Integrin Ligands and Uses Thereof

Abstract

To provide methods of treating disease.SOLUTION: Synthetic αvβ6 integrin ligands of Formula I having serum stability and affinity for integrin αvβ6, which is a receptor expressed in a variety of cell types, are described. The described ligands are useful for delivering cargo molecules, such as RNAi agents or other oligonucleotide-based compounds, to cells that express integrin αvβ6, and thereby facilitating the uptake of the cargo molecules into these cells. Compositions that include αvβ6 integrin ligands and methods of use are also described.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 580,398, filed November 1, 2017, U.S. Provisional Patent Application No. 62 / 646,739, filed March 22, 2018, and U.S. Provisional Patent Application No. 62 / 679,549, filed June 1, 2018, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] background Integrin alpha-v beta-6 (αvβ6), expressed in various cell types, including epithelial cells, is a receptor for the TGF-β latency-associated peptide (LAP) and the extracellular matrix (ECM) proteins fibronectin, vitronectin, and tenascin. While barely detectable in healthy adult epithelia, αvβ6 integrin is upregulated during wound healing and in various cancers (e.g., colon, ovarian, endometrial, and gastric cancers) and is often associated with poor cancer prognosis. αvβ6 integrin has been shown to promote cell invasion and migration in metastasis and inhibit apoptosis. αvβ6 integrin can also regulate matrix metalloproteinase (MMP) expression and activate TGF-β1. Increasing evidence, primarily from in vitro studies, suggests that αvβ6 integrin may promote carcinoma progression. Integrin αvβ6 is therefore attractive as a tumor biomarker and potential therapeutic target, particularly in view of its role in matrix metalloproteinase (MMP) expression and TGF-β1 activation.

[0003] In vivo delivery of therapeutically effective compounds, such as drug compounds, to desired cells and / or tissues remains a common challenge in formulation development. There remains a need for stable and effective targeting ligands that can selectively target cells or tissues and facilitate targeted delivery of cargo molecules (e.g., therapeutically active compounds or components) to specific cells or tissues. Indeed, there is a general need for targeting ligands that can bind to one or more optimal cargo molecules (e.g., one or more pharmaceutical formulations) or other payloads to facilitate delivery of cargo molecules to desired cells or tissues in vivo. Furthermore, there is a need for compounds that target integrin alpha-v beta-6, suitable for binding to cargo molecules, for delivery of cargo molecules to cells expressing integrin alpha-v beta-6 in vivo. For certain cargo molecules, such as therapeutic oligonucleotide-based compounds (e.g., antisense oligonucleotides or RNAi agents), there is a need for targeting ligands capable of targeting integrin alpha-v beta-6 that can bind to the oligonucleotide-based compounds, thereby delivering the therapeutic to cells and / or tissues that express integrin alpha-v beta-6 and facilitating entry of the therapeutic into the cells by receptor-mediated endocytosis, pinocytosis, or other means. Summary of the Invention [Means for solving the problem]

[0004] Abstract Described herein are novel synthetic αvβ6 integrin ligands (also referred to herein as αvβ6 ligands). The αvβ6 integrin ligands disclosed herein are stable in serum and have affinity and can specifically bind to αvβ6 integrin. The αvβ6 integrin ligands can be conjugated to cargo molecules to bind to αvβ6 integrin. This can facilitate delivery of cargo molecules to desired cells or tissues (such as epithelial cells) that express the polypeptide.

[0005] Also disclosed herein are methods for delivering cargo molecules to tissues and / or cells expressing αvβ6 integrin in vivo, comprising administering to a subject one or more of the αvβ6 integrin ligands disclosed herein that are conjugated to one or more cargo molecules.Further disclosed are methods for treating a subject having a disease, condition, or disorder that can be treated in the subject by delivery of a therapeutic cargo molecule (e.g., an active pharmaceutical ingredient) to cells expressing αvβ6 integrin, comprising administering to the subject one or more of the αvβ6 integrin ligands disclosed herein that are conjugated to one or more therapeutic cargo molecules.

[0006] In some embodiments, described herein are methods for inhibiting the expression of a target gene in a cell, comprising administering to the cell an effective amount of one or more αββ integrin ligands linked to one or more oligonucleotide-based compounds (e.g., oligonucleotide-based therapeutic agents) (such as RNAi agents) capable of inhibiting the expression of a target gene in the cell. In some embodiments, described herein are methods for inhibiting the expression of a target gene in a cell of a subject, comprising administering to the subject an effective amount of one or more αββ integrin ligands linked to one or more oligonucleotide-based compounds (such as RNAi agents) capable of inhibiting the expression of a target gene in the cell.

[0007] Further described herein are compositions comprising αvβ6 integrin ligands. The compositions described herein can be pharmaceutical compositions comprising one or more of the αvβ6 integrin ligands disclosed herein linked to one or more therapeutic agents (such as RNAi agents or other cargo molecules).

[0008] In some embodiments, described herein are methods of treating a subject having a disease or disorder mediated at least in part by expression of a target gene, comprising administering to the subject in need an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises one or more αβ integrin ligands disclosed herein linked to one or more oligonucleotide-based compounds (such as RNAi agents).

[0009] In a first aspect, the present disclosure provides synthetic αvβ6 integrin ligands.

[0010] In some embodiments, the αvβ6 integrin ligand disclosed herein has the following formula: [ka] (In the formula, n is an integer from 0 to 7; J is CH or N; Z is for OR 13 , N(R 13 )2, or SR 13 and; R 1 is H, optionally substituted C1-C6 alkyl, OH, COOH, CON(R 5 )2, OR 6 or R 1 comprises a cargo molecule, where each R 5 is, independently, H or C1-C6 alkyl, and R 6 is H or C1-C6 alkyl; R 2 , R P1 , and R P2 are each independently H, halo, optionally substituted cycloalkylene, optionally substituted arylene, optionally substituted heterocycloalkylene, or optionally substituted heteroarylene; or R 2 , R P1 , and R P2 may comprise a cargo molecule; R10 is H or optionally substituted alkyl; R 11 is H or optionally substituted alkyl, or R 11 and R 1 together with the atoms to which they are attached form an optionally substituted heterocycle; R 12 is H or optionally substituted alkyl; Each R 13 are independently H, optionally substituted alkyl, or R 13 comprises a cargo molecule; R 14 is optionally substituted alkyl; where R 1 , R 2 , R 13 , R P1 , and R P2 wherein at least one of the amino acids comprises a cargo molecule) or a pharmaceutically acceptable salt thereof.

[0011] In some embodiments, the αvβ6 integrin ligands disclosed herein can be conjugated to one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30) cargo molecules (e.g., any cargo molecules described herein or known in the art).

[0012] In some embodiments, more than one αvβ6 integrin ligand disclosed herein (e.g., 2, 3, 4, 5, 6, 7, 8, or 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, or 4-5 αvβ6 integrin ligands) can be conjugated to a single cargo molecule (e.g., any cargo molecule described herein or known in the art).

[0013] In another aspect, the present disclosure provides compositions comprising one or more of the αvβ6 integrin ligands described herein. For example, in some embodiments, the compositions comprising one or more of the αvβ6 integrin ligands disclosed herein comprise one or more oligonucleotide-based compound(s) (such as one or more RNAi agent(s)) to be delivered to cells in vivo. In some embodiments, the present disclosure provides compositions for delivering RNAi agents to cells in vivo, wherein the RNAi agents are linked to one or more αvβ6 integrin ligands.

[0014] Compositions comprising one or more αvβ6 integrin ligands are described. In some embodiments, the compositions comprise a pharmaceutically acceptable excipient. In some embodiments, the compositions comprising one or more αvβ6 integrin ligands comprise one or more other pharmaceutical substances or pharmaceutically active ingredients or compounds. In some embodiments, medicaments comprising one or more αvβ6 integrin ligands are described herein.

[0015] Compositions comprising one or more αvβ6 integrin ligands disclosed herein linked to one or more cargo molecules are useful for delivery of cargo molecules to cells expressing integrin αvβ6 in vivo or in vitro. For example, compositions comprising one or more αvβ6 integrin ligands disclosed herein can deliver cargo molecules (such as oligonucleotide-based compounds) to type I and type II alveolar epithelial cells, goblet cells, secretory epithelial cells, ciliated epithelial cells, corneal and conjunctival epithelial cells, dermal epithelial cells, bile duct cells, enterocytes, ductal epithelial cells, glandular epithelial cells, and epithelial tumors (carcinomas) in vivo or in vitro.

[0016] In another aspect, the present disclosure provides methods that involve the use of one or more of the αvβ6 integrin ligands and / or compositions described herein, where desired, the disclosed αvβ6 integrin ligands and / or compositions are in a form suitable for pharmaceutical administration. In other embodiments, the present disclosure provides methods of producing the ligands and compositions (e.g., medicaments) described herein.

[0017] Compositions comprising one or more αvβ6 integrin ligands can be administered to a subject in vivo using any route of administration known in the art to be appropriate for the cargo molecule being administered, including, for example, inhalation (aerosol or dry powder formulation), intranasal, subcutaneous, intravenous, intraperitoneal, intradermal, transdermal, oral, sublingual, topical, or intratumoral administration. In some embodiments, compositions comprising one or more αvβ6 integrin ligands can be administered for systemic delivery, e.g., by intravenous or subcutaneous administration. In some embodiments, compositions comprising one or more αvβ6 integrin ligands can be administered for local delivery, e.g., by inhalation delivery via a dry powder inhaler or nebulizer. In some embodiments, compositions comprising one or more αvβ6 integrin ligands can be administered for local delivery by topical administration.

[0018] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to type I alveolar epithelial cells in vivo, comprising administering to a subject one or more αvβ6 integrin ligands linked to the one or more cargo molecules.

[0019] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to type II alveolar epithelial cells in vivo, comprising administering to a subject one or more αvβ6 integrin ligands linked to the one or more cargo molecules.

[0020] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to goblet cells in vivo, comprising administering to a subject one or more αvβ6 integrin ligands linked to the one or more cargo molecules.

[0021] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to secretory epithelial cells in vivo, comprising administering to a subject one or more αvβ6 integrin ligands linked to the one or more cargo molecules.

[0022] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to ciliated epithelial cells in vivo, comprising administering to a subject one or more αvβ6 integrin ligands linked to the one or more cargo molecules.

[0023] In some embodiments, corneal epithelial cells are transfected in vivo with one or more desired Disclosed herein is a method for delivering a cargo molecule(s) of the present invention, comprising administering to a subject one or more αvβ6 integrin ligands linked to the one or more cargo molecules.

