to a constant NTA-labeled TNFα-6His (100 nM) in an MST assay. (Error bars = SD, n = 2)

[0550] [Table 12]

[0551] Example 24: Cellular uptake of TNFα mediated by bifunctional compounds the purpose Compounds according to the present disclosure are evaluated for binding to TNFα.

[0552] Materials and Methods The cellular uptake of TNFα was examined in HEK293 cells expressing the full-length sortilin receptor using methodology similar to that in Example 3 with the TNFα target.

[0553] Figure 24B shows a mouse anti-TNFα (Invitrogen, MA5-23720) Western blot of cell lysates after 24 hours of incubation of HEK393 / Sortilin with a concentration series of compounds BF040 and BF043 (100 nM to 10 μM) along with TNFα (100 nM). Control cells were incubated without compound and TNFα, or with TNFα alone. An anti-β-actin (Sigma, A5441) Western blot is shown as a control.

[0554] Figure 24C shows the cell-associated FI signal after 24 hours of incubation of HEK293 / Sortilin with a concentration series of BF040, BF043, and BF042 (0.5 nM to 20 μM), and Cy5-TNFalpha (100 nM) in the presence of 80 μM leupeptin. The efficacy of individual bifunctional compounds was assessed as the AUC of TNFalpha internalization (Table 9).

[0555] [Table 13] TIFF2025530891000123.tif36159

[0556] Figure 24D shows the cell-associated FI signal after 24 hours of incubation of HEK293 / Sortilin cells with BF042 (0.5 nM-20 μM) and Cy5-TNFalpha, with or without the addition of leupeptin (80 μM). Inhibition of lysosomal degradation by the addition of leupeptin results in a significant increase in the cell-associated signal.

[0557] In a separate experiment, HEK293 / Sortilin cells were incubated with a compound gradient series and 20 nM TNFα for up to 72 h. After incubation, residual TNFα in the culture supernatant was assessed using a human TNF-α Quantikine ELISA kit (R&D systems, DTA00D).

[0558] Figure 24E shows TNFα levels in culture supernatants of HEK293 / Sortilin cells 72 hours after addition of 20 nM TNFα and the bifunctional molecules BF040 (BF043) or BF042. TNFα is shown as a percentage normalized to the level measured in culture supernatants from cells without added bifunctional molecules. The maximum clearance rates after 72 hours (calculated as added TNFalpha (100%) minus remaining TNFalpha (%)) were 39.4% for BF040, 63.3% for BF043, and 74.4% for BF042.

[0559] To monitor target cytolysis over time, pulse-chase experiments were performed. HEK293 / sortilin cells were preincubated with bifunctional compounds (3 μM) and TNFα (100 nM) for 24 h, after which the culture supernatant was replaced with fresh medium containing neither target nor bifunctional compound. After medium replacement, cells were incubated for up to 24 h, and TNFα levels were assessed at different time points by Western blotting of cell lysates.

[0560] Figure 24F shows anti-TNFα Western blotting of HEK293 / Sortilin cell lysates harvested at the indicated time points (0 h to 24 h) after 24 h of preincubation with BF042 (3 μM) and TNFα (100 nM). The preincubation medium was replaced at the 0 h time point. Anti-β-actin Western blotting is shown as a control. The cell lysate band representing TNFα was most intense at the 0 h time point, significantly decreased at 1 h, and was barely detectable after 7 h of incubation, indicating rapid degradation of TNFα after internalization.

[0561] Figure 24G shows the cell-associated FI signal after 24 h incubation of HEK293 / Sortilin with a concentration series (0.5 nM to 20 μM) of BF042 and Cy5-TNFα (100 nM) with or without the addition of the sortilin binding agent SB013 (1.25, 5.0, and 10 μM) or DMSO.

[0562] Figure 24H shows the cell-associated FI signal after 24 hours of 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.

[0563] conclusion The compounds of the present invention mediate the cellular internalization and lysosomal degradation of TNFα.

[0564] Example 25: In vivo efficacy in a mouse model of acute systemic inflammation the purpose The ability to remove TNFα from plasma is assessed in an animal model of systemic inflammation.

[0565] Materials and Methods The in vivo efficacy of dissolving agents was investigated in a mouse model of lipopolysaccharide (LPS)-induced acute systemic inflammation. In this model, 8-week-old female C57BL / 6J mice were used, and LPS was administered as an IP injection at a final concentration of 0.5 mg / kg in 10 ml / kg of PBS. Thirty minutes to three hours prior to LPS administration, animals were administered the test compound (100–0.3 mg / kg / IP), dexamethasone (Dex) control (0.5 mg / kg / PO), or vehicle control (IP). Untreated (normal) animals were included as a control for LPS-induced TNFα expression. One hour after LPS administration, blood samples were collected, and TNFα levels were assessed by cytometric bead array assay.

[0566] result Figure 25 shows the plasma levels of TNFα in animals treated with vehicle, Dex (0.5 mg / kg), and BF094 (10 or 30 mg / kg) 1 hour after LPS administration. Normal animals served as untreated controls. n=5 (normal / vehicle / Dex) or n=10 per group. A significant decrease in plasma TNFα is 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).

[0567] conclusion The compounds of the present invention can mediate the degradation of target proteins in vivo. The bifunctional compounds can remove target proteins of interest from the plasma of animals. Specifically, these results show that the TNFα bifunctional compounds reduce TNFα levels in the plasma of LPS-stimulated animals. The reduction in TNFα is comparable to the effect produced by dexamethasone, a known anti-inflammatory agent.

[0568] Example S1: Synthesis Protocol General conditions Compounds according to formula (I) can be prepared from readily available starting materials by several synthetic approaches, using both solution-phase and solid-phase chemical protocols, or mixed solution and solid-phase protocols. Examples of synthetic routes are described below in the Examples. All reported yields are unoptimized. Unless otherwise specified, compounds of formula (I) and related formulas obtained as racemic mixtures can be separated to obtain enantiomerically enriched mixtures or pure enantiomers.

[0569] The standard conditions are as follows: All temperatures are in degrees Celsius (°C) and are uncorrected.

[0570] Reagent grade chemicals and anhydrous solvents were purchased commercially and used without further purification unless otherwise stated.

[0571] Silica gel chromatography was performed on a Biotage instrument using prepackaged disposable SiO2 stationary phase columns with an eluent flow rate range of 15–200 mL / min and UV detection (254 and 280 nm).

[0572] Reverse-phase preparative HPLC was performed using a C18 column eluting with a gradient of aqueous MeCN (10 mM NH4HCO3) or aqueous MeCN (0.04% HCl) or aqueous MeCN (0.2% HCOOH) with UV detection (215, 220, and 254 nm).

[0573] Analytical HPLC chromatograms were performed using two main methods: - A Shimadzu 20AB was used (gradient: 10-80% B in 3.00 min, hold at 80% B for 1.0 min, 80-10% B in 0.01 min, then hold at 10% B for 0.50 min (0.01-4.00 min: 0.5 ml / min flow rate, 4.01-4.50 min: 1.0 ml / min flow rate). 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 a Kinetex 5um C18 100A 50*2.1mm. The detection method was a diode array (DAD). - A Shimadzu 20AD was used (gradient: 10-80% B in 3.00 min, hold at 80% B for 0.9 min, 80-10% B in 0.03 min, then hold at 10% for 0.57 min (0.01-3.91 min: 0.8 ml / min flow rate, 3.92-4.50 min: 1.2 ml / min flow rate). Mobile phase A was 10 mM NH4HCO3 aqueous solution, and mobile phase B was 100% acetonitrile. The column used for chromatography was an X-bridgeshield RP18 2.1*50 mm column (5 um particles). The detection method was a diode array (DAD).

[0574] The products were analyzed by LCMS in two main ways: - Agilent 1200 and 6110B were used (gradient: 5% B in 0.40 min, 5-95% B in 0.4-3.0 min, hold at 95% B for 1.00 min, then 95-5% in 0.01 min, flow rate: 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 a Kinetex C18 50*2.1 mm column (5 μm particles). The detection method was diode array (DAD) and positive electrospray ionization. The MS range was 100-1000. -Agilent 1200 and 6110B were used (gradient: 5% B in 0.40 min, 5-95% B from 0.40 to 3.40 min, hold at 95% B for 0.45 min, then 95-5% B in 0.01 min, flow rate: 0.8 ml / min). Mobile phase A was H2O + 10 mM NH4HCO3, and mobile phase B was acetonitrile. The column used for chromatography was an Xbridge Shield RP18 2.1*50 mm column (5 μm particles). The detection method was diode array (DAD) and positive electrospray ionization. The MS range was 100-1000.

[0575] Intermediates were analyzed by LCMS using two main methods: The column used for chromatography on a Shimadzu LC-20AD&MS2020 was a Luna-C18 2.0*30mm (3µm particles). The detection method was a 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 min, 10% B in 0.01 min, 10-80% B (0.01-3.50 min), 80-10% B (3.50-3.80 min), and hold at 10% B for 0.50 min. The flow rates were 0.8 mL / min (0.01–3.80 min) and 1.2 mL / min (3.81–4.30 min). The chromatography was performed using an Agilent 1200 and 6110B (Agilent 1200 and 1956A columns). The column used for chromatography was an Xbridge Shield RP18 2.1*50mm (5µm particles). The detection method was a diode array (DAD). The MS mode was positive electrospray ionization. The MS range was 100-1000. Mobile phase A was 10mM ammonium bicarbonate in water, and mobile phase B was HPLC-grade acetonitrile. The gradient was 10-80% B in 3.00 min, 10% B in 0.00 min, 10-80% B (0.00-2.00 min), hold at 80% B for 0.48 min, 80-10% B (2.48-2.50 min), hold at 10% B for 0.5 min. The flow rates were 1.0 mL / min (0.00-2.48 min) and 1.2 mL / min (2.50-3.00 min).

[0576] 1H NMR spectra were recorded from 11 instruments and the data are different and are shown below: NMR-A:Bruker AVANCE NEO 400MHz / 54mm device (MRCA 400 / 54 / ASC, 16971) NMR-B: Bruker AVANCE III 400 MHz / 54 mm UltraShield Plus, long retention time, instrument (BZH 439'400'70I, D335 / 54-6776) NMR-C: Bruker AVANCE III 400 MHz / 54 mm Ascend instrument (BZH 994'400'70I, D315'54-9223) NMR-D: Varian 400MR 400 MHz / 54 mm instrument (MRCA 400 / 54 / ASC, MRYOO20874) NMR-E: Bruker AVANCE III 400 MHz / 54 mm Ascend instrument (BZH 993'400'70I, D315'54-9213) NMR-F: Bruker AVANCE III 400 MHz / 54 mm Ascend instrument (BZH 1157'400'70I, D315'54-9574) NMR-H: Bruker AVANCE III 400 MHz / 54 mm Ascend instrument (BZH 1126'400'70I, D315'54-9527) NMR-J: Bruker AVANCE NEO 400 MHz / 54 mm Ascend instrument (BZH 1396'400'70I, D315'54-10089) NMR-K: Bruker AVANCE NEO 400 MHz / 54 mm Ascend instrument (BZH 1373'400'70I, D315'54-10026) NMR-S: Varian 400MR 400 MHz / 54 mm instrument (MRCA 400 / 54 / ASC, 20609) NMR-Y: Varian 400MR 400 MHz / 54 mm instrument (MRCA 400 / 54 / ASC, 20188) Chemical shifts are referenced to the solvent peaks, which appear in 1H NMR at 7.27 ppm for CDCl3, 2.50 for DMSO-d6, 4.79 ppm for D2O, and 3.31 ppm for CD3OD.

[0577] SB001 (2S)-5,5-Dimethyl-2-[[6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl]amino]hexanoic acid [ka] Step 1: 5-hydroxy-N-methyl-naphthalene-1-carboxamide To a solution of methanamine (2.67 g, 39.56 mmol, 8 equiv., HCl) in DCE (40 mL), trimethylaluminum (2 M, 24.73 mL, 10 equiv.) was added dropwise at 0°C. After the addition, the mixture was stirred at this temperature for 15 minutes, and then methyl-5-hydroxynaphthalene-1-carboxylate (1 g, 4.95 mmol, 1 equiv.) in DCE (10 mL) was added dropwise at 0°C. The resulting mixture was stirred at 80°C for 16 hours. The reaction mixture was then cooled to 0°C, and DCM (100 mL) was added, followed by 1N HCl solution, until the aluminum salt (100 mL) was solubilized. The mixture was extracted with DCM (100 mL × 3). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give 5-hydroxy-N-methyl-naphthalene-1-carboxamide (500 mg, 50.24% yield, 100% purity) as a white solid.

[0578] Step 2: Methyl 6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylate A mixture of methyl 6-bromopyridine-3-carboxylate (697.35 mg, 3.23 mmol, 1.2 equiv), 5-hydroxy-N-methyl-naphthalene-1-carboxamide (541 mg, 2.69 mmol, 1 equiv), potassium phosphate (570.70 mg, 2.69 mmol, 1 equiv), and CuI (512.04 mg, 2.69 mmol, 1 equiv) in DMSO (10 mL) was degassed and purged with N three times, and then the mixture was stirred at 120 °C under a N atmosphere for 16 h. The mixture was washed with HO (40 mL) and extracted with EA (20 mL × 4). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure.

[0579] The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, elution with a 0–60% ethyl acetate / petroleum ether gradient at 25 mL / min) to afford methyl 6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylate (580 mg, 64.11% yield, 100% purity) as a white solid.

[0580] Step 3: 6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylic acid To a solution of methyl 6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylate (250 mg, 743.29 μmol, 1 equiv.) in a mixture of THF (15 mL) and HO (5 mL) was added LiOH.HO (62.38 mg, 1.49 mmol, 2 equiv.). 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 × 5). The combined organic layers were dried over NaSO, filtered, and concentrated in vacuo. 6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylic acid (200 mg, 83.48% yield) was obtained as a white solid.

[0581] Step 4: 6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl chloride A mixture of 6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylic acid (200 mg, 620.51 μmol, 1 equiv), oxalyl dichloride (157.52 mg, 1.24 mmol, 108.63 μL, 2 equiv), and DMF (4.54 mg, 62.05 μmol, 4.77 μL, 0.1 equiv) in DCM (20 mL) was degassed at 0 °C, and then the mixture was stirred under a N atmosphere at 25 °C for 5 h. 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.