[0024] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to conjunctival epithelial cells in vivo, comprising administering to a subject one or more αvβ6 integrin ligands linked to the one or more cargo molecules.

[0025] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to dermal epithelial cells in vivo, comprising administering to a subject one or more αvβ6 integrin ligands linked to the one or more cargo molecules.

[0026] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to bile duct cells in vivo, comprising administering to a subject one or more αvβ6 integrin ligands linked to the one or more cargo molecules.

[0027] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to intestinal cells in vivo, comprising administering to a subject one or more αvβ6 integrin ligands linked to the one or more cargo molecules.

[0028] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to ductal epithelial cells in vivo, comprising administering to a subject one or more αvβ6 integrin ligands bound to the one or more cargo molecules.

[0029] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to glandular epithelial cells in vivo, comprising administering to a subject one or more αvβ6 integrin ligands linked to the one or more cargo molecules.

[0030] In some embodiments, disclosed herein are methods for delivering one or more desired cargo molecule(s) to an epithelial tumor (carcinoma) in vivo, comprising administering to a subject one or more αvβ6 integrin ligands linked to the one or more cargo molecules.

[0031] In some embodiments, disclosed herein are methods for delivering oligonucleotide-based compounds to type I alveolar epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods for delivering RNAi agents to type I alveolar epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting target gene expression in type I alveolar epithelial cells in vivo, comprising administering to a subject an RNAi agent linked to one or more ligands having affinity for αββ integrin.

[0032] In some embodiments, disclosed herein is a method for delivering an oligonucleotide-based compound to type II alveolar epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more oligonucleotide-based compounds. In some embodiments, disclosed herein is a method for delivering an RNAi agent to type II alveolar epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more RNAi agents. In some embodiments, disclosed herein is a method for inhibiting the expression of a target gene in type II alveolar epithelial cells in vivo, comprising administering to a subject an RNAi agent linked to one or more ligands having affinity for αββ integrin.

[0033] In some embodiments, disclosed herein are methods for delivering oligonucleotide-based compounds to goblet cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods for delivering RNAi agents to goblet cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting target gene expression in goblet cells in vivo, comprising administering to a subject an RNAi agent linked to one or more ligands having affinity for αββ integrin.

[0034] In some embodiments, disclosed herein is a method for delivering an oligonucleotide-based compound to secretory epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more oligonucleotide-based compounds. In some embodiments, disclosed herein is a method for delivering an RNAi agent to secretory epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more RNAi agents. In some embodiments, disclosed herein is a method for inhibiting the expression of a target gene in secretory epithelial cells in vivo, comprising administering to a subject an RNAi agent linked to one or more ligands having affinity for αββ integrin.

[0035] In some embodiments, disclosed herein are methods for delivering oligonucleotide-based compounds to ciliated epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods for delivering RNAi agents to ciliated epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting target gene expression in ciliated epithelial cells in vivo, comprising administering to a subject an RNAi agent linked to one or more ligands having affinity for αββ integrin.

[0036] In some embodiments, disclosed herein are methods for delivering oligonucleotide-based compounds to corneal epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods for delivering RNAi agents to corneal epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more RNAi agents. Disclosed herein in some embodiments is a method of inhibiting expression of a target gene in a corneal epithelial cell in vivo, wherein the method comprises administering to a subject an RNAi agent linked to one or more ligands having affinity for αβ integrin.

[0037] In some embodiments, disclosed herein is a method for delivering an oligonucleotide-based compound to conjunctival epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more oligonucleotide-based compounds. In some embodiments, disclosed herein is a method for delivering an RNAi agent to conjunctival epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more RNAi agents. In some embodiments, disclosed herein is a method for inhibiting the expression of a target gene in conjunctival epithelial cells in vivo, comprising administering to a subject an RNAi agent linked to one or more ligands having affinity for αββ integrin.

[0038] In some embodiments, disclosed herein are methods for delivering oligonucleotide-based compounds to dermal epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods for delivering RNAi agents to dermal epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting target gene expression in dermal epithelial cells in vivo, comprising administering to a subject an RNAi agent linked to one or more ligands having affinity for αββ integrin.

[0039] In some embodiments, disclosed herein is a method for delivering an oligonucleotide-based compound to cholangiocytes in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more oligonucleotide-based compounds. In some embodiments, disclosed herein is a method for delivering an RNAi agent to cholangiocytes in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more RNAi agents. In some embodiments, disclosed herein is a method for inhibiting the expression of a target gene in cholangiocytes in vivo, comprising administering to a subject an RNAi agent linked to one or more ligands having affinity for αββ integrin.

[0040] In some embodiments, disclosed herein are methods for delivering oligonucleotide-based compounds to intestinal cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods for delivering RNAi agents to intestinal cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting target gene expression in intestinal cells in vivo, comprising administering to a subject an RNAi agent linked to one or more ligands having affinity for αββ integrin.

[0041] In some embodiments, the oligonucleotide-based compound is administered to ductal epithelial cells in vivo. Disclosed herein are methods for delivering an RNAi agent to ductal epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods for delivering an RNAi agent to ductal epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting target gene expression in ductal epithelial cells in vivo, comprising administering to a subject an RNAi agent linked to one or more ligands having affinity for αββ integrin.

[0042] In some embodiments, disclosed herein are methods for delivering oligonucleotide-based compounds to glandular epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods for delivering RNAi agents to glandular epithelial cells in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting target gene expression in glandular epithelial cells in vivo, comprising administering to a subject an RNAi agent linked to one or more ligands having affinity for αββ integrin.

[0043] In some embodiments, disclosed herein are methods for delivering oligonucleotide-based compounds to epithelial tumors (carcinomas) in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more oligonucleotide-based compounds. In some embodiments, disclosed herein are methods for delivering RNAi agents to epithelial tumors (carcinomas) in vivo, comprising administering to a subject one or more αββ integrin ligands linked to one or more RNAi agents. In some embodiments, disclosed herein are methods for inhibiting target gene expression in epithelial tumors (carcinomas) in vivo, comprising administering to a subject an RNAi agent linked to one or more ligands having affinity for αββ integrin.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are for illustrative purposes only and are not intended to limit the present invention.

[0045] Other objects, features, aspects, and advantages of the present invention will become apparent from the following detailed description and claims. DETAILED DESCRIPTION OF THE INVENTION

[0046] Detailed Description αvβ6 integrin ligand. Synthetic αvβ6 integrin ligands that have serum stability and affinity for integrin αvβ6 are described herein. The αvβ6 integrin ligands can be used to Cells expressing integrin αvβ6 can be targeted in vitro, in situ, ex vivo, and / or in vivo. In some embodiments, The αvβ6 integrin ligands disclosed herein can be conjugated to one or more cargo molecules and can be used in vitro, in situ, ex vivo, and / or in vivo. The cargo molecule can be preferentially targeted to cells expressing integrin αvβ6 in vivo. In some embodiments, the cargo molecule comprises or consists of a pharmaceutically active compound. In some embodiments, the cargo molecule comprises or consists of an oligonucleotide-based compound, such as an RNAi agent. In some embodiments, the αvβ6 integrin ligand disclosed herein is conjugated to the cargo molecule to target the cargo molecule to epithelial cells in vivo.

[0047] In a first aspect, the present disclosure provides synthetic αvβ6 integrin ligands.

[0048] In some embodiments, the αvβ6 integrin ligand disclosed herein has the following formula: [ka] (In the formula, n is an integer from 0 to 7; J is CH or N; Z is for OR 13 , N(R 13 )2, or SR 13 and; R 1 is H, optionally substituted C1-C6 alkyl, OH, COOH, CON(R 5 )2, OR 6 or R 1 comprises a cargo molecule, where each R 5 are independently H or C1-C6 alkyl, and R 6 is H or C1-C6 alkyl; R 2 , R P1 , and R P2 are each independently H, halo, optionally substituted cycloalkylene, optionally substituted arylene, optionally substituted heterocycloalkylene, or optionally substituted heteroarylene; or R 2 , R P1 , and R P2 may comprise a cargo molecule; R 10 is H or optionally substituted alkyl; R 11 is H or optionally substituted alkyl, or R 11 and R 1 together with the atoms to which they are attached form an optionally substituted heterocycle; R 12 is H or optionally substituted alkyl; Each R 13are independently H, optionally substituted alkyl, or R 13 comprises a cargo molecule; R 14 is optionally substituted alkyl; where R 1 , R 2 , R 13 , R P1 , and R P2 wherein at least one of the amino acids comprises a cargo molecule) or a pharmaceutically acceptable salt thereof.

[0049] In some embodiments, n=3 in Formula I. In some embodiments, n=4 in Formula I.

[0050] In some embodiments of Formula I, R 2 is naphthylene. In some embodiments of Formula I, R 2 is a substituted naphthylene, and R 2 also includes cargo molecules.

[0051] In some embodiments, the αvβ6 integrin ligand disclosed herein has the following formula: [ka] (In the formula, n is an integer from 0 to 7 (i.e., n is 0, 1, 2, 3, 4, 5, 6, or 7); J is CH or N; R 1 is H, C1-C6 alkyl, CH(R 3 )(R 4 ), OH, COOH, CH2CH2CH2NH2, CONHR 5 , OR 6 where R 3 is H or C1-C6 alkyl, and R 4 is H, C1-C6 alkyl, and R 5 is H or C1-C6 alkyl, and R 6is H or C1-C6 alkyl; R 2 is an optionally substituted cycloalkylene, an optionally substituted arylene, an optionally substituted heterocycloalkylene, or an optionally substituted heteroarylene; R 10 is H or optionally substituted alkyl; R 11 is H or optionally substituted alkyl, or R 11 and R 1 together with the atoms to which they are attached form an optionally substituted heterocycle; R 12 is H or optionally substituted alkyl; R 13 is H or optionally substituted alkyl; R 14 is optionally substituted alkyl; where R 1 or R 2 at least one of which comprises a cargo molecule) or a pharmaceutically acceptable salt thereof.

[0052] In some embodiments, in Formula II, n=3. In some embodiments, in Formula II, n=4.

[0053] In some embodiments, the αvβ6 integrin ligand disclosed herein has the following formula: [ka] (In the formula, n is an integer from 1 to 7 (i.e., n is 1, 2, 3, 4, 5, 6, or 7); R 7 comprises one or more cargo molecules; R 8is one or more optionally substituted divalent cyclic moieties having 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, including cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl), cycloalkenyl (e.g., cyclopentenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, or cycloheptenyl), aryl (e.g., phenyl), heteroaryl (e.g., pyridyl, pyrimidinyl, pyridazinyl, pyrrole, pyrazole, imidazole, thiophene, benzothiophene, thiazole, benzothiazole, furan, o or a pharmaceutically acceptable salt thereof.

[0054] In some embodiments, in Formula III, n=3. In some embodiments, in Formula III, n=4.

[0055] In some embodiments, the αvβ6 integrin ligand disclosed herein has the following formula: [ka] (In the formula, n is an integer from 1 to 7 (i.e., n is 1, 2, 3, 4, 5, 6, or 7); R 9 comprises one or more cargo molecules) or a pharmaceutically acceptable salt thereof.

[0056] In some embodiments, in Formula IV, n=3. In some embodiments, in Formula IV, n=4.

[0057] In another aspect, the present invention provides a construct comprising: [ka] (In the formula, n is an integer from 0 to 7; J is CH or N; Z is for OR 13 , N(R 13 )2, or SR 13 and; R 1 is H, optionally substituted C1-C6 alkyl, OH, COOH, CON(R 5 )2, OR 6 or R 1 comprises a linking group attached to a reactive group, where each R 5 are independently H or C1-C6 alkyl, and R 6 is H or C1-C6 alkyl; R 2 , R P1 , and R P2 are each independently H, halo, optionally substituted cycloalkylene, optionally substituted arylene, optionally substituted heterocycloalkylene, or optionally substituted heteroarylene; or R 2 , R P1 , and R P2 may comprise a linking group attached to the reactive group; R 10 is H or optionally substituted alkyl; R 11 is H or optionally substituted alkyl, or R 11 and R 1 together with the atoms to which they are attached form an optionally substituted heterocycle; R 12 is H or optionally substituted alkyl; Each R 13 are independently H, optionally substituted alkyl, or R 13 comprises a linking group attached to the reactive group; R 14is optionally substituted alkyl; where R 1 , R 2 , R 13 , R P1 , and R P2 wherein at least one of the groups comprises a linking group bonded to a reactive group), or a pharmaceutically acceptable salt thereof.

[0058] In some embodiments, the αvβ6 integrin ligands disclosed herein can be conjugated to one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30) cargo molecules (e.g., any cargo molecules described herein or known in the art).

[0059] In some embodiments, more than one αvβ6 integrin ligand disclosed herein (e.g., 2, 3, 4, 5, 6, 7, 8, or 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, or 4-5 αvβ6 integrin ligands) can be conjugated to a single cargo molecule (e.g., any cargo molecule described herein or known in the art).

[0060] In some embodiments, the αvβ6 integrin ligands disclosed herein are attached to one or more cargo molecules, optionally via a linking group (such as, for example, a polyethylene glycol (PEG) group).

[0061] In some embodiments, the αvβ6 integrin ligands disclosed herein are optionally attached to one or more cargo molecules via a scaffold comprising at least one attachment point for each ligand and at least one attachment point for each cargo molecule. In some embodiments, the αvβ6 integrin ligand comprises, consists of, or consists essentially of one cargo molecule. In some embodiments, the αvβ6 integrin ligand comprises, consists of, or consists essentially of more than one cargo molecule.

[0062] In some embodiments, the αvβ6 integrin ligand comprises, consists of, or consists essentially of any of structure 1, structure 2, structure 5, structure 5.1, structure 5.2, structure 6, structure 6.1, structure 6.2, structure 6.3, structure 6.4, structure 7, structure 8, structure 9, structure 10, structure 11, structure 12, structure 13, structure 14, structure 15, structure 16, structure 17, structure 18, structure 19, structure 20, structure 22, structure 23, structure 24, structure 25, structure 27, structure 29, structure 30, structure 31, structure 32, structure 33, structure 34, structure 35, structure 36, or structure 37, each disclosed herein.

[0063] Any of the αvβ6 integrin ligands disclosed herein can be linked to a cargo molecule, a reactive group, and / or a protected reactive group. The αvβ6 integrin ligand disclosed herein can facilitate binding of the cargo molecule to the αvβ6 integrin ligand. The αvβ6 integrin ligands disclosed herein can increase targeting of the cargo molecule to αvβ6 integrin or cells expressing αvβ6 integrin. The cargo molecule can be, but is not limited to, a pharmaceutically active ingredient or compound, a prodrug, or another substance with known therapeutic or diagnostic benefits. In some embodiments, the cargo molecule can be, but is not limited to, a small molecule, an antibody, an antibody fragment, an immunoglobulin, a monoclonal antibody, a label or marker, a lipid, a natural or modified oligonucleotide-based compound (e.g., an antisense oligonucleotide or an RNAi agent), a natural or modified nucleic acid, a peptide, an aptamer, a polymer, a polyamine, a protein, a toxin, a vitamin, polyethylene glycol, a hapten, digoxigenin, biotin, a radioactive atom or molecule, or a fluorophore. In some embodiments, the cargo molecule comprises a pharmaceutically active ingredient or a prodrug. In some embodiments, the cargo molecule comprises an oligonucleotide-based compound as the pharmaceutically active ingredient. In some embodiments, the cargo molecule comprises an RNAi agent as the pharmaceutically active moiety.

[0064] As used herein, the term "alkyl," unless otherwise specified, refers to a straight- or branched-chain saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms. For example, "C1-C6 alkyl" includes alkyl groups having 1, 2, 3, 4, 5, or 6 carbons in a straight- or branched-chain arrangement. Non-limiting examples of alkyl groups include methyl, ethyl, isopropyl, tert-butyl, and n-hexyl. As used herein, the term "aminoalkyl" refers to an alkyl group, as defined above, substituted at any position with one or more amino groups, as permitted by normal valences. The amino groups may be unsubstituted, monosubstituted, or disubstituted. Non-limiting examples of aminoalkyl groups include aminomethyl, dimethylaminomethyl, and 2-aminoprop-1-yl.

[0065] As used herein, unless otherwise specified, the term "cycloalkyl" refers to a saturated or unsaturated non-aromatic hydrocarbon ring group having 3 to 14 carbon atoms. Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, methylcyclopropyl, 2,2-dimethylcyclobutyl, 2-ethylcyclopentyl, and cyclohexyl. Cycloalkyl groups can contain multiple spirocyclic or fused rings. Cycloalkyl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted in any position allowed by normal valences.

[0066] As used herein, the term "cycloalkylene" refers to a divalent radical of a cycloalkyl group, as described herein. Cycloalkylene is a subset of cycloalkyl, referring to the same residues as cycloalkyl, but with two points of substitution. Examples of cycloalkylene include cyclopropylene, [ka] , 1,4-cyclohexylene, [ka] , and 1,5-cyclooxirene [ka] The cycloalkylene groups are optionally mono-, di-, tri-, tetra-, or penta-substituted in any position allowed by normal valences. The cycloalkylene groups may be mono-, bi-, or tricyclic ring systems.

[0067] As used herein, unless otherwise specified, the term "alkenyl" refers to a straight- or branched-chain non-aromatic hydrocarbon radical containing at least one carbon-carbon double bond and having 2 to 10 carbon atoms. Up to five carbon-carbon double bonds may be present in such a group. For example, a "C2-C6" alkenyl is defined as an alkenyl radical having 2 to 6 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, and cyclohexenyl. The straight-chain, branched-chain, or cyclic portions of an alkenyl group may contain double bonds and are optionally mono-, di-, tri-, tetra-, or penta-substituted in any position allowed by normal valences. The term "cycloalkenyl" refers to a monocyclic hydrocarbon group having the specified number of carbon atoms and at least one carbon-carbon double bond.

[0068] As used herein, unless otherwise specified, the term "alkynyl" refers to a straight- or branched-chain hydrocarbon radical containing 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond. Up to five carbon-carbon triple bonds may be present. Thus, "C2-C6 alkynyl" means an alkynyl radical having 2 to 6 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 2-propynyl, and 2-butynyl. The straight- or branched-chain portions of the alkynyl group may be optionally mono-, di-, tri-, tetra-, or penta-substituted in any position allowed by normal valences.

[0069] As used herein, "alkoxyl" or "alkoxy" refers to an -O-alkyl radical having the designated number of carbon atoms. For example, C1-6 alkoxy is intended to include C1, C2, C3, C4, C5, and C6 alkoxy groups. For example, C1-8 alkoxy is intended to include C1, C2, C3, C4, C5, C6, C7, and C8 alkoxy groups. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, s-pentoxy, n-heptoxy, and n-octoxy.

[0070] As used herein, "keto" refers to any alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, or aryl group as defined herein attached via a carbonyl bridge. Examples of keto groups include, but are not limited to, alkanoyl (e.g., acetyl, propionyl, butanoyl, pentanoyl, or hexanoyl), alkenoyl (e.g., acryloyl), alkynoyl (e.g., ethynoyl, propynoyl, butynoyl, pentynoyl, or hexynoyl), aryloyl (e.g., benzoyl), and heteroaryloyl (e.g., pyrroloyl, imidazoloyl, quinolinoyl, or pyridinoyl).

[0071] As used herein, "alkoxycarbonyl" refers to any alkoxy group as defined above attached through a carbonyl bridge (i.e., -C(O)O-alkyl). Examples of alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, iso-propoxycarbonyl, n-propoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, and the like. Examples of alkoxy include, but are not limited to, n-pentoxycarbonyl, n-oxycarbonyl, or n-pentoxycarbonyl.

[0072] As used herein, "aryloxycarbonyl" refers to any aryl group as defined above attached through an oxycarbonyl bridge (i.e., -C(O)O-aryl). Examples of aryloxycarbonyl groups include, but are not limited to, phenoxycarbonyl and naphthyloxycarbonyl.

[0073] As used herein, "heteroaryloxycarbonyl" refers to any heteroaryl group as defined herein attached through an oxycarbonyl bridge (i.e., -C(O)O-heteroaryl). Examples of heteroaryloxycarbonyl groups include, but are not limited to, 2-pyridyloxycarbonyl, 2-oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl, or pyrimidinyloxycarbonyl.

[0074] As used herein, "aryl" or "aromatic" refers to any stable monocyclic or polycyclic carbon ring of up to six atoms in each ring, in which at least one ring is aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, tetrahydronaphthyl, indanyl, and biphenyl. When the aryl substituent is bicyclic and one ring is non-aromatic, attachment is understood to be via the aromatic ring. Aryl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted in any position allowed by normal valences.