[0582] Step 5: (2S)-5,5-Dimethyl-2-[[6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl]amino]hexanoic acid To a mixture of (2S)-2-amino-5,5-dimethyl-hexanoic acid (98.59 mg, 619.20 μmol, 1 equiv.) and TEA (93.98 mg, 928.81 μmol, 129.28 μL, 1.5 equiv.) in DCM (5 mL) was added 6-[[5-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl chloride (211 mg, 619.20 μmol, 1 equiv.) in DCM (5 mL). 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 (13.7 mg, 4.77% yield, 99.0% purity) was obtained as a white solid.

[0583] LCMS (ESI) [M+H] + m / z: theoretical value 464.21, measured value 464.1 1 H NMR (400 MHz, DMSO-d6) : ppm 0.85 (s, 10 H) 1.14 - 1.36 (m, 2 H) 1.63 - 1.87 (m, 2 H) 2.87 (d, J=4.52 Hz, 3 H) 4.21 - 4.38 (m, 1 H) 7.27 (d, J=8.53 Hz, 1 H) 7.38 (d, J=7.28 Hz, 1 H) 7.52 (dd, J=8.41, 7.15 Hz, 1 H) 7.58 - 7.67 (m, 2 H) 7.88 (d, J=8.53 Hz, 1 H) 8.10 (d, J=8.53 Hz, 1 H) 8.33 (dd, J=8.78, 2.51 Hz, 1 H) 8.45 - 8.58 (m, 2 H) 8.64 - 8.74 (m, 1 H)

[0584] SB002 2-[[6-methyl-4-[4-(methylcarbamoyl)phenyl]-2-pyridyl]carbamoyl]-5-(trifluoromethyl)benzoic acid [ka] Step 1: 4-(2-amino-6-methyl-4-pyridyl)-N-methyl-benzamide To a mixture of 4-bromo-6-methyl-pyridin-2-amine (1 g, 5.35 mmol, 1 equiv.) and [4-(methylcarbamoyl)phenyl]boronic acid (1.05 g, 5.88 mmol, 1.1 equiv.) in dioxane / HO (18 mL), NaCO (1.70 g, 16.04 mmol, 3.0 equiv.) and Pd(dppf)Cl (391.21 mg, 534.65 μmol, 0.1 equiv.) were added in one portion under N at 25 °C. The mixture was heated to 90 °C and stirred for 4 h. The mixture 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).

[0585] Step 2: 4-[2-[1,3-dioxo-5-(trifluoromethyl)isoindolin-2-yl]-6-methyl-4-pyridyl]-N-methyl-benzamide To a mixture of 5-(trifluoromethyl)isobenzofuran-1,3-dione (89.57 mg, 414.44 μmol, 1 equiv.) and 4-(2-amino-6-methyl-4-pyridyl)-N-methyl-benzamide (100 mg, 414.44 μmol, 1 equiv.) in AcOH (3 mL) was added under N at 25° C. The mixture was heated to 120° C., stirred at 120° C. for 2 h, and then concentrated in vacuo.

[0586] The residue was purified by column chromatography (SiO, PE: EtOAc = 2:1 to 1:1) and concentrated to give 4-[2-[1,3-dioxo-5-(trifluoromethyl)isoindolin-2-yl]-6-methyl-4-pyridyl]-N-methyl-benzamide (120 mg, 273.11 μmol, 65.90% yield, 100% purity) as a yellow oil.

[0587] Step 3: 2-[[6-methyl-4-[4-(methylcarbamoyl)phenyl]-2-pyridyl]carbamoyl]-5-(trifluoromethyl)benzoic acid To a mixture of 4-[2-[1,3-dioxo-5-(trifluoromethyl)isoindolin-2-yl]-6-methyl-4-pyridyl]-N-methyl-benzamide (120 mg, 273.11 μmol, 1 equiv.) in THF / water (3 mL), LiOH (19.62 mg, 819.33 μmol, 3 equiv.) was added in one portion at 25°C under N2 and stirred at 25°C for 5 h. The mixture was acidified to pH 2 with 2 M HCl. DMF (0.5 mL) was added to dissolve the product. The residue was purified twice by preparative HPLC (HCl condition, column: Boston Green ODS 150*30 mm*5 μm; mobile phase: [water (0.05% HCl)-ACN]; B%: 18%-58%, 9 min). After purification by preparative HPLC and lyophilization, (2-[[6-methyl-4-[4-(methylcarbamoyl)phenyl]-2-pyridyl]carbamoyl]-5-(trifluoromethyl)benzoic acid (18.6 mg, 14.52% yield, 97.5% purity) was obtained as a white solid.

[0588] LCMS (ESI) [M+H] + m / z: theoretical value 458.12, measured value 458.0 1 H NMR (400 MHz, DMSO-d6) : ppm 2.50 (br s, 3 H) 2.81 (d, J=4.40 Hz, 3 H) 7.44 (s, 1 H) 7.76 - 7.87 (m, 3 H) 7.97 - 8.06 (m, 3 H) 8.14 (s, 1 H) 8.33 (br s, 1 H) 8.58 (br d, J=4.52 Hz, 1 H) 11.17 (s, 1 H)

[0589] SB003 (2S)-5,5-Dimethyl-2-[[6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl]amino]hexanoic acid [ka] Step 1: 5-hydroxynaphthalene-2-carboxylic acid To a solution of 5-bromonaphthalene-2-carboxylic acid (5 g, 19.91 mmol, 1 equiv.) in dioxane (50 mL) was added KOH (4.47 g, 79.66 mmol, 4 equiv.) in HO (50 mL). tBuXphosPdG3 (395.94 mg, 497.86 μmol, 0.025 equiv.) was then added under N2, and the reaction was stirred at 110 °C under N2 for 16 h. LCMS showed the reaction was complete. The reaction mixture was acidified to pH 4-5 with 1 M HCl and filtered. Water (50 mL) was added to the filtrate, which was then extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with PE:EtOAc (100:1 to 1:1). 5-Hydroxynaphthalene-2-carboxylic acid (3.2 g, 85.39% yield, 100% purity) was obtained as a yellow solid.

[0590] 1 H NMR (400 MHz, DMSO-d6) δ: ppm 13.01 (1 H, br s) 10.32 (1 H, br s) 8.49 (1 H, d, J=1.5 Hz) 8.20 (1 H, d, J=8.8 Hz) 7.90 (1 H, dd, J=8.8, 1.6 Hz) 7.27 - 7.61 (2 H, m) 7.00 (1 H, dd, J=7.5, 0.8 Hz)

[0591] Step 2: 5-hydroxy-N-methyl-naphthalene-2-carboxamide To 5-hydroxynaphthalene-2-carboxylic acid (2.35 g, 12.49 mmol, 1 equiv) in DCM (30 mL) was added DIEA (4.84 g, 37.46 mmol, 6.53 mL, 3 equiv), EDCI (2.87 g, 14.99 mmol, 1.2 equiv), HOBt (2.02 g, 14.99 mmol, 1.20 equiv), and methanamine (843.17 mg, 12.49 mmol, 1 equiv, HCl) at 20 °C under N. The reaction was stirred at 20 °C for 3 h. The reaction mixture was poured into water (100 mL). The aqueous layer was extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine (50 mL), dried over NaSO, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with (PE: EtOAc = 20: 1 to 1: 3).

[0592] 5-Hydroxy-N-methyl-naphthalene-2-carboxamide (2.4 g, 95.51% yield, 100% purity) was obtained as a red oil.

[0593] LCMS (ESI) [M+H]+ m / z: theoretical value 202.0, measured value 202.2 1 H NMR (400 MHz, CHLOROFORM-d) δ: ppm 8.22 - 8.28 (2 H, m) 7.80 (1 H, dd, J=8.8, 1.5 Hz) 7.51 (1 H, d, J=8.3 Hz) 7.37 (1 H, t, J=7.9 Hz) 6.92 (1 H, d, J=7.3 Hz) 6.31 (1 H, br s) 3.10 (3 H, d, J=5.0 Hz)

[0594] Step 3: Methyl 6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylate A mixture of 5-hydroxy-N-methyl-naphthalene-2-carboxamide (2.4 g, 11.93 mmol, 1 equiv.) methyl 6-bromopyridine-3-carboxylate (3.09 g, 14.31 mmol, 1.2 equiv.), copper(I) iodide (227.15 mg, 1.19 mmol, 0.1 equiv.) and potassium phosphate (5.06 g, 23.85 mmol, 2 equiv.) in DMSO (20 mL) was degassed and purged with N three times, then the mixture was stirred at 120 °C under N atmosphere for 16 h. The reaction mixture was poured into water (50 mL) and extracted with EA (3 × 30 mL). The combined organic layers were washed with brine (30 mL), dried over NaSO, filtered and concentrated 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, yield 33.96%, purity 97.3%) as a yellow solid.

[0595] LCMS (ESI) [M+H]+ m / z: theoretical value 336, measured value 337 1 H NMR (400 MHz, CHLOROFORM-d) δ: 8.70 (1 H, d, J=1.9 Hz) 8.21 - 8.33 (2 H, m) 7.87 (1 H, d, J=8.8 Hz) 7.78 (1 H, d, J=8.3 Hz) 7.71 (1 H, dd, J=8.8, 1.6 Hz) 7.51 (1 H, t, J=7.9 Hz) 7.29 (1 H, dd, J=7.6, 0.8 Hz) 6.98 (1 H, d, J=8.6 Hz) 6.21 (1 H, br s) 3.84 (3 H, s) 3.00 (3 H, d, J=4.8 Hz)

[0596] Step 4: 6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylic acid To a solution of methyl 6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylate (1.57 g, 4.67 mmol, 1 equiv.) in a mixture of THF (45 mL) and HO (5 mL) was added LiOH.HO (391.76 mg, 9.34 mmol, 2 equiv.). 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). The residue was extracted with EA (20 mL × 5). The combined organic layers were dried over NaSO, filtered, and concentrated in vacuo. 6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylic acid (1.3 g, 82.09% yield, 95% purity) was obtained as a white solid.

[0597] LCMS (ESI) [M+H] + m / z: theoretical value 323.10, measured value 323.0 1 H NMR (400 MHz, METHANOL-d4) δ: ppm 8.66 (1 H, d, J=2.26 Hz) 8.44 (1 H, d, J=1.51 Hz) 8.39 (1 H, dd, J=8.66, 2.38 Hz) 7.90 - 7.96 (2 H, m) 7.83 - 7.88 (1 H, m) 7.62 (1 H, t, J=7.91 Hz) 7.39 (1 H, d, J=6.78 Hz) 7.16 (1 H, d, J=8.53 Hz) 2.97 (3 H, s)

[0598] Step 5: 6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl chloride A mixture of 6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carboxylic acid (200 mg, 620.51 μmol, 1 equiv), oxalyl chloride (118.14 mg, 930.77 μmol, 81.48 μL, 1.5 equiv), and DMF (4.54 mg, 62.05 μmol, 4.77 μL, 0.1 equiv) in DCM (10 mL) was degassed at 0° C., and then the mixture was stirred under a N atmosphere at 25° C. for 2 h. The reaction mixture was concentrated under reduced pressure to afford 6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl chloride (215 mg, crude) as a yellow gum, which was used directly without further purification.

[0599] LCMS (ESI) [M+OMe] + m / z: theoretical value 337.11, measured value 337.1

[0600] Step 6: (2S)-5,5-Dimethyl-2-[[6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl]amino]hexanoic acid [ka] To a mixture of (2S)-2-amino-5,5-dimethyl-hexanoic acid (100 mg, 628.04 μmol, 1 equiv.) and NaCO (232.98 mg, 2.20 mmol, 3.5 equiv.) in HO (16 mL), dioxane (6 mL), 6-[[6-(methylcarbamoyl)-1-naphthyl]oxy]pyridine-3-carbonyl chloride (214.01 mg, 628.04 μmol, 1 equiv.) was added. The mixture was stirred at 20 °C for 2 h. The reaction mixture was poured into water (40 mL) and extracted with EtOAc (40 mL × 3). The combined aqueous layers were 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 as a white solid.

[0601] LCMS (ESI) [M+H] + m / z: Calculated value 464.21, Measured value 464.2 1 H NMR (400 MHz, DMSO-d6) δ: ppm 8.61 - 8.72 (2 H, m) 8.48 - 8.56 (2 H, m) 8.34 (1 H, dd, J=8.53, 2.51 Hz) 7.96 (1 H, d, J=8.28 Hz) 7.80 - 7.93 (2 H, m) 7.64 (1 H, t, J=7.91 Hz) 7.43 (1 H, d, J=7.03 Hz) 7.29 (1 H, d, J=8.53 Hz) 4.16 - 4.41 (1 H, m) 2.83 (2 H, d, J=4.52 Hz) 2.74 - 2.98 (1 H, m) 1.62 - 1.90 (2 H, m) 1.09 - 1.45 (2 H, m) 0.86 (9 H, s)

[0602] SB004 2-((6-methyl-4-(methylcarbamoyl)pyridin-2-yl)carbamoyl)-5-(trifluoromethyl)benzoic acid [ka] Step 1: 2-chloro-N,6-dimethylisonicotinamide To 2-chloro-6-methyl-pyridine-4-carboxylic acid (5 g, 29.14 mmol, 1 equiv.) in DMF (200 mL) was added methanamine (9.84 g, 145.70 mmol, 5 equiv., HCl), TEA (14.74 g, 145.70 mmol, 20.28 mL, 5 equiv.), and HATU (22.16 g, 58.28 mmol, 2 equiv.). The reaction was stirred under N at 20 °C for 2 h. The reaction was quenched with HO (800 mL). The aqueous layer was extracted with EtOAc (3 × 200 mL). The combined organic layers were washed with brine (2000 mL), dried over NaSO, filtered, and concentrated in vacuo. The residue was 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) was obtained as a yellow solid.