[0075] As used herein, the term "arylene" refers to a divalent radical of the aryl group described herein. Arylene is a subset of aryl and refers to the same residue as aryl, but has two substitution points. Examples of arylene include phenylene, which refers to a divalent phenyl group. Arylene groups are optionally mono-, di-, tri-, tetra-, or penta-substituted at any position allowed by normal valence.

[0076] As used herein, the term "halo" refers to a halogen radical. For example, "halo" can refer to a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) radical.

[0077] As used herein, the term "heteroaryl" refers to a stable monocyclic or bicyclic ring of up to seven atoms in each ring, where at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N, and S. Examples of heteroaryl groups include, but are not limited to, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, benzimimidazolonyl, benzoxazolonyl, quinolinyl, isoquinolinyl, dihydroisoindolonyl, imidazopyridinyl, isoindolonyl, indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinoline. "Heteroaryl" is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl. If a heteroaryl substituent is bicyclic and one ring is non-aromatic or does not contain a heteroatom, it is understood to be attached via the aromatic ring or the heteroatom-containing ring. Heteroaryl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted in any position allowed by normal valences.

[0078] As used herein, the term "heteroarylene" refers to any heteroaryl group as defined herein. Heteroarylene refers to a divalent radical of an aryl group. Heteroarylene is a subset of heteroaryl and refers to the same residue as heteroaryl, but with two points of substitution. Examples of heteroaryl include pyridinylene, pyrimidinylene, and pyrrolylene. Heteroarylene groups are optionally mono-, di-, tri-, tetra-, or penta-substituted in any position allowed by normal valences.

[0079] As used herein, the terms "heterocycle," "heterocyclic," or "heterocyclyl" refer to a 3- to 14-membered aromatic or non-aromatic heterocycle (including polycyclic groups) containing 1 to 4 heteroatoms selected from the group consisting of O, N, and S. As used herein, the term "heterocyclic" is also considered synonymous with the terms "heterocycle" and "heterocyclyl," and are understood to have the same definition as set forth herein. "Heterocyclyl" includes heteroaryls as defined above, as well as dihydro and tetrahydro analogs thereof.Examples of heterocyclyl groups include azetidinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthopyridinyl, oxadiazolyl, and oxooxazolidinyl. , oxazolyl, oxazoline, oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyridinonyl, pyrimidyl, pyrimidinonyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, tetrazolyl, tetrazolopyridyl, thiazolin ... Diazolyl, thiazolyl, thienyl, triazolyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, Heterocyclyl groups include, but are not limited to, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dihydrothiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and N-oxides thereof. Attachment of heterocyclyl substituents can occur via carbon atoms or heteroatoms. Heterocyclyl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted at any position permitted by normal valences.

[0080] As used herein, the term "heterocycloalkyl" means a 3- to 14-membered non-aromatic heterocycle (including polycyclic groups) containing 1 to 4 heteroatoms selected from the group consisting of O, N, and S. Examples of heterocyclyl groups include azetidinyl, oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, oxetanyl, pyranyl, pyridinonyl, pyrimidinonyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrofuranyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dioxidethiomorpholinyl, and tetrahydrothienyl. Heterocycloalkyl groups include, but are not limited to, heterocycloalkyl, cycloalkyl groups, and N-oxides thereof. Attachment of a heterocycloalkyl substituent can occur via a carbon atom or a heteroatom. Heterocyclyl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted in any position allowed by normal valences.

[0081] As used herein, the term "heterocycloalkylene" refers to a divalent radical of a heterocycloalkyl group as described herein. Heterocycloalkylene is a subset of heterocycloalkyl and refers to the same residue as heterocycloalkyl, but with two points of substitution. Examples of heterocycloalkylene include piperidinylene, azetidinylene, and tetrahydrofuranylene. Heterocycloalkylene groups are optionally mono-, di-, tri-, tetra-, or penta-substituted in any position allowed by normal valences.

[0082] As used herein, the terms "treat" and "treatment" and the like refer to a method or process employed to obtain a reduction or alleviation of the number, severity, and / or frequency of one or more symptoms of a disease in a subject. As used herein, "treat" and "treatment" can include prevention, management, prophylactic treatment, and / or suppression of the number, severity, and / or frequency of one or more symptoms of a disease in a subject.

[0083] As used herein, the phrase "introduction into a cell," when referring to an RNAi agent, refers to functional delivery of the RNAi agent into a cell. The phrase "functional delivery" refers to delivery of the RNAi agent to a cell in such a manner that the RNAi agent can have the expected biological activity (e.g., sequence-specific inhibition of gene expression).

[0084] Unless otherwise stated, symbols used herein [ka] The use of means that any group or groups can be attached to this symbol according to the scope of the invention described herein.

[0085] As used herein, the term "isomers" refers to compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or in the spatial arrangement of their atoms. Isomers that differ in the spatial arrangement of their atoms are called "stereoisomers." Stereoisomers that are not mirror images of one another are called "diastereoisomers," and stereoisomers that are non-superimposable mirror images are called "enantiomers" or sometimes optical isomers. A carbon atom bonded to four non-identical substituents is called a "chiral center." When a compound described herein contains an olefinic double bond or other center of geometric asymmetry where the isomeric structure is not specifically defined, it is intended that the compound may contain both E and Z geometric isomers, individually or in mixtures. A compound of Formula I or a pharmaceutically acceptable salt thereof is intended to include, for example, all possible isomers, as well as racemic and optically pure forms thereof. Similarly, all tautomeric forms are intended to be included unless expressly stated otherwise.

[0086] As used herein, a linking group is one or more atoms that connect one molecule or portion of a molecule to another second molecule or second portion of a molecule. In the art, the terms linking group and spacer are sometimes used interchangeably. Similarly, as used in the art, the term scaffold is sometimes used interchangeably with linking group. In some embodiments, a linking group can include a peptide cleavable linking group. In some embodiments, a linking group can be a peptide cleavable linking group. The linking group may comprise or consist of the peptide phenylalanine-citrulline-phenylalanine-proline. In some embodiments, the linking group may comprise or consist of a PEG group.

[0087] As used herein, the term "linked," when referring to a connection between two molecules, means that the two molecules are joined together by a covalent bond, or that the two molecules are associated through a non-covalent bond (e.g., a hydrogen bond or an ionic bond). In some instances, when the term "linked" refers to an association between two molecules through a non-covalent bond, the K of association between two different molecules is used.D is 1 × 10 in a physiologically acceptable buffer (e.g., phosphate-buffered saline). -4 Less than M (e.g., 1 × 10 -5 Less than M, 1 x 10 -6 Less than M or 1 x 10 -7 M or less). Unless otherwise indicated, the term linked, as used herein, may refer to a connection between a first compound and a second compound with or without any intervening atoms or groups of atoms.

[0088] Those skilled in the art will readily understand and appreciate that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state depending on the environment in which the compound or composition is placed. Thus, as used herein, it is contemplated that the structures disclosed herein may have certain functional groups, such as, for example, OH, SH, or NH, that may be protonated or deprotonated. The disclosure herein is intended to cover the disclosed compounds and compositions regardless of their protonation state based on the pH of the environment, as will be readily understood by those skilled in the art.

[0089] Structures can be depicted as having "dangling" bonds on the ring structure to indicate bonds to any carbon atom or heteroatom on the ring as allowed by valence. For example, the structure [ka] indicates that R can replace any hydrogen atom at any of the five available positions on the ring. A "floating" bond can also be used in bicyclic structures to indicate a bond to any position on either ring of the bicycle as allowed by valence. In the case of a bicycle, the bond will be shown as "floating" on both rings (e.g., [ka] indicates that R can replace any hydrogen atom at any of the seven available positions on the ring).

[0090] When used in a claim, the phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. When used in a claim, the phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps, and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0091] Description for targeting and delivering cargo molecules to cells expressing αvβ6 integrin The use of αvβ6 integrin ligands of the following is described herein: Cargo molecules can be delivered to cells in vitro, in situ, ex vivo, or in vivo.

[0092] In some embodiments of Formula Ib, the linking group is a PEG group containing 2 to 20 ethylene glycol units.

[0093] In some embodiments of Formula Ib, the reactive group is azide.

[0094] In some embodiments, the αv6 integrin ligand is: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] (In the formula, [ka] indicates the point of attachment to the moiety containing the cargo molecule), or a pharmaceutically acceptable salt thereof.

[0095] In some embodiments, the αvβ6 integrin ligands disclosed herein can be conjugated to one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30; or 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30) cargo molecules (e.g., any cargo molecules described herein or known in the art).

[0096] In some embodiments, more than one αvβ6 integrin ligand disclosed herein (e.g., 2, 3, 4, 5, 6, 7, 8, or 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, or 4-5 αvβ6 integrin ligands) can be conjugated to a single cargo molecule (e.g., any cargo molecule described herein or known in the art).

[0097] In some embodiments, the αvβ6 integrin ligands disclosed herein are attached to one or more cargo molecules, optionally via a linking group (such as, for example, a polyethylene glycol (PEG) group).

[0098] In some embodiments, the αvβ6 integrin ligands disclosed herein are optionally attached to one or more cargo molecules via a scaffold comprising at least one attachment point for each ligand and at least one attachment point for each cargo molecule. In some embodiments, the αvβ6 integrin ligand comprises, consists of, or consists essentially of one cargo molecule. In some embodiments, the αvβ6 integrin ligand comprises, consists of, or consists essentially of more than one cargo molecule.

[0099] In some embodiments, the αβ integrin ligand is Structure 1, Structure 2, Structure 5, Structure 5.1, Structure 5.2, Structure 6, or Structure 6.1, each of which is disclosed herein. 1, Structure 6.2, Structure 6.3, Structure 6.4, Structure 7, Structure 8, Structure 9, Structure 10, Structure 11, Structure 12, Structure 13, Structure 14, Structure 15, Structure 16, Structure 17, Structure 18, Structure 19, Structure 20, Structure 22, Structure 23, Structure 24, Structure 25, Structure 27, Structure 29, Structure 30, Structure 31, Structure 32, Structure 33, Structure 34, Structure 35, Structure 36, or Structure 37.

[0100] Any of the αvβ6 integrin ligands disclosed herein can be linked to a cargo molecule, a reactive group, and / or a protected reactive group. The reactive group can be used to facilitate the binding of the αvβ6 integrin ligand to the cargo molecule. The αvβ6 integrin ligands disclosed herein can increase targeting of the cargo molecule to αvβ6 integrin or cells expressing αvβ6 integrin. The cargo molecule can be, but is not limited to, a pharmaceutically active ingredient or compound, a prodrug, or another substance with known therapeutic benefits. In some embodiments, the cargo molecule can be, but is not limited to, a small molecule, an antibody, an antibody fragment, an immunoglobulin, a monoclonal antibody, a label or marker, a lipid, a natural or modified oligonucleotide-based compound (e.g., an antisense oligonucleotide or an RNAi agent), a natural or modified nucleic acid, a peptide, an aptamer, a polymer, a polyamine, a protein, a toxin, a vitamin, polyethylene glycol, a hapten, digoxigenin, biotin, a radioactive atom or molecule, or a fluorophore. In some embodiments, the cargo molecule comprises a pharmaceutically active ingredient or a prodrug. In some embodiments, the cargo molecule comprises an oligonucleotide-based compound as the pharmaceutically active ingredient. In some embodiments, the cargo molecule comprises an RNAi agent as the pharmaceutically active ingredient.

[0101] In one aspect, the present invention provides a structure comprising an αvβ6 integrin ligand described herein, a linking group, and a scaffold, wherein the scaffold is bound to a cargo molecule. In some embodiments, the structure may comprise a monodentate ligand. In some embodiments, the structure may comprise a bidentate ligand. In some embodiments, the structure may comprise a tridentate ligand. In some embodiments, the structure may comprise a tetradentate ligand.

[0102] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0103] In some embodiments, the αvβ6 integrin ligand of structure 1 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0104] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0105] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0106] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0107] In some embodiments, the αvβ6 integrin ligand of structure 2 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0108] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0109] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0110] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0111] In some embodiments, the αvβ6 integrin ligand of structure 5 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0112] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0113] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0114] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0115] In embodiments, the length of the PEG in the PEG-azide reactive group can vary. In some embodiments, the αvβ6 integrin ligand of structure 5.1 can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, including the following structure: [ka]

[0116] The reactive group (or protected reactive group) is used to attach the molecule of interest (e.g., cargo molecule (directly) The binding of the αvβ6 integrin ligand to the αvβ6 integrin ligand may be facilitated via one or more scaffolds and / or linkers.

[0117] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0118] In some embodiments, the αvβ6 integrin ligand of structure 5.2 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0119] In some embodiments, the PEG-azide reactive group can be replaced with an alkyl-azide reactive group. In some embodiments, the αβ integrin ligand can be synthesized to include an alkyl-azide reactive group, including the following structure: [ka]

[0120] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0121] In some embodiments, the αvβ6 integrin ligand of structure 6 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0122] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0123] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0124] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0125] In some embodiments, the αvβ6 integrin ligand of structure 6.1 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0126] In embodiments, the length of the PEG in the PEG-azide reactive group can vary. In some embodiments, the αβ integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group, including the following structure: [ka]

[0127] The reactive group (or protected reactive group) can be used to facilitate attachment of the αvβ6 integrin ligand to a molecule of interest (e.g., a cargo molecule (directly or via one or more scaffolds and / or linkers)).

[0128] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0129] In some embodiments, the αvβ6 integrin ligand of structure 6.2 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0130] In some embodiments, the PEG-azide reactive group can be replaced with an alkyl-azide reactive group. In some embodiments, the αβ integrin ligand can be synthesized to include an alkyl-azide reactive group, including the following structure: [ka]

[0131] The reactive group (or protected reactive group) can be used to facilitate attachment of the αvβ6 integrin ligand to a molecule of interest (e.g., a cargo molecule (directly or via one or more scaffolds and / or linkers)).

[0132] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0133] In some embodiments, the αvβ6 integrin ligand of structure 6.3 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0134] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule. In some embodiments, the αvβ6 integrin ligand can be synthesized to include an azide reactive group, including the following structure: [ka]

[0135] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0136] In some embodiments, the αvβ6 integrin ligand of structure 6.4 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0137] In some embodiments, αvβ6 integrin ligands can be synthesized to include azide reactive groups and include the following structures: [ka]

[0138] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0139] In some embodiments, the αvβ6 integrin ligand of structure 7 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0140] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0141] In some embodiments, αvβ6 integrin ligands can be synthesized to include PEG-azide reactive groups and include the following structures: [ka]

[0142] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0143] In some embodiments, the αvβ6 integrin ligand of structure 8 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0144] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0145] In some embodiments, αvβ6 integrin ligands can be synthesized to include PEG-azide reactive groups and include the following structures: [ka]

[0146] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0147] In some embodiments, the αvβ6 integrin ligand of structure 9 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0148] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0149] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0150] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0151] In some embodiments, the αvβ6 integrin ligand of structure 10 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0152] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0153] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0154] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0155] In some embodiments, the αvβ6 integrin ligand of structure 11 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0156] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0157] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0158] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0159] In some embodiments, the αvβ6 integrin ligand of structure 12 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0160] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0161] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0162] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0163] In some embodiments, the αvβ6 integrin ligand of structure 13 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0164] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0165] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0166] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0167] In some embodiments, the αvβ6 integrin ligand of structure 14 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0168] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0169] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0170] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0171] In some embodiments, the αvβ6 integrin ligand of structure 15 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0172] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0173] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0174] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0175] In some embodiments, the αvβ6 integrin ligand of structure 16 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0176] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0177] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0178] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0179] In some embodiments, the αvβ6 integrin ligand of structure 17 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0180] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0181] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0182] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0183] In some embodiments, the αvβ6 integrin ligand of structure 18 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0184] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0185] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0186] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0187] In some embodiments, the αvβ6 integrin ligand of structure 19 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0188] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0189] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0190] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0191] In some embodiments, the αvβ6 integrin ligand of structure 20 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0192] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0193] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0194] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0195] In some embodiments, the αvβ6 integrin ligand of structure 22 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0196] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0197] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0198] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0199] In some embodiments, the αvβ6 integrin ligand of structure 23 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0200] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0201] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0202] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0203] In some embodiments, the αvβ6 integrin ligand of structure 24 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0204] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0205] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0206] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0207] In some embodiments, the αvβ6 integrin ligand of structure 25 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0208] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0209] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0210] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0211] In some embodiments, the αvβ6 integrin ligand of structure 25 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0212] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0213] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0214] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure:

[0215] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0216] In some embodiments, the αvβ6 integrin ligand of structure 25 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0217] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0218] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0219] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0220] In some embodiments, the αvβ6 integrin ligand of structure 25 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0221] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0222] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0223] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0224] In some embodiments, the αvβ6 integrin ligand of structure 25 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0225] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0226] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0227] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0228] In some embodiments, the αvβ6 integrin ligand of structure 25 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0229] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0230] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0231] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0232] In some embodiments, the αvβ6 integrin ligand of structure 25 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0233] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0234] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0235] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0236] In some embodiments, the αvβ6 integrin ligand of structure 25 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0237] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0238] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0239] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0240] In some embodiments, the αvβ6 integrin ligand of structure 25 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0241] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0242] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0243] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0244] In some embodiments, the αvβ6 integrin ligand of structure 25 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0245] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0246] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0247] In some embodiments, the αvβ6 integrin ligand disclosed herein comprises the following structure: [ka]

[0248] In some embodiments, the αvβ6 integrin ligand of structure 25 is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0249] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, including the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0250] In some embodiments, the αvβ6 integrin ligand can be synthesized to include a polyethylene glycol (PEG)-azide reactive group and includes the following structure: [ka]

[0251] Structure 1a, Structure 1b, Structure 2a, Structure 2b, Structure 5a, Structure 5b, Structure 6a, Structure 6b, Structure 7a, Structure 7b, Structure 8a, Structure 8b, Structure 9a, Structure 9b, Structure 10a, Structure 10b, Structure 11a, Structure 11b, Structure 12a, Structure 12b, Structure Structure 13a, Structure 13b, Structure 14a, Structure 14b, Structure 15a, Structure 15b, Structure 16a, Structure 16b, Structure 17a, Structure 17b, Structure 18a, Structure 18b, Structure 19a, Structure 19b, Structure 20a, Structure 20b, Structure 22a, Structure 22b, Structure The disclosed reactive groups on any of structures 23a, 23b, 24a, 24b, 25a, 25b, 27a, 27b, 29a, 29b, 30a, 30b, 31a, 31b, 32a, 32b, 33a, 33b, 34a, 34b, 35a, 35b, 36a, 36b, 37a, or 37b can be used to attach the αvβ6 integrin ligand to a molecule of interest (i.e., to a cargo molecule such as an RNAi agent). The cargo molecule can be any molecule desired to target αvβ6 integrin-expressing cells. Multilocus αvβ6 integrin ligands and scaffolds

[0252] As disclosed herein, in some embodiments, one or more αvβ6 integrin ligands can be linked to one or more cargo molecules. In some embodiments, only one αvβ6 integrin ligand is attached to the cargo molecule (referred to herein as a "monodentate" or "monovalent" ligand). In some embodiments, two αvβ6 integrin ligands are attached to the cargo molecule (referred to herein as a "bidentate" or "divalent" ligand). In some embodiments, three αvβ6 integrin ligands are attached to the cargo molecule (referred to herein as a "tridentate" or "bivalent" ligand). In some embodiments, four αvβ6 integrin ligands are attached to the cargo molecule (referred to herein as "tetradentate" or "tetravalent" ligands). In some embodiments, there may be more than four αvβ6 integrin ligands attached to the cargo molecule.

[0253] In some embodiments, when only one αvβ6 integrin ligand is bound to a cargo molecule (referred to herein as a "monodentate" ligand), the αvβ6 integrin ligand can be directly bound to the cargo molecule. In some embodiments, the αvβ6 integrin ligands disclosed herein can be bound to the cargo molecule via a scaffold or other linker structure.

[0254] In some embodiments, the αvβ6 integrin ligands disclosed herein comprise one or more scaffolds. Scaffolds (sometimes referred to in the art as linking groups or linkers) can be used to facilitate the attachment of one or more cargo molecules to one or more αvβ6 integrin ligands disclosed herein. Useful scaffolds compatible with the ligands disclosed herein are generally known in the art. Non-limiting examples of scaffolds that can be used with the αvβ6 integrin ligands disclosed herein include, but are not limited to, polymers and polyamino acids (e.g., bis-glutamic acid, poly-L-lysine, etc.). In some embodiments, the scaffold comprises a cysteine ​​linker or group, DBCO-PEG. 1-24 -NHS, propargyl-PEG 1-24 -NHS, and / or multidentate DBCO and / or propargyl moieties.