[0603] LCMS (ESI) [M+H] + m / z: theoretical value 185.0, measured value 185.4 H NMR (400 MHz, CHLOROFORM-d) : ppm 7.47 (1 H, s) 7.43 1 H, s) 7.14 (1 H, br s) 2.97 (3 H, d, J=4.9 Hz) 2.54 (3 H, s)

[0604] Step 2: 2-((diphenylmethylene)amino)-N,6-dimethylisonicotinamide To 2-chloro-N,6-dimethyl-pyridine-4-carboxamide (3.4 g, 18.42 mmol, 1 equiv.) and diphenylmethanamine (4.34 g, 23.94 mmol, 4.02 mL, 1.3 equiv.) in toluene (85 mL) was added t-BuONa (4.42 g, 46.04 mmol, 2.5 equiv.), [1-(2-diphenylphosphanyl-1-naphthyl)-2-naphthyl]-diphenyl-phosphane (344.01 mg, 552.48 μmol, 0.03 equiv.), and Pd(dba) (168.64 mg, 184.16 μmol, 0.01 equiv.). The reaction was stirred at 90° C. under N 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) to give 2-(benzhydridoneamino)-N,6-dimethyl-pyridine-4-carboxamide (600 mg, 90% yield, 90% purity) as a yellow solid.

[0605] LCMS (ESI) [M+H] + m / z: theoretical value 330.1, measured value 330.1

[0606] Step 3: 2-Amino-N,6-dimethylisonicotinamide 2-(Benzhydridoneamino)-N,6-dimethyl-pyridine-4-carboxamide (600 mg, 1.82 mmol, 1 equiv) in THF (5 mL) and HCl / HO (5 mL, 10% purity) was stirred at 20 °C for 16 h. The reaction was washed with EtOAc (10 mL). The residue was adjusted to pH 10 with saturated aqueous NaHCO and the aqueous layer was concentrated in vacuo. DCM (10 mL) was added to the residue and stirred for 10 min. 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) as a yellow solid.

[0607] LCMS (ESI) [M+H] + m / z: theoretical value 166.2, measured value 166.1

[0608] Step 4: 2-(1,3-dioxo-5-(trifluoromethyl)isoindolin-2-yl)-N,6-dimethylisonicotinamide 2-Amino-N,6-dimethyl-pyridine-4-carboxamide (170 mg, 1.03 mmol, 1 equiv.) and 5-(trifluoromethyl)isobenzofuran-1,3-dione (222.40 mg, 1.03 mmol, 1 equiv.) in AcOH (4 mL) were stirred at 120 °C under N for 16 h. 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 as a yellow solid.

[0609] LCMS (ESI) [M+H] + m / z: theoretical value 364.1, measured value 364.0

[0610] Step 5: 2-((6-methyl-4-(methylcarbamoyl)pyridin-2-yl)carbamoyl)-5-(trifluoromethyl)benzoic acid To 2-[1,3-dioxo-5-(trifluoromethyl)isoindolin-2-yl]-N,6-dimethyl-pyridine-4-carboxamide (210 mg, 578.05 μmol, 1 equiv.) in THF (4 mL) and HO (4 mL) was added LiOH.HO (72.77 mg, 1.73 mmol, 3 equiv.) and stirred under N at 20 °C for 1 h. The reaction was concentrated, and the residue was purified by preparative HPLC (column: Phenomenex Gemini-NX 150*30 mm*5 μm; mobile phase: [water (0.05% ammonium hydroxide v / v)-ACN]; B%: 4%-44%, 14 min). After preparative HPLC purification, the eluent was lyophilized to give 2-[[6-methyl-4-(methylcarbamoyl)-2-pyridyl]carbamoyl]-5-(trifluoromethyl)benzoic acid (14 mg, 5.88% yield, 96.7% purity) as a white solid.

[0611] LCMS (ESI) [M+H] + m / z: theoretical value 382.1, measured value 382.0 1 H NMR (400 MHz, DMSO-d6) : ppm 13.09 (1 H, br s) 8.66 (1 H, br d, J=4.3 Hz) 8.39 (1 H, s) 7.88 - 8.01 (2 H, m) 7.74 (1 H, br d, J=8.0 Hz) 7.30 (3 H, s) 2.79 (3 H, d, J=4.4 Hz) 2.45 (3 H, s)

[0612] SB007 (2S)-5,5-Dimethyl-2-[[6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carbonyl]amino]hexanoic acid [ka] Step 1: Methyl 6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carboxylate A mixture of 3-hydroxy-N-methyl-benzamide (400 mg, 2.65 mmol, 1 equiv.), methyl 6-bromopyridine-3-carboxylate (571.66 mg, 2.65 mmol, 1 equiv.), potassium phosphate (561.70 mg, 2.65 mmol, 1 equiv.), and CuI (503.96 mg, 2.65 mmol, 1 equiv.) in DMSO (10 mL) was degassed and purged with N three times, and then the mixture was stirred at 120 °C under N atmosphere for 16 h. The mixture was washed with HO (40 mL) and extracted with EA (20 mL × 4). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, elution with a 0-100% ethyl acetate / petroleum ether gradient at 25 mL / min) to afford methyl 6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carboxylate (73.92% yield, 100% purity) as a white solid.

[0613] Step 2: 6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carboxylic acid To a solution of methyl 6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carboxylate (460 mg, 1.61 mmol, 1 equiv.) in a mixture of THF (15 mL) and HO (5 mL) was added LiOH.HO (134.84 mg, 3.21 mmol, 2 equiv.). 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 × 5). The combined organic layers were dried over NaSO, filtered, and concentrated in vacuo. 6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carboxylic acid (82.29% yield) was obtained as a white solid.

[0614] Step 3: 6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carbonyl chloride A mixture of 6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carboxylic acid (0.1 g, 367.30 μmol, 1 equiv.), oxalyl chloride (69.93 mg, 550.95 μmol, 48.23 μL, 1.5 equiv.), and DMF (2.68 mg, 36.73 μmol, 2.83 μL, 0.1 equiv.) in DCM (5 mL) was degassed at 0° C., and the mixture was then stirred under a N atmosphere at 25° C. for 2 h. The reaction mixture was concentrated under reduced pressure. 6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carbonyl chloride (91.29 mg, crude) was obtained as a yellow gum, which was used directly without further purification.

[0615] Step 4: (2S)-5,5-Dimethyl-2-[[6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carbonyl]amino]hexanoic acid To a mixture of (2S)-2-amino-5,5-dimethyl-hexanoic acid (50 mg, 314.02 μmol, 1 equiv.) and NaCO (116.49 mg, 1.10 mmol, 3.5 equiv.) in dioxane (8 mL), HO (3 mL), 6-[3-(methylcarbamoyl)phenoxy]pyridine-3-carbonyl chloride (91.29 mg, 314.02 μmol, 1 equiv.) was added. The mixture was stirred at 20 °C for 2 h. The reaction mixture was poured into water (40 mL) and extracted with EtOAc (40 mL × 3). The combined aqueous layers were lyophilized in vacuo to give 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.

[0616] LCMS (ESI) [M+H] + m / z: theoretical value 413.20, measured value 414.1 1 H NMR (400 MHz, DMSO-d6) δ: ppm 0.87 (s, 9 H) 1.17 - 1.35 (m, 2 H) 1.65 - 1.85 (m, 2 H) 2.77 (d, J=4.5 Hz, 3 H) 4.31 (td, J=8.4, 5.3 Hz, 1 H) 7.17 (d, J=8.5 Hz, 1 H) 7.34 (dd, J=8.0, 1.8 Hz, 1 H) 7.53 (t, J=7.9 Hz, 1 H) 7.61 (t, J=1.9 Hz, 1 H) 7.72 (d, J=7.8 Hz, 1 H) 8.31 (dd, J=8.7, 2.4Hz, 1H) 8.50 (br d, J=4.5 Hz, 1 H) 8.63 (d, J=2.3 Hz, 1 H) 8.71 (d, J=7.8 Hz, 1 H)

[0617] SB008 (2S)-2-[[6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid [ka] Step 1: 3-[1-(2-methoxyethyl)triazol-4-yl]phenol To a solution of 3-ethynylphenol (576.00 mg, 4.88 mmol, 1.1 equiv), 1-azido-2-methoxyethane (450 mg, 4.45 mmol, 1 equiv), and TEA (450.37 mg, 4.45 mmol, 619.48 μL, 1 equiv) in tert-butanol (10 mL) and HO (10 mL), CuSO4.5HO (277.82 mg, 1.11 mmol, 0.25 equiv), sodium (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-olate (440.86 mg, 2.23 mmol, 0.5 equiv) were added, and the mixture was stirred at 20 °C for 2 h. The reaction mixture was filtered through Celite, poured into water (40 mL), and extracted with EtOAc (40 mL × 3). The combined organic layers were washed with brine (40 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo to give a crude residue. The residue was 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) was obtained as a white solid.

[0618] 1 H NMR (400 MHz, CDCl3) δ: ppm 7.87 (1 H, s) 7.62 (1 H, br s) 7.12 - 7.33 (2 H, m) 6.82 - 6.98 (1 H, m) 5.31 (1 H, s) 4.57 (2 H, t, J=4.89 Hz) 3.79 (2 H, t, J=5.02 Hz) 3.37 (3 H, s)

[0619] Step 2: 6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylate A mixture of 3-[1-(2-methoxyethyl)triazol-4-yl]phenol (800 mg, 3.65 mmol, 1 equiv.), methyl 6-bromopyridine-3-carboxylate (788.00 mg, 3.65 mmol, 1 equiv.), CuI (69.49 mg, 364.90 μmol, 0.1 equiv.), and KPO (1.55 g, 7.30 mmol, 2 equiv.) in DMSO (10 mL) was degassed and purged with N three times, then the mixture was stirred at 120 °C under a N atmosphere for 16 h. The reaction was poured into water (40 mL) and extracted with EtOAc (40 mL × 3). The combined organic layers were washed with brine (40 mL), dried over NaSO, 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.

[0620] 1 H NMR (400 MHz, CDCl3) δ: ppm 8.83 (1 H, d, J=2.01 Hz) 8.29 (1 H, dd, J=8.66, 2.38 Hz) 7.91 (1 H, s) 7.62 - 7.81 (2 H, m) 7.49 (1 H, t, J=7.91 Hz) 7.09 - 7.20 (1 H, m) 6.98 (1 H, d, J=8.78 Hz) 4.50 - 4.66 (2 H, m) 3.92 (3 H, s) 3.73 - 3.85 (2 H, m) 3.37 (3 H, s)

[0621] Step 3: 6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylic acid To a solution of methyl 6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylate (170 mg, 479.74 μmol, 1 equiv.) in a mixture of THF (3 mL) and HO (1 mL), LiOH.HO (40.26 mg, 959.48 μmol, 2 equiv.) was added, and 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 × 5). The combined organic layers were dried over NaSO, filtered, and concentrated in vacuo. 6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylic acid (140 mg, 84.03% yield, 98% purity) was obtained as a yellow solid.

[0622] 1 H NMR (400 MHz, METHANOL-d4) : ppm 8.74 (1 H, d, J=2.01 Hz) 8.31 - 8.39 (2 H, m) 7.71 - 7.78 (1 H, m) 7.61 - 7.67 (1 H, m) 7.52 (1 H, t, J=7.91 Hz) 7.15 (1 H, ddd, J=8.16, 2.38, 0.75 Hz) 7.07 (1 H, d, J=8.53 Hz) 4.61 (2 H, t, J=5.02 Hz) 3.76 - 3.99 (2 H, m) 3.35 (3 H, s)

[0623] Step 4: 6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carbonyl chloride A mixture of 6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylic acid (140 mg, 411.36 μmol, 1 equiv.), oxalyl chloride (78.32 mg, 617.04 μmol, 54.01 μL, 1.5 equiv.), and DMF (3.01 mg, 41.14 μmol, 3.17 μL, 0.1 equiv.) in DCM (5 mL) was degassed at 0 °C, and then the mixture was stirred under a N atmosphere at 25 °C for 2 h. 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.

[0624] Step 5: (2S)-2-[[6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid To a mixture of (2S)-2-amino-5,5-dimethyl-hexanoic acid (50 mg, 314.02 μmol, 0.75 equiv.) and NaCO (155.09 mg, 1.46 mmol, 3.5 equiv.) in HO (8 mL) and dioxane (3 mL), 6-[3-[1-(2-methoxyethyl)triazol-4-yl]phenoxy]pyridine-3-carbonyl chloride (150 mg, 418.09 μmol, 1 equiv.) was added. The mixture was stirred at 20 °C for 2 h. The reaction mixture was poured into water (40 mL) and extracted with EtOAc (40 mL × 3). The combined aqueous layers were 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.

[0625] LCMS (ESI) [M+H] + m / z: theoretical value 482.2, measured value 482.2 1H NMR (400 MHz, DMSO-d6) δ: ppm 8.70 (1 H, d, J=7.83 Hz) 8.65 (1 H, d, J=1.96 Hz) 8.61 (1 H, s) 8.32 (1 H, dd, J=8.62, 2.51 Hz) 7.73 - 7.80 (1 H, m) 7.63 - 7.67 (1 H, m) 7.52 (1 H, t, J=7.89 Hz) 7.11 - 7.20 (2 H, m) 4.57 (2 H, t, J=5.20 Hz) 4.23 - 4.43 (1 H, m) 3.77 (2 H, t, J=5.20 Hz) 3.26 (3H, s) 1.64 - 1.92 (2 H, m) 1.16 - 1.38 (2 H, m) 0.87 (9 H, s)

[0626] SB009 [ka] Step 1: (3-Acetoxyphenyl)acetate To benzene-1,3-diol (1 g, 9.08 mmol, 1.52 mL, 1 equiv.) in AcO (14.83 g, 145.31 mmol, 13.61 mL, 16 equiv.) was added p-toluenesulfonyl chloride (173.14 mg, 908.18 μmol, 0.1 equiv.) under N at 20 °C. The reaction was stirred at 20 °C for 1.5 h. The reaction was quenched with 10% NaOH / HO (45 mL) and extracted with DCM (3 × 30 mL). The combined organic layers were washed with brine (50 mL), dried over NaSO, filtered, and concentrated 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.

[0627] LCMS (ESI) [M+H] + m / z: theoretical value 195.0, measured value 194.8 1H NMR (400 MHz, CHLOROFORM-d) : ppm 7.38 (1 H, t, J=8.1 Hz) 6.99 (2 H, dd, J=8.2, 2.2 Hz) 6.85 - 6.95 (1 H, m) 2.22 - 2.36 (6 H, m)

[0628] Step 2: (3-hydroxyphenyl)acetate To a mixture of (3-acetoxyphenyl)acetate (1.6 g, 8.24 mmol, 1 equiv.) and benzene-1,3-diol (907.27 mg, 8.24 mmol, 1.37 mL, 1 equiv.) in DMSO (10 mL) was added K2CO3 (1.14 g, 8.24 mmol, 1 equiv.) in one portion at 20 °C under N2. The mixture was stirred at 20 °C for 4 h. TLC showed the reaction was complete. The mixture was poured into water (20 mL), and the aqueous phase was extracted with ethyl acetate (10 mL × 6). The combined organic phases were washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether / ethyl acetate = 10 / 1 to 2 / 1). (3-hydroxyphenyl)acetate (1.9 g, 75.78% yield) was obtained as a colorless oil.

[0629] 1 H NMR (400 MHz, CHLOROFORM-d) : ppm 7.21 (1 H, t, J=8.1 Hz) 6.61 - 6.72 (2 H, m) 6.56 (1 H, t, J=2.3 Hz) 2.27 - 2.36 (3 H, m)

[0630] Step 3: 3-(2-methoxyethoxy)phenol To a mixture of (3-hydroxyphenyl)acetate (1.7 g, 11.17 mmol, 1 equiv.) and 2-methoxyethyl 4-methylbenzenesulfonate (2.32 g, 10.06 mmol, 0.9 equiv.) in DMF (30 mL) was added K2CO3 (3.40 g, 24.58 mmol, 2.2 equiv.) in one portion under N2 at 20 °C. The mixture was stirred at 80 °C for 4 h. To the reaction was added NaOH (20%, 32 mL), and the mixture was stirred at 0 °C for 15 min. The aqueous layer was basified to pH 5 with HCl / HO (1 M, 240 mL) and extracted with DCM (3 × 30 mL). The combined organic layers were washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was combined with the residue of ES17843-53-P1A and purified by silica gel chromatography eluting with (PE:EA=100:1 to 3:1) to give 3-(2-methoxyethoxy)phenol (1.04 g, yield 55.34%) as a yellow oil.

[0631] 1 H NMR (400 MHz, CHLOROFORM-d) : ppm 7.12 (1 H, t, J=8.0 Hz) 6.22 - 6.55 (3 H, m) 5.24 (1 H, br s) 4.10 (2 H, dd, J=5.4, 3.9 Hz) 3.70 - 3.82 (2 H, m) 3.41 - 3.51 (3H, m)

[0632] Step 4: Methyl 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carboxylate and 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carboxylic acid To a mixture of 3-(2-methoxyethoxy)phenol (916 mg, 5.45 mmol, 1 equiv.) and methyl 6-bromopyridine-3-carboxylate (1.18 g, 5.45 mmol, 1 equiv.) in DMSO (12 mL) was added CuI (103.72 mg, 544.62 μmol, 0.1 equiv.) and KPO (2.31 g, 10.89 mmol, 2 equiv.) under N. The reaction was stirred at 120 °C for 16 h. To the reaction was added 100 mL of HO, then the mixture was filtered and the filter cake was washed with 30 mL of EtOAc. The filtrate was extracted with EtOAc (3 × 30 mL). The combined organic layers were washed with brine (20 mL), dried over NaSO, filtered, and concentrated in vacuo. The crude product was purified by silica gel chromatography eluting with (PE: EtOAc = 20:1 to 1:1) to give methyl 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carboxylate (260 mg, 857.21 μmol, 15.74% yield) as a yellow oil. The remaining aqueous layer was adjusted to pH 2 with 1 M HCl / HO and then extracted with EtOAc (3 × 30 mL). The combined organic layers were washed with brine (30 mL), dried over anhydrous SO, filtered, and concentrated in vacuo to give crude 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carboxylic acid (500 mg, 31.74% yield).

[0633] 1 H NMR (400 MHz, CHLOROFORM-d) : ppm 8.84 (1 H, dd, J=2.3, 0.6 Hz) 8.27 (1 H, dd, J=8.6, 2.4 Hz) 7.30 - 7.38 (1 H, m) 7.05 - 7.19 (1 H, m) 6.81 - 6.97 (1 H, m) 6.70 - 6.78 (1 H, m) 6.37 - 6.56 (1 H, m) 4.05 - 4.19 (2 H, m) 3.93 (2 H, s) 3.75 (2 H, td, J=4.7, 2.2 Hz) 3.46 (3 H, d, J=1.7 Hz)

[0634] Step 5: 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carboxylic acid To a mixture of methyl 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carboxylate (260 mg, 857.21 μmol, 1 equiv.) in THF (4.5 mL) and HO (1.5 mL) was added LiOH.HO (71.94 mg, 1.71 mmol, 2 equiv.). 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 with HCl (1 M). The residue was extracted with EtOAc (20 mL × 5). The combined organic layers were dried over NaSO, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with (DCM:MeOH = 50:1 to 10:1) to afford 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carboxylic acid (247.9 mg, 99.97% yield) as a white oil.

[0635] LCMS (ESI) [M+H] + m / z: theoretical value 290.1, measured value 289.9 1 H NMR (400 MHz, CHLOROFORM-d) : ppm 8.94 (1 H, d, J=2.3 Hz) 8.34 (1 H, dd, J=8.7, 2.4 Hz) 7.30 - 7.41 (1 H, m) 6.97 (1 H, d, J=8.7 Hz) 6.85 - 6.91 (1 H, m) 6.71 - 6.83 (2 H, m) 4.10 - 4.17 (2 H, m) 3.78 (2 H, dd, J=5.4, 4.0 Hz) 3.43 - 3.51 (3 H, m)

[0636] Step 6: 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carbonyl chloride To 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carboxylic acid (100 mg, 345.68 μmol, 1 equiv) in DCM (4 mL) was added DMF (2.53 mg, 34.57 μmol, 2.66 μL, 0.1 equiv) and oxalyl chloride (65.82 mg, 518.52 μmol, 45.39 μL, 1.5 equiv) under N at 0° C. The reaction was stirred at 20° C. for 2 h. 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.

[0637] LCMS (ESI) [M-Cl+OMe] + m / z: theoretical value 304.0, measured value 304.0

[0638] Step 7: (2S)-2-[[6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid To a solution of (2S)-2-amino-5,5-dimethyl-hexanoic acid (50.00 mg, 314.02 μmol, 0.912 equiv.) and NaCO (127.78 mg, 1.21 mmol, 3.5 equiv.) in dioxane (3 mL) and HO (8 mL), 6-[3-(2-methoxyethoxy)phenoxy]pyridine-3-carbonyl chloride (106 mg, 344.46 μmol, 1 equiv.) was added. The reaction was stirred at 20 °C for 2 h. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (10 mL × 3). The combined aqueous layers were lyophilized in vacuo to give a crude residue. The crude product was combined with the product of (ES17843-67-P1A) and purified by preparative HPLC (column: Boston Green ODS 150*30mm*5μm; mobile phase: [water (0.05% HCl)-ACN]; B%: 29%~69%, 9 min). 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, yield 8.00%, purity 98.8%) as a white solid.

[0639] LCMS (ESI) [M+H] + m / z: theoretical value 431.2, measured value 431.2 1 H NMR (400 MHz, DMSO-d6) : ppm 8.68 (1 H, d, J=7.8 Hz) 8.64 (1 H, d, J=2.3 Hz) 8.29 (1 H, dd, J=8.6, 2.4 Hz) 7.32 (1 H, t, J=8.2 Hz) 7.08 (1 H, d, J=8.7 Hz) 6.83 (1 H, dd, J=8.2, 2.1 Hz) 6.76 (1 H, t, J=2.3 Hz) 6.72 (1 H, dd, J=7.8, 1.8 Hz) 4.23 - 4.38 (1 H, m) 4.23 - 4.38 (1 H, m) 4.06 - 4.11 (2H, m) 3.64 - 3.66 (2 H, m) 3.29 (3 H, s) 1.62 - 1.85 (2 H, m) 1.13 - 1.38 (2 H, m) 0.87 (9 H, s)

[0640] SB010 (2S)-2-[[6-[3-[1-(2-acetamidoethyl)triazol-4-yl]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid [ka] Step 1: Tert-butyl N-[2-[4-(3-hydroxyphenyl)triazol-1-yl]ethyl]carbamate To a solution of 3-ethynylphenol (600 mg, 5.08 mmol, 1.1 equiv), tert-butyl N-(2-azidoethyl)carbamate (859.80 mg, 4.62 mmol, 1 equiv), and TEA (467.22 mg, 4.62 mmol, 642.67 μL, 1 equiv) in tert-butanol (10 mL) and HO (10 mL) was added CuSO4.5HO (288.22 mg, 1.15 mmol, 0.25 equiv), sodium (2R)-2-[(1S)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-olate (457.37 mg, 2.31 mmol, 0.5 equiv), and the mixture was stirred at 20 °C for 12 h. The reaction mixture was filtered through Celite, poured into water (40 mL), and extracted with EtOAc (40 mL × 3). The combined organic layers were washed with brine (40 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1 to 3 / 1). Tert-butyl N-[2-[4-(3-hydroxyphenyl)triazol-1-yl]ethyl]carbamate (1.2 g, 85.39% yield) was obtained as a white solid.

[0641] LCMS (ESI) [M+H] + m / z: theoretical value 304.1, measured value 305.1

[0642] Step 2: 3-[1-(2-aminoethyl)triazol-4-yl]phenol To a solution of tert-butyl N-[2-[4-(3-hydroxyphenyl)triazol-1-yl]ethyl]carbamate (1 g, 3.29 mmol, 1 equiv.) in DCM (10 mL) was added TFA (3.75 g, 32.86 mmol, 2.43 mL, 10 equiv.). The reaction mixture was stirred under nitrogen at 20° C. for 17 h. The reaction mixture was directly concentrated under reduced pressure. 3-[1-(2-aminoethyl)triazol-4-yl]phenol was obtained as a white solid.

[0643] Step 3: N-[2-[4-(3-hydroxyphenyl)triazol-1-yl]ethyl]acetamide To a solution of 3-[1-(2-aminoethyl)triazol-4-yl]phenol (700 mg, 3.43 mmol, 1 equiv) and TEA (1.73 g, 17.14 mmol, 2.39 mL, 5 equiv) in DCM (10 mL) was added acetyl chloride (269.05 mg, 3.43 mmol, 244.59 μL, 1 equiv) dropwise under N at 0 °C. The reaction mixture was warmed to 20 °C and stirred for 2 h. The reaction was poured into water (40 mL) and extracted with DCM (40 mL × 3). The combined organic layers were washed with brine (40 mL), dried over NaSO, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC (basic conditions), and the remaining 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.

[0644] 1H NMR (400 MHz, DMSO-d6) δ: ppm 8.26 - 8.67 (m, 1 H) 8.05 (br t, J=5.5 Hz, 1 H) 7.27 (d, J=1.0 Hz, 1 H) 7.20 - 7.24 (m, 2 H) 6.63 - 6.76 (m, 1 H) 4.42 (t, J=5.9 Hz, 2 H) 3.51 (q, J=5.8 Hz, 2 H) 1.67 - 1.88 (m, 3 H).

[0645] Step 4: Methyl 6-[3-[1-(2-acetamidoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylate A mixture of methyl 6-bromopyridine-3-carboxylate (105.27 mg, 487.28 μmol, 1.2 equiv.), N-[2-[4-(3-hydroxyphenyl)triazol-1-yl]ethyl]acetamide (100 mg, 406.07 μmol, 1 equiv.), potassium phosphate (86.19 mg, 406.07 μmol, 1 equiv.), and CuI (77.34 mg, 406.07 μmol, 1 equiv.) in DMSO (4 mL) was degassed and purged with N three times. The mixture was then stirred at 120 °C under a N atmosphere for 16 h. The reaction mixture was extracted with EA (20 mL × 4). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® silica flash column, elution with a 0-100% ethyl acetate / petroleum ether gradient at 25 mL / min) to afford methyl 6-[3-[1-(2-acetamidoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylate (90.40% yield) as a white solid.

[0646] 1 H NMR (400 MHz, DMSO-d6) δ: ppm 8.71 (d, J=2.0 Hz, 1 H) 8.61 (s, 1 H) 8.34 (dd, J=8.5, 2.5 Hz, 1 H) 8.04 (br t, J=5.8 Hz, 1 H) 7.76 (d, J=7.8 Hz, 1 H) 7.63 - 7.66 (m, 1 H) 7.53 (t, J=7.9 Hz, 1 H) 7.13 - 7.21 (m, 2 H) 4.43 (t, J=6.0 Hz, 2 H) 3.86 (s, 3 H) 3.50 (q, J=6.0 Hz, 2 H) 1.76 - 1.79 (m, 3H)

[0647] Step 5: 6-[3-[1-(2-acetamidoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylic acid To a solution of methyl 6-[3-[1-(2-acetamidoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylate (100 mg, 262.20 μmol, 1 equiv.) in a mixture of THF (0.6 mL) and HO (0.2 mL), LiOH.HO (22.01 mg, 524.41 μmol, 2 equiv.) 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 × 5). The combined organic layers were dried over NaSO, filtered, and concentrated in vacuo to give 6-[3-[1-(2-acetamidoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylic acid (93.44% yield, 90% purity) as a white solid.

[0648] 1 H NMR (400 MHz, DMSO-d6) δ: ppm 8.68 (d, J=2.0 Hz, 1 H) 8.61 (s, 1 H) 8.31 (dd, J=8.7, 2.4 Hz, 1 H) 8.05 (br s, 1 H) 7.75 (d, J=8.0 Hz, 1 H) 7.63 (s, 1 H) 7.53 (t, J=7.9 Hz, 1 H) 7.16 (br d, J=8.8 Hz, 2 H) 4.43 (br t, J=5.8 Hz, 2 H) 3.51 (q, J=5.8 Hz, 2 H) 1.78 (s, 3 H)

[0649] Step 6: (2,5-Dioxopyrrolidin-1-yl)6-[3-[1-(2-acetamidoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylate To a solution of 6-[3-[1-(2-acetamidoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylic acid (80 mg, 217.77 μmol, 1 equiv.) in DMF (2 mL) was added 1-hydroxypyrrolidine-2,5-dione (62.66 mg, 544.43 μmol, 2.5 equiv.) and EDCI (62.62 mg, 326.66 μmol, 1.5 equiv.), and the mixture was degassed three times. N2The 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 to give (2,5-dioxopyrrolidin-1-yl)6-[3-[1-(2-acetamidoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylate (100 mg, crude) as a yellow oil.