[0255] In some embodiments, the scaffold used to link one or more αvβ6 integrin ligands disclosed herein to one or more cargo molecules has the following structure: [ka]

[0256] Scaffold 1, for example, facilitates efficient conjugation of both αvβ6 integrin ligand monomers and one or more cargo molecules. Scaffold 1 contains an amine-reactive p-nitrophenol (also called 4-nitrophenol) ester, an amine bridge, three units of PEG2, and a terminal alkyne. The 4-nitrophenol ester can be coupled to a primary amine on a cargo molecule (such as a primary amine on an RNA trigger created by amide formation with a terminal amine group (e.g., NH2-C6)). The terminal alkyne can be coupled to azide-modified ligands (both peptides and small molecules) via copper-catalyzed click chemistry.

[0257] In some embodiments, the cargo molecule is an RNAi agent. In some embodiments, scaffold 1 can be attached to a terminus of the RNAi agent (such as to the 5' end of the sense strand of the RNAi agent). For example, the 5' end of the sense strand of the RNAi agent can be modified to include a C6 amine (-C6-NH2) attached to the 5' end of the 5' terminal nucleotide of the RNAi agent. An RNAi agent having such a C6 amine modification (or another modification that results in a terminal amine) can be readily attached to scaffold 1, as shown by the following structural representation: [ka] (In the formula, [ka] indicates an RNAi agent).

[0258] The alkyne group of structure 380 above can then be attached to an αvβ6 integrin ligand disclosed herein to form a tridentate αvβ6 integrin ligand.

[0259] In some embodiments, the scaffold can be synthesized using DBCO (dibenzocyclooctyne), which can be represented by the following structure: [ka] (In the formula, [ka] indicates attachment to a moiety containing a reactive group or cargo molecule).

[0260] In some embodiments, a triazole group is formed between the RNAi agent and the αvβ6 integrin ligand disclosed herein, as shown in the following general structure: [ka] (In the formula, [ka] represents any suitable scaffold or linking group that can be used to link a ligand to an RNAi agent; [ka] indicates an RNAi agent).

[0261] In some embodiments, the scaffold can be synthesized as a phosphoramidite compound, allowing the tridentate ligand to be readily coupled to the 5' end of the sense strand of the RNAi agent via phosphoramidite synthesis, as shown in the following structure: [ka]

[0262] To attach the compound of structure 400 to the 5' end of the sense strand of an RNAi agent after synthesis, the terminal alkyne can then be linked to an αvβ6 integrin ligand disclosed herein.

[0263] In some embodiments, the αvβ6 integrin ligand disclosed herein is selected from the group consisting of Structure 1, Structure 2, Structure 5, Structure 5.1, Structure 5.2, Structure 6, Structure 6.1, Structure Structure 6.2, Structure 6.3, Structure 6.4, Structure 7, Structure 8, Structure 9, Structure 10, Structure 11, Structure 12, Structure 13, Structure 14, Structure 15, Structure 16, Structure 17, Structure 18, Structure 19, Structure 20, Structure 22, Structure 23, Structure 24, Structure 25, Structure 27, Structure 29, Structure 30, Structure 31, Structure 32, Structure 33, Structure 34, Structure 35, Structure 36, Structure 37, wherein the αvβ6 integrin ligand is a tridentate ligand linked via a scaffold.

[0264] In some embodiments, the αvβ6 integrin ligands disclosed herein comprise structure 2 in a tridentate form and can be represented by the following structure: [ka]

[0265] In some embodiments, the αvβ6 integrin ligands disclosed herein comprise structure 6.1 in a tridentate form and can be represented by the following structure: [ka]

[0266] In some embodiments, the αvβ6 integrin ligands disclosed herein comprise structure 6.1 in a tridentate form and can be represented by the following structure: [ka]

[0267] In some embodiments, the αβ integrin ligand disclosed herein comprises structure 6.1 in a tridentate form including a glutaric acid linker and can be represented by the following structure: [ka]

[0268] In some embodiments, the αβ integrin ligand disclosed herein comprises structure 6.1 in a tridentate form bound to an RNAi agent and can be represented by the following structure: [ka] (In the formula, [ka] indicates an RNAi agent).

[0269] In some embodiments, the αvβ6 integrin ligands disclosed herein comprise structure 6.1 in a tridentate form and can be represented by the following structure: [ka] (In the formula, [ka] denotes any suitable scaffold that can be used to link the ligand and cargo molecule).

[0270] In some embodiments, the αβ integrin ligand disclosed herein comprises structure 6.1 in a tridentate form bound to an RNAi agent and can be represented by the following structure: [ka] (In the formula, [ka] indicates any suitable scaffold that can be used to link the ligand and RNAi agent; [ka] indicates an RNAi agent). Reactive groups and protected reactive groups.

[0271] Reactive groups are well known in the art and form a covalent linkage between two molecules or reactants. Reactive groups suitable for use within the present invention include, but are not limited to, amino groups, amide groups, carboxylic acid groups, azides, alkynes, propargyl groups, B CN (bicyclo[6.1.0]nonyne), DBCO (dibenzocyclooctyne) thiol, maleimide group, aminooxy group, N-hydroxysuccinimide (NHS) or other activated esters (e.g., PNP, TFP, PFP), bromo group, aldehyde, carbonate, tosylate, tetrazine, trans-cyclooctene (TCO), hydrazide, hydroxyl group, disulfide, and orthopyridyl disulfide group.

[0272] The incorporation of a reactive group can facilitate the conjugation of the αvβ6 integrin ligand disclosed herein to a cargo molecule. Conjugation reactions are well known in the art and form a covalent bond between two molecules or reactants. Conjugation reactions suitable for use within the scope of the present invention include, but are not limited to, amide coupling reactions, Michael addition reactions, hydrazone formation reactions, and click chemistry cycloaddition reactions.

[0273] In some embodiments, the αββ integrin targeting ligands disclosed herein can be synthesized as tetrafluorophenyl (TFP) esters, which can be substituted with a reactive amino group for attachment to a cargo molecule. In some embodiments, the integrin targeting ligands disclosed herein can be synthesized as azides, which can be coupled to a propargyl or DBCO group, for example, via a click chemistry cycloaddition reaction, for attachment to a cargo molecule.

[0274] Protected reactive groups are also commonly used in the art. Protecting groups temporarily convert reactive groups into groups that do not react under conditions that would cause the unprotected group to react, thereby, for example, providing chemical selectivity in subsequent chemical reactions. Protected reactive groups suitable for use within the scope of the present invention include, but are not limited to, BOC group (t-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), carboxybenzyl (CBZ) group, benzyl ester, and PBF (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl). Cargo molecules (including RNAi agents)

[0275] The cargo molecule is any molecule that is believed to have a desirable effect on cells containing the αvβ6 integrin receptor when detached from the αvβ6 integrin ligand described herein. The cargo molecule can be, but is not limited to, a pharmaceutical component, a pharmaceutical agent, a prodrug, a substance with known therapeutic benefits, a small molecule, an antibody, an antibody fragment, an immunoglobulin, a monoclonal antibody, a label or marker, a lipid, a natural or modified nucleic acid or polynucleotide, a peptide, a polymer, a polyamine, a protein, an aptamer, a toxin, a vitamin, PEG, a hapten, digoxigenin, biotin, a radioactive atom or molecule, or a fluorophore. In some embodiments, one or more cargo molecules (e.g., the same or different cargo molecules) are linked to one or more αvβ6 integrin ligands so that the cargo molecules target cells expressing αvβ6 integrin.

[0276] In some embodiments, the one or more cargo molecules are pharmaceutical compositions or compositions. In some embodiments, the one or more cargo molecules are oligonucleotide-based compounds. As used herein, an "oligonucleotide-based compound" refers to a compound having a molecular weight of about 10 to 50 (e.g., 10 to 48, 10 to 46, 10 to 44, 10 to 42, 10 to 40, 10 to 38, 10 to 36, 10 to 34, 10 to 32, 10 to 30, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 14, 10 to 12, 12 to 50, 12 to 48, 12 to 46, 12 to 44, 12 to 42, 12 to 40, 12 to 38, 12 to 36, 12 to 34, 12 to 32, 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20, 12 to 18, 12 to 16 ... 14、14~50、14~48、14~46、14~44、14~42、14~40、14~38、14~36、14~34、14~32、14~30、14~28、14~26、14~24、14~22、14~20、14~18、14~16、16~50、16~48、16~46、16~44、16~42、16~40、16~38、16~36、16~34、16~32、16~30、16~28、16~26、16~24、16~22、16~20、16~18、18~50、18~48、18~46、18~44、18~42、18~40、18~38、18~36、18~34、18~32、18~30、18~28、18~26、18~24、18~22、18~20、20~50、20~48、20~46、20~44、20~42、20~40、20~38、20~36、20~34、20~32、20~30、20~28、20~26、20~24、20~22、22~50、22~48、22~46、22~44、22~42、22~40、22~38、22~36、22~34、22~32、22~30、22~28、22~26、22~24、24~50、24~48、24~46、24~44、24~42、24~40、24~38、24~36、24~34、24~32、24~30、24~28、24~26、26~50、26~48、26~46、26~44、26~42、26~40、26~38、26~36、26~34、26~32、26~30、26~28、28~50、28~48、28~46、28~44、28~42、28~40、28~38、28~36、28~34、28~32、28~30、30~50、30~48、30~46、30~44、30~42、30~40、30~38、30~36、30~34、30~32、32~50、32~48、32~46、32~44、32~42、32~40、32~38、32~36、32~34、34~50、34~48、34~46、34~44、34~42、34~40、34~38、34~36、36~50、36~48、36~46、36~44、36~42、36~40、36~38、38~50、38~48、38~46、38~44、38~42、38~40、40~50、40~48、40~46、40~44、40~42、42~50、42~48、42~46、42~44、44~50、The oligonucleotide-based compound is a nucleotide sequence comprising 44-48, 44-46, 46-50, 46-48, or 48-50 nucleotides or nucleotide base pairs. In some embodiments, the oligonucleotide-based compound has a nucleobase sequence that is at least partially complementary to a coding sequence in an expressed target nucleic acid or target gene in a cell. In some embodiments, the oligonucleotide-based compound can inhibit expression of the underlying gene upon delivery to a cell that expresses the gene, and this compound is referred to herein as an "expression-inhibiting oligonucleotide-based compound." Gene expression can be inhibited in vitro or in vivo.