[0650] Step 7: (2S)-2-[[6-[3-[1-(2-acetamidoethyl)triazol-4-yl]phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid A mixture of (2,5-dioxopyrrolidin-1-yl)-6-[3-[1-(2-acetamidoethyl)triazol-4-yl]phenoxy]pyridine-3-carboxylate (80 mg, 172.25 μmol, 1 equiv.), (2S)-2-amino-5,5-dimethyl-hexanoic acid (27.43 mg, 172.25 μmol, 1 equiv.), and DIEA (33.39 mg, 258.38 μmol, 45.01 μL, 1.5 equiv.) in DCM (0.4 mL), DMF (2.4 mL), and HO (1.2 mL) was degassed and purged with N three times, and then the mixture was stirred under N atmosphere at 20° C. for 4 h. The reaction mixture was concentrated in vacuo to give a crude residue, which was purified by preparative HPLC (HCl conditions). The remaining aqueous solution was lyophilized to give (2S)-2-[[6-[3-[1-(2-acetamidoethyl)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.

[0651] LCMS (ESI) [M+H] + m / z: theoretical value 508.24, measured value 509.3 1H NMR (400 MHz, DMSOd6) δ: 8.71 (d, J=7.8 Hz, 1 H) 8.65 (d, J=2.5 Hz, 1 H) 8.62 (s, 1 H) 8.32 (dd, J=8.5, 2.5 Hz, 1 H) 8.07 (br t, J=5.6 Hz, 1 H) 7.74 (d, J=8.0 Hz, 1 H) 7.60 - 7.65 (m, 1 H) 7.52 (t, J=7.9 Hz, 1 H) 7.11 - 7.19 (m, 2 H) 4.43 (t, J=5.9 Hz, 2 H) 4.31 (td, J=8.4, 5.0 Hz, 3H) 3.50 (q, J=6.0 Hz, 2 H) 1.78 (s, 3 H) 1.16 - 1.36 (m, 2 H) 0.86 (s, 9 H)

[0652] SB011 (2S)-2-[[6-[3-(2-acetamidoethoxy)phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid [ka] Step 1: [3-[2-(tert-butoxycarbonylamino)ethoxy]phenyl]acetate A mixture of (3-hydroxyphenyl)acetate (1.7 g, 11.17 mmol, 1.52 mL, 1 equiv.), tert-butyl N-(2-hydroxyethyl)carbamate (1.80 g, 11.18 mmol, 1.73 mL, 1 equiv.), and PPh (3.52 g, 13.41 mmol, 1.2 equiv.) in THF (30 mL) was degassed and purged with N three times. DIAD (2.71 g, 13.41 mmol, 2.61 mL, 1.2 equiv.) was added at 0 °C, and the mixture was then stirred at 20 °C for 16 h under a N atmosphere. The reaction mixture was poured into water (40 mL) and extracted with EtOAc (40 mL × 3). The combined organic layers were washed with brine (40 mL), dried over NaSO, filtered, and concentrated 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, yield 54.70%, purity 95%) as a colorless oil.

[0653] 1 H NMR (400 MHz, CDCl3) : ppm 7.29 (1 H, t, J=8.28 Hz) 6.78 (1 H, dd, J=8.16, 2.13 Hz) 6.71 (1 H, dd, J=8.16, 1.38 Hz) 6.65 (1 H, t, J=2.26 Hz) 4.01 (2 H, t, J=5.02 Hz) 3.54 (2 H, br d, J=4.27 Hz) 2.31 (3 H, s) 1.47 (9 H, s)

[0654] Step 2: Tert-butyl N-[2-(3-hydroxyphenoxy)ethyl]carbamate To a mixture of [3-[2-(tert-butoxycarbonylamino)ethoxy]phenyl]acetate (1.7 g, 5.76 mmol, 1 equiv.) in MeOH (30 mL), KCO (1.59 g, 11.51 mmol, 2 equiv.) and HO (6 mL) were added, and the mixture was stirred at 20 °C for 2 h. The reaction mixture was poured into 10% aqueous HCl (80 mL) and extracted with EtOAc (40 mL × 3). The combined organic layers were washed with brine (40 mL), dried over NaSO, filtered, and concentrated in vacuo. The residue was purified by column chromatography (SiO, 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.

[0655] 1 H NMR (400 MHz, CDCl3) : ppm 7.12 (1 H, t, J=8.13 Hz) 6.40 - 6.54 (3 H, m) 3.98 (2 H, t, J=5.07 Hz) 3.52 (2 H, br d, J=5.01 Hz) 1.47 (9 H, s)

[0656] Step 3: Methyl 6-[3-[2-(tert-butoxycarbonylamino)ethoxy]phenoxy]pyridine-3-carboxylate A mixture of tert-butyl N-[2-(3-hydroxyphenoxy)ethyl]carbamate (100 mg, 394.80 μmol, 1 equiv.), methyl 6-fluoropyridine-3-carboxylate (74.00 mg, 477.03 μmol, 1.21 equiv.), and CsCO (385.90 mg, 1.18 mmol, 3 equiv.) in DMF (3 mL) was degassed and purged with N three times, and then the mixture was stirred under a N atmosphere at 20 °C for 16 h. The reaction mixture was poured into water (50 mL) and extracted with EA (3 × 30 mL). The combined organic layers were washed with brine (30 mL), dried over NaSO, filtered, and concentrated 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, yield 70.43%, purity 90%) as a yellow solid.

[0657] LCMS (ESI) [M+H-100] + m / z: theoretical value 289.1, measured value 289.0 1 H NMR (400 MHz, CDCl3) : ppm 8.84 (1 H, d, J=2.20 Hz) 8.29 (1 H, dd, J=8.62, 2.38 Hz) 7.24 - 7.40 (1 H, m) 6.95 (1 H, d, J=8.68 Hz) 6.78 (2 H, ddd, J=12.93, 8.22, 1.96 Hz) 6.69 - 6.73 (1 H, m) 4.02 (2 H, t, J=5.07 Hz) 3.93 (3 H, s) 3.47 - 3.58 (2 H, m) 1.46 (9 H, s)

[0658] Step 4: Methyl 6-[3-(2-aminoethoxy)phenoxy]pyridine-3-carboxylate A mixture of methyl 6-[3-[2-(tert-butoxycarbonylamino)ethoxy]phenoxy]pyridine-3-carboxylate (120 mg, 308.95 μmol, 1 equiv.), HCl / dioxane (4 M, 772.37 μL, 10 equiv.) in dioxane (3 mL) was stirred for 2 h at 20° C. The mixture was concentrated in vacuo to afford 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.

[0659] 1 H NMR (400 MHz, DMSO-d6) δ: ppm 8.70 (1 H, d, J=2.26 Hz) 8.32 (4 H, br dd, J=8.66, 2.38 Hz) 7.39 (1 H, t, J=8.16 Hz) 7.14 (1 H, d, J=8.78 Hz) 6.91 (1 H, dd, J=8.16, 1.88 Hz) 6.77 - 6.86 (2 H, m) 4.20 (2 H, t, J=5.14 Hz) 3.86 (3 H, s) 3.15 - 3.26 (2 H, m)

[0660] Step 5: Methyl 6-[3-(2-acetamidoethoxy)phenoxy]pyridine-3-carboxylate To a mixture of methyl 6-[3-(2-aminoethoxy)phenoxy]pyridine-3-carboxylate (90 mg, 312.18 μmol, 1 equiv.) in THF (3 mL), AcO (38.24 mg, 374.61 μmol, 35.09 μL, 1.2 equiv.) and EtN (94.77 mg, 936.53 μmol, 130.35 μL, 3 equiv.) were added, and the mixture was stirred at 20 °C for 12 h. The reaction mixture was poured into water (40 mL) and extracted with EtOAc (40 mL × 3). The combined organic layers were washed with brine (40 mL), dried over anhydrous SO, filtered, and concentrated in vacuo to afford methyl 6-[3-(2-acetamidoethoxy)phenoxy]pyridine-3-carboxylate (80 mg, 77.58% yield) as a yellow oil, which was used directly without purification.

[0661] 1 H NMR (400 MHz, DMSO-d6) δ: ppm 8.71 (1 H, d, J=1.96 Hz) 8.31 (1 H, dd, J=8.56, 2.45 Hz) 8.04 - 8.22 (1 H, m) 7.35 (1 H, t, J=8.13 Hz) 7.12 (1 H, d, J=8.68 Hz) 6.86 (1 H, dd, J=8.31, 1.83 Hz) 6.73 - 6.82 (2 H, m) 3.98 (2 H, t, J=5.69 Hz) 3.86 (3 H, s) 3.39 (2 H, q, J=5.62 Hz) 1.82 (3 H, s)

[0662] Step 6: 6-[3-(2-acetamidoethoxy)phenoxy]pyridine-3-carboxylic acid To a solution of methyl 6-[3-(2-acetamidoethoxy)phenoxy]pyridine-3-carboxylate (80 mg, 242.18 μmol, 1 equiv.) in a mixture of THF (3 mL) and HO (1 mL), LiOH.HO (20.32 mg, 484.36 mol, 2 equiv.) 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 with HCl (1 M). The residue was extracted with EA (20 mL × 5), and the combined organic layers were dried over NaSO, filtered, and concentrated in vacuo to give 6-[3-(2-acetamidoethoxy)phenoxy]pyridine-3-carboxylic acid (70 mg, 24% yield, 90% purity) as a yellow oil.

[0663] 1H NMR (400 MHz, DMSO-d6) δ: ppm 8.68 (1 H, d, J=2.26 Hz) 8.29 (1 H, dd, J=8.66, 2.38 Hz) 8.02 - 8.15 (1 H, m) 7.35 (1 H, t, J=8.16 Hz) 7.09 (1 H, d, J=8.53 Hz) 6.85 (1 H, dd, J=8.28, 2.01 Hz) 6.73 - 6.80 (2 H, m) 3.96 - 4.01 (2 H, m) 3.40 - 3.42 (2 H, m) 1.82 (3 H, s)

[0664] Step 7: (2,5-Dioxopyrrolidin-1-yl)6-[3-(2-acetamidoethoxy)phenoxy]pyridine-3-carboxylate To a solution of 1-hydroxypyrrolidine-2,5-dione (63.67 mg, 553.26 μmol, 2.5 equiv) in DMF (5 mL) were added 6-[3-(2-acetamidoethoxy)phenoxy]pyridine-3-carboxylic acid (70 mg, 221.30 μmol, 1 equiv) and EDCI (63.64 mg, 331.95 μmol, 1.5 equiv), and the mixture was degassed three times and stirred under N for 3 h at 20° C. The reaction mixture was poured into water (40 mL) and extracted with EtOAc (40 mL × 3). The combined organic layers were washed with brine (40 mL), dried over anhydrous SO, filtered, and concentrated in vacuo to afford (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.

[0665] LCMS (ESI) [M+H] + m / z: theoretical value 414.1, measured value 414.0

[0666] Step 8: (2S)-2-[[6-[3-(2-acetamidoethoxy)phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid A mixture of (2,5-dioxopyrrolidin-1-yl)-6-[3-(2-acetamidoethoxy)phenoxy]pyridine-3-carboxylate (85 mg, 205.62 μmol, 1 equiv), (2S)-2-amino-5,5-dimethyl-hexanoic acid (33.15 mg, 208.20 μmol, 1.01 equiv), and DIEA (26.58 mg, 205.62 μmol, 35.82 uL, 1 equiv) in DCM (0.5 mL), HO (1.5 mL), and DMF (3 mL) was degassed and purged with N three times, then the mixture was stirred under N atmosphere at 25 °C for 16 h. The reaction mixture 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 min. (2S)-2-[[6-[3-(2-acetamidoethoxy)phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (16.5 mg, yield 15.79%, purity 90%) was obtained as a white solid.

[0667] LCMS (ESI) [M+H] + m / z: Calculated value 458.22, Measured value 458.2 1 H NMR (400 MHz, DMSO-d6) δ: ppm 8.68 (1 H, d, J=7.83 Hz) 8.64 (1 H, d, J=2.08 Hz) 8.29 (1 H, dd, J=8.62, 2.51 Hz) 8.03 - 8.13 (1 H, m) 7.33 (1 H, t, J=8.13 Hz) 7.09 (1 H, d, J=8.93 Hz) 6.80 - 6.92 (1 H, m) 6.70 - 6.77 (2 H, m) 4.31 (1 H, td, J=8.47, 5.20 Hz) 3.97 (2 H, t, J=5.69 Hz) 3.39 (2 H, q, J=5.62 Hz) 1.81 (3 H, s) 1.77 (1 H, br t, J=5.14 Hz) 1.11 - 1.37 (3 H, m) 0.87 (9 H, s)

[0668] SB012 (2S)-2-[[6-[3-(acetamidomethyl)phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid [ka] Step 1: N-[(3-hydroxyphenyl)methyl]acetamide To a solution of 3-(aminomethyl)phenol (800 mg, 6.50 mμoλ, 1 equiv.) and EtN (1.64 g, 16.2 mmol, 2.26 mL, 2.5 equiv.) in DCM (20 mL) was added acetyl chloride (509 mg, 6.50 mmol, 463 μL, 1.0 equiv.) dropwise under N at 0°C. The reaction mixture was warmed to 20°C and stirred for 2 h. 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% ammonia hydroxide v / v)-ACN; column: YMC Triart C18 250*50 mm*7 μm). The product was lyophilized to give N-[(3-hydroxyphenyl)methyl]acetamide (720 mg, 65% yield, 97% purity) as a colorless oil.

[0669] 1 H NMR (400 MHz, CDCl3) δ: 7.10 (t, J = 7.82 Hz, 1 H), 6.76 (s, 1 H), 6.66 - 6.74 (m, 2 H), 5.96 (br s, 1 H), 4.30 (d, J = 6.00 Hz, 2H), 1.95 (s, 3 H).

[0670] Step 2: Methyl 6-[3-(acetamidomethyl)phenoxy]pyridine-3-carboxylate A mixture of N-[(3-hydroxyphenyl)methyl]acetamide (620 mg, 3.75 mmol, 1 equiv.), methyl 6-bromopyridine-3-carboxylate (810 mg, 3.75 mmol, 1 equiv.), potassium phosphate (796.7 mg, 3.75 mmol, 1 equiv.), and CuI (714 mg, 3.75 mmol, 1.0 equiv.) in DMSO (15 mL) was degassed and purged with N three times, and then the mixture was stirred at 120 °C under a N atmosphere for 16 h. The mixture was washed with HO (40 mL) and extracted with ethyl acetate (20 mL × 4). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® silica flash column, elution with a 0-100% ethyl acetate / petroleum ether gradient at 25 mL / min) to give methyl 6-[3-(acetamidomethyl)phenoxy]pyridine-3-carboxylate (54% yield, 86% purity) as a yellow oil.

[0671] 1 H NMR (400 MHz, CDCl3) δ: 8.70 - 8.77 (m, 1 H), 8.22 (dd, J = 8.63, 2.38 Hz, 1 H), 7.33 (t, J = 7.75 Hz, 1 H), 7.11 (d, J = 7.63 Hz, 1 H), 6.97 - 7.05 (m, 2 H), 6.89 (d, J = 8.75 Hz, 1 H), 5.83 (br s, 1 H), 4.40 (d, J = 5.75 Hz, 2 H), 3.85 (s, 3 H), 2.89 (s, 2 H), 2.81 (s, 2 H) 1.97 (s, 3 H).

[0672] Step 3: 6-[3-(acetamidomethyl)phenoxy]pyridine-3-carboxylic acid To a solution of methyl 6-[3-(acetamidomethyl)phenoxy]pyridine-3-carboxylate (610 mg, 2.0 mmol, 1 equiv.) in a mixture of THF (15 mL) and HO (5 mL) was added LiOH.HO (170 mg, 4.0 mmol, 2.0 equiv.). 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 ethyl acetate (20 mL × 5), and the combined organic layers were dried over NaSO, filtered, and concentrated in vacuo to give 6-[3-(acetamidomethyl)phenoxy]pyridine-3-carboxylic acid (500 mg, 86% yield, 100% purity) as a yellow oil.

[0673] 1 H NMR (400 MHz, METHANOL-d4) δ: 8.84 (d, J = 1.75 Hz, 1 H), 8.31 (dd, J = 8.63, 2.38 Hz, 1 H), 7.40 (t, J = 7.82 Hz, 1 H), 7.18 (d, J = 7.63 Hz, 1 H), 7.04 - 7.11 (m, 2 H), 6.98 (d, J = 8.76 Hz, 1 H), 5.95 (br s, 1 H), 4.47 (d, J = 5.75 Hz, 2 H), 2.05 (s, 3 H), 1.26 (t, J = 7.13 Hz, 2 H).

[0674] Step 4: (2,5-Dioxopyrrolidin-1-yl)6-[3(acetamidomethyl)phenoxy]pyridine-3-carboxylate To a solution of 6-[3-(acetamidomethyl)phenoxy]pyridine-3-carboxylic acid (50 mg, 174 μmol, 1 equiv.) in DMF (2 mL) was added 1-hydroxypyrrolidine-2,5-dione (50 mg, 436 μmol, 2.5 equiv.) and EDCI (50 mg, 261 μmol, 1.5 equiv.), and the mixture was degassed three times and stirred under N at 20° C. for 3 h. 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 × 2), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give (2,5-dioxopyrrolidin-1-yl)-6-[3-(acetamidomethyl)phenoxy]pyridine-3-carboxylate (50 mg, 68% yield, 92% purity) as a yellow oil.

[0675] LCMS (ESI) [M+H] + m / z: theoretical value 383.1, measured value 384.0

[0676] Step 5: (2S)-2-[[6-[3-(acetamidomethyl)phenoxy]pyridine-3carbonyl]amino]-5,5-dimethyl-hexanoic acid A mixture of (2,5-dioxopyrrolidin-1-yl) 6-[3-[1-(2-acetamidomethyl)phenoxy]pyridine-3-carboxylate (50 mg, 130 μmol, 1 equiv.), (2S)-2-amino-5,5-dimethyl-hexanoic acid (21 mg, 130 μmol, 1 equiv.), and DIEA (25 mg, 195 μmol, 34 μL, 1.5 equiv.) in DCM (0.2 mL), DMF (1.2 mL), and HO (0.6 mL) was degassed and purged with N three times, and then the mixture was stirred under N atmosphere at 20° C. for 4 h. The reaction mixture was concentrated in vacuo to give a residue that was purified by preparative HPLC (water (0.225% FA)-ACN, YMC Triart C18 250*50 mm*7 μm). The remaining aqueous solution was lyophilized to give (2S)-2-[[6-[3-(acetamidomethyl)phenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid (41 mg, 71% yield, 96% purity) as a white solid.

[0677] 1 H NMR (400 MHz, CDCl3) δ: 8.59 (br s, 1 H), 8.13 (br d, J = 7.75 Hz, 1 H), 7.32 - 7.41 (m, 1 H), 7.21 (br s, 1 H), 7.14 (br d, J = 7.25 Hz, 1 H), 6.99 - 7.09 (m, 2 H), 6.90 (br d, J = 8.00 Hz, 1 H), 6.51 (br s, 1 H), 4.70 (br d, J = 5.00 Hz, 1 H), 4.44 (br s, 2 H), 1.91 - 2.10 (m, 4 H), 1.79 (br d, J = 6.25 Hz, 1 H), 1.14 - 1.39 (m, 3 H), 0.87 (s, 9 H).

[0678] BF021 (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 [ka] Step 1: Synthesis of (2,5-dioxopyrrolidin-1-yl)5-bromonaphthalene-2-carboxylate To a solution of 5-bromonaphthalene-2-carboxylic acid (1.0 g, 3.98 mmol, 1 equiv.) in DMF (10 mL) was added 1-hydroxypyrrolidine-2,5-dione (1.15 g, 9.96 mmol, 2.5 equiv.) and EDCI (1.15 g, 5.97 mmol, 1.5 equiv.), and the mixture was degassed three times and stirred under N at 20 °C for 3 h. The reaction was poured into HO (20 mL) and filtered. The filter cake was washed with HO (3 × 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.

[0679] 1 H NMR (400 MHz, DMSO) δ: ppm 8.93 (1 H, d, J=0.8 Hz) 8.33 (1 H, t, J=8.0 Hz) 8.22 (1 H, d, J=0.8 Hz) 8.14 (1 H, d, J=4.8 Hz) 7.62 (1 H, t, J=8.0 Hz) 2.90 (4H, s)

[0680] 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 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 equiv.), (2,5-dioxopyrrolidin-1-yl)5-bromonaphthalene-2-carboxylate (729.38 mg, 2.10 mmol, 1 equiv.), and DIEA (812.28 mg, 6.29 mmol, 1.09 mL, 3 equiv.) in DMF (20 mL) was degassed and purged with N three times, and then the mixture was stirred under N atmosphere at 20 °C for 16 h. 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.

[0681] 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 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 equiv.) in dioxane (30 mL) was added KOH (432.07 mg, 7.70 mmol, 4 equiv.) in HO (30 mL). Then, tBuXPhos Pd G3 (76.46 mg, 96.26 μg, 0.05 equiv.) was added under N2. The reaction was stirred at 110 °C under N2 for 16 h. The reaction was extracted with EtOAc (50 mL × 3). The aqueous layer was adjusted to pH 3–4 with 1 M aqueous HCl and extracted with EtOAc (150 mL × 3). The organic layer was dried over NaSO 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 pale red solid.

[0682] 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 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-2-carboxamide (120 mg, 262.84 μmol, 1 equiv.) and CsCO (128.46 mg, 394.26 μmol, 1.5 equiv.) in DMF (3 mL) was added methyl 6-fluoropyridine-3-carboxylate (44.85 mg, 289.12 μmol, 1.1 equiv.). The reaction was stirred at 25 °C for 16 h. HO (10 mL) was added to the solution, and the solution was extracted with EtOAc (50 mL × 8). The organic layer was dried over NaSO and concentrated. 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.

[0683] LCMS (ESI) [M+H] + m / z: theoretical value 591.28, measured value 592.3

[0684] 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 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-carboxylate (110 mg, 185.91 μmol, 1 equiv.) in THF (3 mL), MeOH (2 mL), and HO (1 mL) was added LiOH.HO (15.60 mg, 371.82 μmol, 2 equiv.). The reaction was stirred at 25 °C for 2 h. MeOH and THF were removed in vacuo, and the aqueous layer was adjusted to pH 4–5 with 1 N aqueous HCl and extracted with EtOAc (20 mL × 2). The organic layer was dried over Na2SO4 and concentrated to give 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.

[0685] LCMS (ESI) [M+Na] + m / z: theoretical value 577.2, measured value 578.1 1H NMR (400 MHz, DMSO-d6) δ: ppm 8.81 (1 H, br t, J=5.3 Hz) 8.58 (2 H, d, J=1.8 Hz) 8.34 (1 H, dd, J=8.7, 2.4 Hz) 8.09 (1 H, br t, J=5.0 Hz) 7.91 - 7.99 (2 H, m) 7.83 (1 H, d, J=8.8 Hz) 7.65 (1 H, t, J=7.9 Hz) 7.46 (1 H, d, J=7.5 Hz) 7.29 (1 H, d, J=8.5 Hz) 6.42 (1 H, br d, J=2.0 Hz) 4.23 (1 H, dd, 2.08 (2 H, br t, J=7.4 Hz) 1.38 - 1.58 (4 H, m) 1.21 - 1.35 (3 H, m)

[0686] 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 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 equiv.) and EDCI (54.76 mg, 285.64 μmol, 1.5 equiv.) in DMF (5 mL) was added 1-hydroxypyrrolidine-2,5-dione (54.79 mg, 476.07 μmol, 2.5 equiv.). The reaction was stirred at 25° C. for 3 h. The reaction was concentrated to afford (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 directly without purification.

[0687] 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 (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 in DCM (0.5 mL), DMF (3.0 mL), and HO (1.5 mL). A mixture of carboxylate (128.4 mg, 190.30 μμολ, 1 equiv.), (2S)-2-amino-5,5-dimethyl-hexanoic acid (30.30 mg, 190.30 μmol, 1 equiv.), and DIEA (36.89 mg, 285.45 μmol, 49.72 μL, 1.5 equiv.) was degassed and purged with N three times, then the mixture was stirred under N atmosphere at 25 °C for 3 h. 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 min) 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.

[0688] LCMS (ESI) [M+H] + m / z: theoretical value 718.3, measured value 719.1 1H NMR (400 MHz, DMSO-d6) δ: ppm 8.60 - 8.71 (2 H, m) 8.47 - 8.55 (2 H, m) 8.34 (1 H, dd, J=8.6, 2.5 Hz) 7.81 - 7.99 (4 H, m) 7.64 (1 H, t, J=7.9 Hz) 7.43 (1 H, dd, J=7.5, 0.8 Hz) 7.25 - 7.30 (1 H, m) 6.39 (2 H, br s) 4.30 (1 H, td, J=8.5, 5.2 Hz) 4.24 (1 H, dd, J=7.7, 4.5 Hz) 4.04 (1 H, dd, J=7.6, 4.5 Hz) 3.36 (2 H, br d, J=6.0 Hz) 3.25 (2 H, q, J=6.0 Hz) 2.96 - 3.04 (1 H, m) 2.74 (1 H, dd, J=12.4, 5.1 Hz) 2.52 - 2.58 (1 H, m) 2.07 (2 H, t, J=7.3 Hz) 1.67 - 1.85 (2 H, m) 1.40 - 1.61 (4 H, m) 1.19 - 1.36 (4 H, m) 0.83 - 0.87 (9 H, m)

[0689] BF027 (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 [ka] Step 1: Synthesis of (2,5-dioxopyrrolidin-1-yl)5-bromonaphthalene-1-carboxylate To a solution of 5-bromonaphthalene-1-carboxylic acid (1 g, 3.98 mmol, 1 equiv.) in DMF (10 mL) was added 1-hydroxypyrrolidine-2,5-dione (1.15 g, 9.96 mmol, 2.5 equiv.) and EDCI (1.15 g, 5.97 mmol, 1.5 equiv.), and the mixture was degassed three times and stirred under N at 20 °C for 3 h. The reaction was poured into HO (20 mL) and filtered. The filter cake was washed with HO (3 × 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.

[0690] 1 H NMR (400 MHz, DMSO-d6) δ: ppm 8.64 - 8.60 (2 H, m) 8.43 (1 H, m) 8.10 (1H, m), 7.90 (1 H, m), 7.70-7.65 (1 H, m), 2.95 (4 H, s).

[0691] 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 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 equiv.) in DMF (60 mL) was added DIPEA (902.54 mg, 6.98 mmol, 1.22 mL, 2 equiv.) and (2,5-dioxopyrrolidin-1-yl)-5-bromonaphthalene-1-carboxylate (1.22 g, 3.49 mmol, 1 equiv.). The mixture was stirred at 20 °C for 16 h. The reaction was filtered, and the filter cake was washed with HO (3 × 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.

[0692] LCMS (ESI) [M+H] + m / z: theoretical value 520.5, measured value 521.2

[0693] 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 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-bromo-naphthalene-1-carboxamide (1 g, 1.93 mmol, 1 equiv.) in dioxane (20 mL) and HO (20 mL), tBuXPhos Pd G3 (76.46 mg, 96.26 μmol, 0.05 equiv.) and KOH (432.04 mg, 7.70 mmol, 4 equiv.) were added. The mixture was stirred at 110 °C for 16 h. The reaction mixture was concentrated under reduced pressure and washed with MTBE (30 mL × 2). The solid was acidified to pH 5 with 1 M 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) as a brown solid, which was used directly without further purification.

[0694] LCMS (ESI) [M+H] + m / z: theoretical value 457.5, measured value 457.3

[0695] Step 4: 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 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 equiv.) in DMF (10 mL) was added methyl 6-fluoropyridine-3-carboxylate (271.82 mg, 1.75 mmol, 1 equiv.) and CsCO (1.14 g, 3.50 mmol, 2 equiv.). The reaction mixture was stirred at 20 °C for 16 h. The reaction mixture was washed with HO (40 mL) and extracted with EA (40 mL × 3). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give 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 gray solid, which was used without purification.

[0696] LCMS (ESI) [M+H] + m / z: theoretical value 591.7, measured value 592.1

[0697] Step 5: Synthesis 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 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-carboxylate (800 mg, 1.35 mmol, 1 equiv.) in a mixture of THF (12 mL) and HO (4 mL), LiOH.HO (113.48 mg, 2.70 mmol, 2 equiv.) was added. The mixture was stirred at 25 °C for 4 h. 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.