[0277] "Oligonucleotide-based compounds" include, but are not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, small interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), ribozymes, interfering RNA molecules, and Dicer substrates. In some embodiments, the oligonucleotide-based compounds are single-stranded oligonucleotides (such as antisense oligonucleotides). In some embodiments, the oligonucleotide-based compounds are double-stranded oligonucleotides. In some embodiments, the oligonucleotide-based compounds are double-stranded oligonucleotides that are RNAi agents.

[0278] In some embodiments, one or more cargo molecules are "RNAi agents," which are defined herein as compositions comprising RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that are capable of degrading or inhibiting translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence-specific manner. As used herein, an RNAi agent refers to an agent that induces RNA interference via an RNA interference mechanism (i.e., induction of RNA interference via interaction with the RNA interference pathway mechanism (RNA-induced silencing complex, or RISC) in mammalian cells) or any other mechanism(s) or pathway. RNAi agents, as the term is used herein, can operate through (singular or plural). While RNAi agents, as this term is used herein, are primarily thought to operate through RNA interference mechanisms, the disclosed RNAi agents are not bound or limited to any particular pathway or mechanism of action. The RNAi agents disclosed herein are composed of a sense strand and an antisense strand, and include, but are not limited to, short (or small) interfering RNAs (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and Dicer substrate. The antisense strand of the RNAi agent described herein is at least partially complementary to the mRNA to be targeted. The RNAi agent may contain one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0279] Typically, an RNAi agent can be composed of at least a sense strand (also called a passenger strand) comprising a first sequence and an antisense strand (also called a guide strand) comprising a second sequence. The sense and antisense strands of an RNAi agent can each be 16-49 nucleotides in length. In some embodiments, the sense and antisense strands of an RNAi agent are independently 17-26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 19-26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21-26 nucleotides in length. In some embodiments, the sense and antisense strands are independently 21-24 nucleotides in length. The sense and antisense strands can be either the same length or different lengths. The RNAi agent comprises an antisense strand sequence that is at least partially complementary to a sequence in a target gene, and when delivered to a cell expressing the target, the RNAi agent can inhibit expression of one or more target genes in vivo or in vitro.

[0280] Oligonucleotide-based compounds (generally) and RNAi agents (specifically) can be composed of modified nucleotides and / or one or more non-phosphodiester bonds.As used herein, "modified nucleotide" refers to a nucleotide other than ribonucleotides (2'-hydroxyl nucleotides).In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2'-modified nucleotides, 3'-3'-linked (reverse) nucleotides, nucleotides containing unnatural bases, bridged nucleotides, peptide nucleic acids, 2',3'-seconucleotide mimics (unlocked nucleobase analogs, locked nucleotides, 3'-O-methoxy (2' internucleoside linked) nucleotides, 2'-F-arabinonucleotides, 5'-Me, 2'-fluoronucleotides, morpholino nucleotides, vinylphosphonate deoxyribonucleotides, vinylphosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleotides. 2'-modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-deoxy nucleotides, 2'-methoxyethyl (2'-O-2-methoxylethyl) nucleotides, 2'-amino nucleotides, and 2'-alkyl nucleotides.

[0281] Additionally, one or more nucleotides of an oligonucleotide-based compound (such as an RNAi agent) can be linked by a non-standard linkage or backbone (i.e., a modified internucleoside linkage or a modified backbone). It may be a non-sulfate containing covalent internucleoside linkage. Modified internucleoside linkages or backbones include, but are not limited to, 5'-phosphorothioate groups, chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g., methylphosphonates or 3'-alkylenephosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidate, aminoalkylphosphoramidate, or thionophosphoramidate), thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates of opposite polarity in which pairs of adjacent nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.

[0282] It is not necessary for all positions in a given compound to be uniformly modified; conversely, more than one modification can be incorporated into a single oligonucleotide-based compound, or even into a single nucleotide thereof.

[0283] In some embodiments, the cargo molecule is an RNAi agent for inhibiting αENaC gene expression. The cargo molecule may be an RNAi agent described in International Patent Application No. PCT / US18 / 40874 (the entirety of which is incorporated herein by reference).

[0284] The sense strand and antisense strand of RNAi agent can be synthesized and / or modified by methods known in the art.For example, the disclosure of the RNAi agent that is related to the inhibition of α-ENaC expression can be found, for example, in International Publication No. WO2008 / 152131 (its entirety is incorporated herein by reference).Further disclosure of RNAi agent, for example, in the disclosure of modification, can be found, for example, in Arrowhead Pharmaceuticals, Inc.'s International Patent Application No. PCT / US2017 / 0455446 (its entirety is also incorporated herein by reference). In some embodiments, one or more cargo molecule(s) may comprise or consist of a PEG moiety, which may act as a pharmacokinetic (PK) modulator. In some embodiments, one or more cargo molecules may comprise or consist of about 20-900 ethylene oxide (CH2-CH2-O) units (e.g., 20-850, 20-800, 20-750, 20-700, 20-650, 20-600, 20-550, 20-500, 20-450, 20-400, 20-350, 20-300, 20-250, 20-200, 20-150, 20-100, 20-75, 20-50, 100-850, 100-800, 100-7 50, 100-700, 100-650, 100-600, 100-550, 100-500, 100-450, 100-400, 100-350, 100-300, 100-250, 100-200, 100-150, 200-850, 200-800, 200-750, 200-700, 200-650, 200-600, 200-550, 200-500, 200-450, 200-400, 200-350, 200-300, 200-250, 250-9 00, 250-850, 250-800, 250-750, 250-700, 250-650, 250-600, 250-550, 250-500, 250-450, 250-400, 250-350, 250-300, 300-900, 300-850, 300-800, 300-750, 300-700, 300-650, 300-600, 300-550, 300-500, 300-450, 300-400, 300-350, 350-900, 350-8 50, 350-800, 350-750, 350-700, 350-650, 350-600, 350-550, 350-500, 350-450, 350-400, 400-900, 400-850, 400-800, 400-750, 400-700, 400-650, 400-600, 400-550, 400-500, 400-450, 450-900, 450-850, 450-800, 450-750, 450-700, 450-650, 450-6 00, 450-550, 450-500, 500-900, 500-850, 500-800, 500-750, 500-700, 500-650, 500-600, 500-550, 550-900, 550-850, 550-800, 550-750, 550-700, 550-650, 550-600, 600-900, 600-850, 600-800, 600-7 The cargo molecule(s) may comprise a PEG moiety having approximately 50, 600-700, 600-650, 650-900, 650-850, 650-800, 650-750, 650-700, 700-900, 700-850, 700-800, 700-750, 750-900, 750-850, 750-800, 800-900, 850-900, or 850-900 ethylene oxide units. In some embodiments, one or more cargo molecule(s) comprise a PEG moiety having approximately 455 ethylene oxide units (molecular weight of about 20 kilodaltons (kDa)). In some embodiments, the molecular weight of the PEG moiety is about 2 kilodaltons. In some embodiments, the molecular weight of the PEG moiety is about 20 kilodaltons. In some embodiments, the molecular weight of the PEG moiety is about 40 kilodaltons. The PEG moiety described herein can be linear or branched. The PEG moiety can be discrete (monodisperse) or non-disperse (polydisperse). PEG moieties for use as PK-enhancing cargo molecules can be commercially purchased. In some embodiments, one or more cargo molecules include a PEG moiety that can act as a PK modulator or enhancer, and a different cargo molecule, such as a pharmaceutically active ingredient or compound.

[0285] The αvβ6 integrin ligand described herein includes its salt or solvate.The solvate of αvβ6 integrin ligand is understood to mean the adduct of inert solvent molecules on αvβ6 integrin ligand formed by mutual attractive force.Solvate is for example monohydrate or dihydrate, or the adduct of compound with alcohol (for example, methanol or ethanol).

[0286] A free amino group or a free hydroxyl group can be provided as a substituent on the αvβ6 integrin ligand with a corresponding protecting group.

[0287] αvβ6 integrin ligands also include, for example, derivatives (ie, αvβ6 integrin ligands modified with, for example, alkyl or acyl groups, sugars, or oligopeptides that are cleaved either in vitro or in vivo).

[0288] In some embodiments, the αvβ6 integrin ligands disclosed herein facilitate delivery of cargo molecules into the cytosol of cells that display αvβ6 integrin on their surface via ligand-mediated endocytosis, pinocytosis, or by other means. In some embodiments, the αvβ6 integrin ligands disclosed herein facilitate delivery of cargo molecules to the plasma membrane of cells that display αvβ6 integrin. Pharmaceutical Composition

[0289] In some embodiments, the present disclosure provides pharmaceutical compositions comprising, consisting of, or consisting essentially of one or more αvβ6 integrin ligands disclosed herein.

[0290] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of an active pharmaceutical ingredient (API), and optionally one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (vehicle) is a substance other than the active pharmaceutical ingredient (API, therapeutic agent) that is intentionally included in a drug delivery system. An excipient does not exert, or is not intended to exert, a therapeutic effect at the intended dosage. An excipient a) aids in the processing of the drug delivery system during manufacturing; and b) protects or supports the stability, bioavailability, or patient tolerability of the API. a) enhance or augment the properties of the API, b) aid in product identification, and / or d) may act to enhance any other attribute of the overall safety, efficacy, delivery of the API during storage or use. Pharmaceutically acceptable excipients may or may not be inert substances.

[0291] Excipients include, but are not limited to, absorption enhancers, anti-adherents, anti-foaming agents, antioxidants, binders, buffers, carriers, coatings, dyes, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, water repellents, and wetting agents.

[0292] The pharmaceutical compositions described herein may contain other components commonly found in pharmaceutical compositions. In some embodiments, the additional component is a pharmaceutically active material. Pharmaceutically active materials include, but are not limited to, antipruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.), small molecule drugs, antibodies, antibody fragments, aptamers, and / or vaccines.

[0293] Pharmaceutical compositions may also include preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for varying osmotic pressure, buffers, coating agents, or antioxidants. Pharmaceutical compositions may also include other agents with known therapeutic benefits.

[0294] The pharmaceutical composition can be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated. It can be administered by any method commonly known in the art, including, but not limited to, topical (e.g., by transdermal patch), pulmonary (e.g., by inhalation or insufflation of powder or aerosol (including nebulizer, intratracheal, intranasal)), epidermal, transdermal, oral, or parenteral. Parenteral administration includes, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal (e.g., by implanted device), intracranial, intraparenchymal, intrathecal, and intraventricular administration. In some embodiments, the pharmaceutical composition described herein is administered by subcutaneous injection. The pharmaceutical composition can be orally administered, for example, in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, liquids, emulsions, or suspensions. Administration can also be rectal (eg, using a suppository); topical or transdermal (eg, using an ointment, cream, gel, or liquid); or parenteral (eg, using an injectable solution).