[0698] LCMS (ESI) [M+Na] + m / z: theoretical value 577.2, measured value 578.3

[0699] Step 6: (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 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 equiv) in DMF (2 mL) was added 1-hydroxypyrrolidine-2,5-dione (49.81 mg, 432.79 μmol, 2.5 equiv) and EDC (49.78 mg, 259.67 μmol, 1.5 equiv), and the mixture was degassed three times and stirred under N at 20 °C for 4 h. 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.

[0700] 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 (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 in DCM (0.4 mL), DMF (2.4 mL), and HO (1.2 mL). A mixture of 2S-carboxylate (100 mg, 148.21 μmol, 1 equiv), (2S)-2-amino-5,5-dimethyl-hexanoic acid (23.60 mg, 148.21 μmol, 1 equiv), and DIEA (28.73 mg, 222.31 μμολ, 38.72 uL, 1.5 equiv) was degassed and purged with N three times, then the mixture was stirred under N atmosphere at 20 °C for 4 h. 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.

[0701] 1H NMR (400 MHz, DMSO-d6) δ: ppm 8.65 - 8.70 (1 H, m) 8.58 - 8.64 (1 H, m) 8.55 (1 H, m) 8.33 (1H, dd, J=8.8, 2.4 Hz) 8.13 (1 H, d, J=8.4 Hz) 7.90 (1 H, m) 7.88 (1 H, d, J=8. Hz) 7.61 - 7.68 (2 H, m) 7.52 - 7.56 (1 H, m) 7.38 (1 H, d, J=6.8 Hz) 7.26 (1 H, d, J=8.4 Hz) 6.40 (1 H, br s) 6.33 (1 H, br s) 4.24 - 4.31 (2 H, m) 4.05 - 4.09 (1 H, m) 3.25 - 3.35 (4 H, m) 3.04 - 3.08 (1 H, m) 2.75 (1 H, dd, J=12.4, 5.1 Hz) 2.07 (2 H, t, J=7.3 Hz) 1.32 - 1.81 (5 H, m) 1.15 - 1.30 (5 H, m) 0.85 (9 H, s) LCMS (ESI) [M+H] + m / z: theoretical value 718.31, measured value 719.2

[0702] BF022 (S)-5,5-Dimethyl-2-(6-(3-(2-(2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethoxy)ethoxy)ethoxy)phenoxy)nicotinamido)hexanoic acid [ka] Step 1: 2-[2-[2-(Tert-butoxycarbonylamino)ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate 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 EtN (1.83 g, 18.05 mmol, 2.51 mL, 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 DCM (20 mL × 3). The combined organic phase was washed with brine (20 mL), dried over NaSO, 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 / 1-5 / 1) to give 2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (2.3 g, yield 94.74%) as a brown oil.

[0703] LCMS (ESI) [M+Na] + m / z: theoretical value 426.1, measured value 426.1 1 H NMR (400 MHz, CHLOROFORM-d) : ppm 7.81 (2 H, d, J=8.3 Hz) 7.35 (2 H, d, J=8.0 Hz) 4.94 (1 H, br s) 4.13 - 4.23 (2 H, m) 3.68 - 3.73 (2 H, m) 3.48 - 3.61 (6 H, m) 3.30 (2 H, br s) 2.36 - 2.55 (3 H, m) 1.44 (9 H, s)

[0704] Step 2: Tert-butyl (2-(2-(2-(3-hydroxyphenoxy)ethoxy)ethoxy)ethyl)carbamate To a solution of 2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate (2.0 g, 4.96 mmol, 1.0 equiv.) and (3-hydroxyphenyl)acetate (904.99 mg, 5.95 mmol, 1.2 equiv.) in DMF (40 mL) was added K2CO3 (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 × 2). The combined organic phase was washed with brine (50 mL), dried over Na2SO4, 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 / 1-1 / 1) to give tert-butyl N-[2-[2-[2-(3-hydroxyphenoxy)ethoxy]ethoxy]ethyl]carbamate (1.0 g, yield 59.1%) as a pale yellow oil.

[0705] LCMS (ESI) [M+Na] + m / z: theoretical value 341.2, measured value 364.2

[0706] Step 3: Methyl 6-[3-[2-[2-[2-(Tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate To a mixture of tert-butyl N-[2-[2-[2-(3-hydroxyphenoxy)ethoxy]ethoxy]ethyl]carbamate (1.0 g, 2.93 mmol, 1 equiv.) and methyl 6-fluoropyridine-3-carboxylate (545.26 mg, 3.51 mmol, 1.2 equiv.) in DMF (20 mL) was added CsCO (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 × 3). The combined organic phase was washed with brine (60 mL), dried over NaSO, 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 / 1-2 / 1) to give methyl 6-[3-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate (900 mg, yield 64.4%) as a pale yellow oil.

[0707] LCMS (ESI) [M+Na] + m / z: theoretical value 477.2, measured value 499.3

[0708] Step 4: Methyl 6-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)phenoxy)nicotinate 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 equiv) in DCM (20 mL) was added TFA (11.96 g, 104.93 mmol, 7.77 mL, 50 equiv). The mixture was stirred at 20 °C for 3 h. The mixture was concentrated in vacuo, dissolved in DCM (200 mL), basified to pH 8 with saturated aqueous NaHCO3, and then extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo to give methyl 6-[3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate (900 mg, 94.9% yield) as a yellow oil.

[0709] LCMS (ESI) [M+H] + m / z: theoretical value 377.2, measured value 377.1

[0710] Step 5: Methyl 6-(3-(2-(2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)methylpentanamido)ethoxy)ethoxy)ethoxy)phenoxy)nicotinate 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 equiv.) in DMF (15 mL), HATU (1.21 g, 3.19 mmol, 1.5 equiv.) and DIEA (824.07 mg, 6.38 mmol, 1.11 mL, 3.0 equiv.) were added under N. The mixture was stirred at 20 °C for 30 min, and then methyl 6-[3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate (800 mg, 2.13 mmol, 1 equiv.) was added. The reaction was stirred for 1 h, and then the mixture was poured into water (100 mL). The aqueous phase was extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with brine (50 mL), dried over Na2SO4, 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 / 1-1 / 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) as a yellow solid.

[0711] LCMS (ESI) [M+H]+ m / z: theoretical value 603.2, measured value 603.4 1H NMR (400 MHz, CHLOROFORM-d) δ: ppm 8.82 (1 H, d, J=1.8 Hz) 8.28 (1 H, dd, J=8.5, 2.3 Hz) 7.33 (1 H, t, J=8.2 Hz) 6.94 (1 H, d, J=8.3 Hz) 6.83 (1 H, d, J=1.8 Hz) 6.69 - 6.80 (2 H, m) 6.63 (1 H, br t, J=5.4 Hz) 6.19 (1 H, br s) 4.49 (1 H, dd, J=7.7, 4.6 Hz) 4.29 (1 H, dd, J=7.7, 4.6 Hz) 4.12 - 4.16 (3 H, m) 3.93 (3 H, s) 3.82 - 3.88 (2 H, m) 3.69 - 3.74 (2 H, m) 3.63 - 3.68 (2 H, m) 3.54 - 3.61 (2 H, m) 3.40 - 3.47 (2 H, m) 3.08 - 3.14 (1 H, m) 2.89 (1 H, dd, J=12.8, 5.0 Hz) 2.73 (1 H, d, J=12.8 Hz) 2.21 (2 H, td, J=7.3, 2.5 Hz) 1.99 (1 H, br s) 1.60 - 1.75 (4 H, m) 1.34 - 1.50 (4 H, m)

[0712] Step 6: 6-(3-(2-(2-(2-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)methylpentanamido)ethoxy)ethoxy)ethoxy)phenoxy)nicotinic acid To a mixture of 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 equiv.) in THF (10 mL), MeOH (5 mL), and HO (5 mL) was added LiOH.HO (187.99 mg, 4.48 mmol, 3.0 equiv.). The mixture was stirred at 20 °C for 2 h. The mixture was concentrated in vacuo to remove MeOH, and the aqueous layer was then acidified to pH 1–2 with 1 M aqueous HCl and extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with brine (50 mL), dried over Na2SO4, 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 / 1-1 / 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.

[0713] LCMS (ESI) [M+H]+ m / z: Theoretical value 589.2 Measured value 589.1

[0714] 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)methylpentanamido)ethoxy)ethoxy)ethoxy)phenoxy)nicotinate To 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 equiv.) and 1-hydroxypyrrolidine-2,5-dione (146.63 mg, 1.27 mmol, 1.5 equiv.) in DMF (10 mL) was added EDC (244.24 mg, 1.27 mmol, 1.5 equiv.) under N2. The mixture was stirred at 20 °C for 2 h. The mixture was poured into water (50 mL) and saturated aqueous NaHCO3 (10 mL). The aqueous phase was extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with brine (30 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo to afford (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 pale yellow oil, which was used directly without further purification.

[0715] LCMS (ESI) [M+H]+ m / z: theoretical value 686.3, measured value 686.5

[0716] 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)pentanamido)ethoxy)ethoxy)ethoxy)phenoxy)nicotinamido)hexanoic acid To 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 mol, 1 equiv.) and (2S)-2-amino-5,5-dimethylhexanoic acid (69.66 mg, 437.48 umol, 1 equiv.) in DMF (10 mL) was added DIEA (169.62 mg, 1.31 mmol, 228.60 μL, 3.0 equiv.) under N. 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), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by preparative HPLC (column: Boston Green ODS 150*30mm*5μm, mobile phase: [water (0.05%HCl)-ACN], B%: 22%~62%, 9 min) 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, yield 26.84%, HCl salt) as a white solid.

[0717] LCMS (ESI) [M+H]+ m / z: theoretical value 730.3, measured value 730.3 1H NMR (400 MHz, DMSO-d6) : ppm 8.69 (1 H, d, J=7.8 Hz) 8.64 (1 H, d, J=2.3 Hz) 8.28 (1 H, dd, J=8.5, 2.5 Hz) 7.83 (1 H, t, J=5.4 Hz) 7.33 (1 H, t, J=8.2 Hz) 7.09 (1 H, d, J=8.5 Hz) 6.84 (1 H, dd, J=7.9, 2.1 Hz) 6.70 - 6.80 (2 H, m) 6.41 (1 H, br s) 4.26 - 4.38 (2 H, m) 4.07 - 4.17 (3 H, m) 3.71 - 3.77 (2 H, m) 3.56 - 3.61 (2 H, m) 3.50 - 3.54 (3 H, m) 3.18 (2 H, q, J=5.9 Hz) 3.06 - 3.11 (1 H, m) 2.81 (1 H, dd, J=12.4, 5.1 Hz) 2.51 - 2.56 (3 H, m) 2.01 - 2.09 (3 H, m) 1.68 - 1.83 (2 H, m) 1.55 - 1.65 (1 H, m) 1.39 - 1.53 (3 H, m) 1.20 - 1.35 (4 H, m) 0.87 (9 H, s) 1H NMR (400 MHz, CHLOROFORM-d) : ppm 8.67 (1 H, s) 8.27 (1 H, br d, J=7.1 Hz) 7.78 (1 H, br d, J=7.4 Hz) 7.30 - 7.38 (2 H, m) 6.93 - 7.05 (1 H, m) 6.80 (2 H, br t, J=7.8 Hz) 6.74 (1 H, br s) 6.41 (1 H, br s) 4.73 (1 H, br d, J=3.9 Hz) 4.54 (1 H, br s) 4.36 (1 H, br s) 4.13 - 4.21 (2 H, m) 3.85 (2 H, br s) 3.58 - 3.75 (4 H, m) 3.50 - 3.57 (2 H, m) 3.36 (2 H, br s) 3.19 (1 H, br s) 2.94 (1 H, br d, J=9.8 Hz) 2.74 (1 H, br d, J=12.6 Hz) 2.12 (3 H, br d, J=7.1 Hz) 1.96 - 2.05 (5 H, m) 1.32 - 1.45 (5 H, m) 0.89 (9 H, s)

[0718] BF025 2-[[6-[3-[2-[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]ethoxy]ethoxyphenoxy]pyridine-3-carbonyl]amino]-5,5-dimethyl-hexanoic acid [ka] Step 1: 2-[2-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl 4-methylbenzenesulfonate tert-Butyl N-[2-[2-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate (100 mg, 262.15 μμολ, 1 equiv.) was dissolved in THF (1 mL). NaOH (20.97 mg, 524.30 μmol, 2 equiv.) dissolved in HO (0.2 mL) was added. The mixture was cooled to 0°C, and 4-methylbenzenesulfonyl chloride (59.97 mg, 314.58 μmol, 1.2 equiv.) dissolved in THF (1 mL) was added dropwise. The reaction mixture was warmed to 20°C and stirred for 16 h. TLC (KMnO, DCM:MeOH = 10:1) showed the reaction was complete. The THF was evaporated, and 50 mL of HO was added, followed by extraction with 5 mL of CHCl (twice) and washing with 5 mL of brine. The product was dried over MgSO and concentrated. The residue was purified by silica gel chromatography eluting with DCM:MeOH (50:1 to 10:1) to afford 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.

[0719] 1 H NMR (400 MHz, CHLOROFORM-d) : ppm 7.82 (2 H, d, J=8.1 Hz) 7.36 (2 H, d, J=8.0 Hz) 5.06 (1 H, br s) 4.11 - 4.26 (2 H, m) 3.50 - 3.80 (21 H, m) 3.33 (2 H, br s) 2.47 (3 H, s) 1.46 (9 H, s)

[0720] Step 2: Tert-butyl N-[2-[2-[2-[2-[2-[2-(3-hydroxyphenoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate 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 equiv.) and (3-hydroxyphenyl)acetate (272.68 mg, 1.79 mmol, 1.2 equiv.) in DMF (10 mL) was added CsCO (1.46 g, 4.48 mmol, 3 equiv.). The reaction mixture was stirred at 80 °C under N for 16 h. TLC (DCM:MeOH = 10:1) showed the reaction was complete. The reaction mixture was concentrated to a residue, to which HO (20 mL) was added and extracted with EtOAc (20 mL × 3). The combined organic layer was washed with brine (25 mL), dried over anhydrous NaSO, filtered, and concentrated. The crude product was purified by silica gel chromatography eluting with (PE: EtOAc = 100: 1 to 1: 3) to give tert-butyl N-[2-[2-[2-[2-[2-[2-(3-hydroxyphenoxy) ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethyl] carbamate (360 mg, yield 45.56%, purity 89.5%) as a yellow oil.

[0721] LCMS (ESI) [M+Na] + m / z: theoretical value 496.2, measured value 496.2 Step 3: Methyl 6-[3-[2-[2-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate To a solution of tert-butyl N-[2-[2-[2-[2-[2-[2-(3-hydroxyphenoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]carbamate (360 mg, 760.21 μmol, 1 equiv.) and methyl 6-fluoropyridine-3-carboxylate (141.51 mg, 912.25 μmol, 1.2 equiv.) in DMF (8 mL) was added CsCO (743.07 mg, 2.28 mmol, 3 equiv.). The reaction was stirred at 80 °C for 20 h. LCMS showed the reaction was complete. The reaction mixture was concentrated to a residue, to which HO (15 mL) was added and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over anhydrous NaSO, filtered, and concentrated. The crude product was purified by silica gel chromatography eluting with (DCM:MeOH=100:1 to 10:1) to give methyl 6-[3-[2-[2-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate (247 mg, 48.03% yield, 89.98% purity) as a yellow oil.

[0722] LCMS (ESI) [M+Na] + m / z: theoretical value 631.3, measured value 631.1

[0723] Step 4: Methyl 6-[3-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate To a solution of methyl 6-[3-[2-[2-[2-[2-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate (247 mg, 405.80 μmol, 1 equiv) in DCM (10 mL) was added TFA (925.41 mg, 8.12 mmol, 600.91 μL, 20 equiv). The reaction was stirred at 20 °C for 20 h. TLC showed the reaction was complete. The reaction was basified to pH 8 with saturated aqueous NaHCO3 and then extracted with DCM (3 × 10 mL). The combined organic layers were washed with brine (15 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo to afford methyl 6-[3-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate (206 mg, 84.95% yield, 85.1% purity) as a yellow oil, which was used directly without further purification.

[0724] LCMS (ESI) [M+Na] + m / z: theoretical value 509.2, measured value 509.3

[0725] Step 5: Methyl 6-[3-[2-[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]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate To a solution of 5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid (74.94 mg, 306.75 μmol, 1 equiv) in DMF (1 mL) was added HATU (174.95 mg, 460.12 μmol, 1.5 equiv) and DIPEA (118.94 mg, 920.25 μmol, 160.29 μL, 3 equiv). After stirring at 20° C. for 30 minutes, 6-[3-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]phenoxy]pyridine-methyl 3-carboxylate (156 mg, 306.75 μmol, 1 equiv.) was added. The resulting mixture was stirred for 20 minutes. ℃ The mixture was stirred at rt for 1 h and then concentrated to a residue. HO (20 mL) was added, followed by extraction with ethyl acetate (20 mL × 3). The combined organic phase was washed with brine (30 mL), dried over NaSO, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography eluting with (EtOAc:MeOH = 50:1 to 10:1) to afford 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]ethoxy]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate (88.73% yield) as a yellow oil.

[0726] LCMS (ESI) [M+H] + m / z: theoretical value 735.3, measured value 735.3 1H NMR (400 MHz, DMSO-d6) : ppm 8.71 (1 H, d, J=2.4 Hz) 8.31 (1 H, dd, J=8.6, 2.5 Hz) 7.83 (1 H, br t, J=5.6 Hz) 7.35 (1 H, t, J=8.1 Hz) 7.12 (1 H, d, J=8.8 Hz) 6.86 (1 H, dd, J=8.1, 2.1 Hz) 6.80 (1 H, t, J=2.3 Hz) 6.76 (1 H, dd, J=7.9, 1.8 Hz) 6.42 (1 H, s) 6.36 (1 H, s) 4.25 - 4.33 (1 H, m) 4.07 - 4.15 (3 H, m) 3.86 (3 H, s) 3.70 - 3.77 (2 H, m) 3.56 - 3.61 (2 H, m) 3.47 - 3.55 (13 H, m) 3.36 - 3.43 (2 H, m) 3.14 - 3.21 (4 H, m) 3.05 - 3.12 (1 H, m) 2.82 (1 H, dd, J=12.4, 5.1 Hz) 2.06 (2 H, t, J=7.4 Hz) 1.56 - 1.67 (1 H, m) 1.38 - 1.55 (3 H, m) 1.15 - 1.36 (2 H, m)

[0727] Step 6: 6-[3-[2-[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]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylic acid To a solution of methyl 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]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylate (150 mg, 204.12 μmol, 1 equiv.) in THF (1 mL), MeOH (0.5 mL), and HO (0.5 mL) was added LiOH.HO (25.70 mg, 612.37 μmol, 3 equiv.). The mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated to a residue, acidified with 1 N HCl to pH 1, and then extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine (15 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo to give 6-[3-[2-[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]ethoxy]ethoxy]phenoxy]pyridine-3-carboxylic acid (121 mg, 77.69% yield, 94.47% purity) as a yellow oil.

[0728] LCMS (ESI) [M+H] + m / z: theoretical value 721.3, measured value 721.2 1H NMR (400 MHz, METHANOL-d4) : ppm 8.64 (1 H, d, J=1.9 Hz) 8.25 (1 H, dd, J=8.8, 2.4 Hz) 7.25 (1 H, t, J=8.2 Hz) 6.91 (1 H, d, J=8.6 Hz) 6.77 (1 H, dd, J=8.3, 1.7 Hz) 6.57 - 6.72 (2 H, m) 4.34 - 4.43 (1 H, m) 4.19 (1 H, dd, J=7.9, 4.4 Hz) 4.01 - 4.08 (2 H, m) 3.70 - 3.78 (2 H, m) 3.58 - 3.66 (3H, m) 3.48 - 3.56 (13 H, m) 3.37 - ...

Claims

1. Bifunctional compounds containing the structure of formula (I): T Ｌ - L Ｉ - S Ｌ (I) (In the formula, S Ｌ This is the part that binds to soltirin with a dissociation constant (KD) of 50 μM or less; L Ｉ is a linker or bond; T Ｌ This is the portion that binds to extracellular target molecules with a dissociation constant (KD) of 50 μM or less. or a pharmaceutically acceptable salt thereof.

2. S Ｌ However, the bifunctional compound according to claim 1 having a structure according to formula III: 【Chemistry 1】 (where Q １ is a bond or -CH ２ -; R ３ This is the expression of formula (IIIa), formula (IIIb), or formula B, 【Chemistry 2】 In the formula, R ３ａ H, halogen, alkoxy, -CF ３ , and C which is replaced by any choice １ -C ４ Selected from the group consisting of alkyl groups; In the formula, * indicates combination with formula (III), R Ｌ is, L Ｉ (This represents a combination with [another entity].)

3. S Ｌ However, equation (IIIi) 【Transformation 3】 This is due to the formula, where R Ｌ However, L Ｉ A bifunctional compound according to claim 2, which represents a bond with [another compound].

4. S Ｌ The bifunctional compound according to claim 2, wherein the compound is of formula (IIIk) or formula (IIIn): 【Chemistry 4】 【Transformation 5】 (In the formula, R Ｌ is, L Ｉ (This represents a combination with [another entity].)

5. S Ｌ However, the bifunctional compound according to claim 2 is of formula (D-I) or formula (D-II): 【Transformation 6】 【Transformation 7】 (In the formula, R Ｌ However, L Ｉ (This represents a connection to)

6. S Ｌ However, the bifunctional compound according to claim 1 comprises a peptide containing the following sequence: X ５ -X １ X ２ X ３ X ４ (Sequence No. 16) (In the formula, X ５ This is an optional amino acid residue or peptide containing 2 to 30 amino acid residues or bonds. X １ is R, P, F, Y, L, K, G, or H; X ２ is Q, Y, L, E, or G; X ３ is Y, F, L, I, Q, E, or N; X ４ is a conservative substitution of M, K, L, or L; L Ｉ (It is conjugated at the N-terminus.)

7. X １ However, it is F or R, X² is Q, X3 is Q, and X 4 is L. The bifunctional compound according to claim 6.

8. The bifunctional compound according to claim 1, wherein the extracellular target molecule is selected from the group consisting of: TNF-α, ANCEPTL-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, choline sterase, 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, Lyrein-1, lipoprotein lipase, human matrix metalloptidase 1, macrophage migration inhibitor, transforming factor-p (TGF-p), thrombospondin-1 (TSP-T), CD40 ligand, urokinase-type plasminogen activator, plasminogen activator tissue type (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 C1.

9. The bifunctional compound according to claim 1, wherein the extracellular target molecule is TNFα.

10. T Ｌ However, the bifunctional compound according to claim 1 is as follows: I. Formula XVIIa 【Transformation 8】 (In the formula, R ＸＶＩＩａ is, formula 【Chemistry 9】 It has, and here, R ＸＶＩＩｄ and R ＸＶＩＩｅ Independently, C １ -C ５ Alkyl, C １ -C ５ Alkoxy, cyano, halogen, and Ｃ１ Selected from the group consisting of haloalkyls, each of which is independently substituted by any choice; X is an atom selected from N or CH; * represents a bond with formula (XVIIa); R ＸＶＩＩｂ and R ＸＶＩＩｂ’ These are H and C, respectively, independently. １ -C ３ Selected from alkyl groups; R ＸＶＩＩｃ R Ｌ Either or selected from the group consisting of formula XVIIa-1 and formula XVIIa-2: 【Chemistry 10】 Here, R ＸＶＩＩｆ C is replaced by an optional substitution. １ -C ５ Selected from the group consisting of alkyl groups, where one or more methylene groups are substituted with a group selected from the group consisting of carbonyl, ester, amide, -NH-, or -O-, where * represents a bond with formula XVIIa; R Ｌ is, L Ｉ (Represents a combination with); II. Formula XVIIb 【Chemistry 11】 (wherein R XVIIi is selected from bonded and optionally substituted piperazine groups; R XVIIj is, 【Chemistry 12】 Selected from the group consisting of, where * represents combination with formula XVIIb; III. Formula XVIIc 【Chemistry 13】 (wherein R XVIIg is a C1-C4 alkyl group, and R XVIIh is optionally substituted with one or more groups selected from halogens, haloalkyl groups, cyano, hydroxyl, amino, hydroxyl, alkoxy groups, C3-C6 cycloalkyl groups, and C3-C6 heterocycloalkyl groups); or IV. Formula XVIId; 【Chemistry 14】 Here, R L represents a combination with L I.

11. T Ｌ The bifunctional compound according to claim 1, wherein the compound is based on formulas XVIIa-3, XVIIa-4, and XVIIa-5: 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 (In the formula, R Ｌ is, L Ｉ (This represents a combination with [another entity].)

12. T Ｌ The bifunctional compound according to claim 1, wherein the compound is based on formula XVIIb-1 or formula XVIIb-2: [Chemistry 18] 【Chemistry 19】 (In the formula, R Ｌ is, L Ｉ (This represents a combination with [another entity].)

13. T Ｌ The bifunctional compound according to claim 1, wherein the compound is based on formula XVIIc-1 or formula XVIIc-2: 【Chemistry 20】 【Chemistry 21】 (In the formula, R Ｌ is, L Ｉ (This represents a combination with [another entity].)

14. The difunctional compound according to claim 1, wherein the difunctional compound is one of the formulas XVIIIa, XVIIIb, XVIIIb-1, XVIIIc, XVIIId, XVIIId-1, XVIIIe, XVIIIf, XVIIIf-1, XVIIIg, XVIIIh, and XVIIIh-1: 【Chemistry 22】 【change】 【change】 (In the formula, Q ３ is a bond or -CH ２ - and; R ３ａ H, halogen, alkoxy, -CF ３ , and C which is replaced by any choice １ - ４ Selected from the group consisting of alkyl groups; L Ｉ (This indicates a linker.)

15. The difunctional compound according to claim 1, wherein the difunctional compound is one of formulas D-III, D-IV, D-V, and D-VI: 【Chemistry 23】 。

16. L Ｉ However, the bifunctional compound according to claim 1 having the following structure: 【Chemistry 24】 (In the formula, * is T Ｌ or S Ｌ Represents a connection point to any of the following: L １ and L ２ Each is independent, bonded, -C(H ２ )-, -O-, -N(H)-; functional groups selected from carbonyl, ester, amide, and carbamate; and C １ -C ３ Selected from the group consisting of hydrocarbon chains (in which one or more methylene groups are individually and optionally replaced with carbonyl, ester, amide, carbamate, thiourea, urea sulfonamide, and triazole); Z is divalent, saturated or unsaturated, straight-chain or branched-chain, C １－ C ３０ selected from the group consisting of hydrocarbon chains, wherein one or more methylene groups are individually and optionally replaced by one or more of the following: -O-, -N(H)-, -N(R Ｌ１ ), -OC(=O)-, -C(=O)O-, -C(=O)-, -N(H)C(=O)-, -N(R Ｌ１ ), -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 ); optionally substituted divalent aromatic groups, optionally substituted carbocycles, optionally substituted heterocycles, optionally substituted divalent aromatic heterocycles; 【Chemistry 25】 R Ｌ１ C １－５ Selected from the group consisting of alkyl groups; n and w are each integers from 1 to 9.

17. L Ｉ However, one of the following equations (XVIa) to (XVIbw) is selected: 【Chemistry 26】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 In the formula, * represents a combination with either S L or T L. A bifunctional compound according to any one of claims 1 to 16.

18. The bifunctional compound according to claim 1, wherein the bifunctional compound is selected from any one of compounds BF030 to BF144: 【Chemistry 27】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 。

19. A pharmaceutical for use in the treatment of a subject requiring treatment of a disorder or condition associated with abnormal TNF-α levels, wherein the pharmaceutical is a bifunctional compound of formula (I): T L - L I - S L (I) (In the formula, S L is the part that binds to soltirin with a dissociation constant (K D) of 50 μM or less; L I is a linker or bond; T L is the portion that binds to extracellular target molecules with a dissociation constant (K D) of 50 μM or less. or containing a pharmaceutically acceptable salt thereof, A drug in which the aforementioned disorder or condition is an inflammatory disease, autoimmune disease, or cancer.

20. The pharmaceutical product for use according to claim 19, wherein the disorder or condition is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, graft-versus-host disease, ankylosing spondylitis, psoriasis, suppurative dacryoadenitis, refractory asthma, systemic erythrocytosis, diabetes mellitus, and the induction of cachexia.

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