[0295] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile solutions or suspensions for injection. For intravenous administration, suitable carriers include physiological saline, bacteriostatic aqueous solution, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline. The pharmaceutical composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, the maintenance of the required particle size in the case of dispersions, and the use of surfactants. In many cases, it will be preferable to include isotonic agents (e.g., sugars, polyalcohols (e.g., mannitol, sorbitol), and sodium chloride) in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0296] Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent, as required by the addition of the ingredients enumerated above. Can be prepared by incorporating one or combination of them, and then filter sterilization.Generally, dispersion is prepared by incorporating active compound into sterile vehicle that contains basic dispersion medium and other necessary ingredients from the above-listed ingredients.For the sterile powder that is used to prepare sterile injection solution, preparation method includes vacuum drying and freeze-drying, which can obtain powder of active ingredient and any other desired ingredients from the solution of active ingredient that is previously filter sterilized.

[0297] Formulations suitable for intra-articular administration may be in the form of a sterile aqueous preparation of any of the ligands described herein, which may be in microcrystalline form (e.g., in the form of an aqueous microcrystalline suspension). Liposomal formulations or biodegradable polymer systems may also be used to provide any of the ligands described herein for both intra-articular and intraocular administration.

[0298] Active compounds can be prepared with carriers that prevent the compound from rapid elimination from the body, such as controlled release formulations (including implants and microencapsulated delivery systems).Biodegradable biocompatible polymers (such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid) can be used.The method for preparing such formulations will be clear to those skilled in the art.Liposomal suspensions can also be used as pharmaceutically acceptable carriers.They can be prepared according to methods known to those skilled in the art (for example, as described in U.S. Patent No. 4,522,811).

[0299] The pharmaceutical composition may contain other additional ingredients commonly found in pharmaceutical compositions. Such additional ingredients include, but are not limited to, antipruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.). As used herein, a "pharmacologically effective amount," a "therapeutically effective amount," or simply an "effective amount" refers to the amount of a pharmaceutically active agent to achieve a pharmacological, therapeutic, or prophylactic result.

[0300] Pharmaceuticals comprising an αvβ6 integrin ligand are also an object of the present invention, as are processes for producing such pharmaceuticals, wherein the process comprises the steps of bringing one or more compounds comprising an αvβ6 integrin ligand, and, if desired, one or more other substances having known therapeutic benefits, into a pharmaceutically acceptable form.

[0301] The αvβ6 integrin ligands described herein and pharmaceutical compositions comprising the αvβ6 integrin ligands disclosed herein can be packaged or included in a kit, container, pack, or dispenser. The αvβ6 integrin ligands and pharmaceutical compositions comprising the αvβ6 integrin ligands can be packaged in pre-filled syringes or vials. Cells, tissues, and non-human organisms

[0302] Contemplated herein are cells, tissues, and non-human organisms that comprise at least one of the αvβ6 integrin ligands described herein. The cells, tissues, or non-human organisms are produced by delivering the αvβ6 integrin ligand to the cells, tissues, or non-human organisms by any means available in the art. In some embodiments, the cells are mammalian cells, including but not limited to human cells. Targeting Groups, Linking Groups, Pharmacokinetic (PK) Modulators, and Delivery Vehicles

[0303] In some embodiments, the αvβ6 ligand is conjugated to one or more non-nucleotide groups, including, but not limited to, a linking group, a pharmacokinetic (PK) modulator, a delivery polymer, or a delivery vehicle. The non-nucleotide group can enhance targeting, delivery, or attachment of the cargo molecule. Targeting groups and Examples of linking groups are provided in Table 6. The non-nucleotide group can be covalently linked to the 3' and / or 5' end of either the sense strand and / or the antisense strand. In embodiments where the cargo molecule is an RNAi agent, the RNAi agent comprises a non-nucleotide group linked to the 3' and / or 5' end of the sense strand. In some embodiments, the non-nucleotide group is linked to the 5' end of the sense strand of the RNAi agent. The αvβ6 ligand can be linked to the cargo molecule directly or indirectly via a linker / linking group. In some embodiments, the αvβ6 ligand is linked to the cargo molecule via a labile, cleavable, or reversible bond or linker.

[0304] In some embodiments, the non-nucleotide group enhances the pharmacokinetic or biodistribution properties of the RNAi agent or conjugate to which the RNAi agent is attached, such that the conjugate has improved cell- or tissue-specific distribution and cell-specific uptake. In some embodiments, the non-nucleotide group enhances endocytosis of the RNAi agent.

[0305] Targeting groups or targeting moieties enhance the pharmacokinetic or biodistribution properties of the cargo molecule to which they are attached, improving cell-specific (including, in some cases, organ-specific) distribution and cell-specific (or organ-specific) uptake of the cargo molecule. In some embodiments, the targeting group may comprise an αβ ligand described herein. In some embodiments, the targeting group comprises a linker. In some embodiments, the targeting group comprises a PK modulator. In some embodiments, the αβ ligand is linked to the cargo molecule using a linker, such as a PEG linker, or one, two, or three abasic and / or ribitol (abasic ribose) residues, which may serve as a linker.

[0306] Cargo molecule can be synthesized with reactive group such as amino group (also referred to herein as amine).In the embodiment where cargo molecule is RNAi agent, reactive group can be linked to 5'-end and / or 3'-end.Then, reactive group can be used to attach to αvβ6 ligand by using the typical method in the art.

[0307] For example, in some embodiments, an RNAi agent is synthesized with an NH2-C6 group at the 5'-end of the sense strand of the RNAi agent. The terminal amino group can then be reacted to form a conjugate with, for example, a group comprising an αvβ6 integrin targeting ligand. In some embodiments, an RNAi agent is synthesized with one or more alkyne groups at the 5'-end of the sense strand of the RNAi agent. The terminal alkyne group(s) can then be reacted to form a conjugate with, for example, a group comprising an αvβ6 integrin targeting ligand.

[0308] In some embodiments, a linking group is attached to the αvβ6 ligand. The linking group facilitates the covalent linking of the αvβ6 ligand to a cargo molecule, a pharmacokinetic modulator, a delivery polymer, or a delivery vehicle. Examples of linking groups include, but are not limited to: Alk-SMPT-C6, Alk-SS-C6, DBCO-TEG, Me-Alk-SS-C6, and C6-SS-Alk-Me, reactive groups such as primary amine groups and alkyne groups, alkyl groups, abasic residues / nucleotides, amino acids, trialkyne functional groups, ribitol, and / or PEG groups.

[0309] A linker or linking group is a connection between two atoms that connects one chemical group or segment of interest (such as an RNAi agent) to another chemical group or segment of interest (such as an αβ ligand, a pharmacokinetic modulator, or a delivery polymer) via one or more covalent bonds. A labile linkage includes a labile bond. The linkage may optionally include a spacer that increases the distance between the two joined atoms. A spacer may be used to link the Spacers may further add flexibility and / or length to the bond. Spacers include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups; each of which may contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides. Spacer groups are well known in the art, and the preceding list is not meant to limit the scope of the description.

[0310] In some embodiments, αvβ6 ligand is linked to cargo molecule without using additional linker.In some embodiments, αvβ6 ligand is designed with linker that can be easily linked to cargo molecule.In some embodiments, when two or more RNAi agents are included in composition, two or more RNAi agents can be linked to their respective targeting groups using the same linker.In some embodiments, when two or more RNAi agents are included in composition, two or more RNAi agents are linked to their respective targeting groups using different linkers.

[0311] Examples of certain linking groups are provided in Table A. [Table A-1] [Table A-2] [Table A-3] [Table A-4] [Table A-5] [Table A-6] [Table A-7] [Table A-8] [Table A-9] [Table A-10] [Table A-11] (In the formula, [ka] indicates the point of attachment to the cargo molecule).

[0312] Alternatively, other linking groups known in the art can be used.

[0313] The embodiments and items provided above will now be illustrated with the following non-limiting examples. [Example]

[0314] The following examples do not limit the invention, but are intended to illustrate certain embodiments disclosed herein. Example 1. Synthesis of αvβ6 integrin ligands

[0315] Some abbreviations used in the synthetic experimental details of the following examples are defined as follows: h or hr = hour(s); min = minute(s); mol = mole(s); mmol = millimole(s); M = molar; μM = micromolar; g = gram(s); μg = microgram(s); rt or RT = room temperature; L = liter(s); mL = milliliter(s); wt = weight; EtO = diethyl ether; THF = tetrahydrofuran; DMSO = dimethyl sulfoxide; EtOAc = ethyl acetate; EtN or TEA = triethylamine; i-PrNEt or DIPEA or DIEA = diisopropylethylamine; CHCl or DCM = methylene chloride; CHCl = chloroform; CDCl = deuterated chloroform; CCl = carbon tetrachloride; MeOH = methanol; EtOH = ethanol; DM F = dimethylformamide; BOC = t-butoxycarbonyl; CBZ = benzyloxycarbonyl; TBS = t-butyldimethylsilyl; TBSCl or TBDMSCl = t-butyldimethylsilyl chloride; TFA = trifluoroacetic acid; DMAP = 4-dimethylaminopyridine; NaN3 = sodium azide; Na2SO4 = sodium sulfate; NaHCO3 = sodium bicarbonate; NaOH = sodium hydroxide; MgSO4 = magnesium sulfate; K2CO3 = potassium carbonate; KOH = potassium hydroxide; NH4OH = ammonium hydroxide; NHCl = ammonium chloride; SiO2 = silica; Pd-C = palladium on carbon; HCl = hydrogen chloride or hydrochloric acid; NMM = N-methylmorpholine; H2 = hydrogen gas; KF = potassium fluoride; EDC-HCl = N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; MTBE = methyl tert-butyl ether; Ar = argon; N2 = nitrogen; R T = retention time.

[0316] Chemical names for structures 1-37 were generated automatically using ChemDraw® software. Synthesis of structure 1b ((14S,17S)-1-azido-14-(5-((4-methylpyridin-2-yl)amino)pentanamido)-17-(4-(naphthalen-1-yl)phenyl)-15-oxo-3,6,9,12-tetraoxa-16-azanonadecanoic acid). [ka]

[0317] Compound 1 (methyl (S)-(-)-1-tritylaziridine-2-carboxylate (4.204 g, 12.24 mmol, 1.0 equiv.) and triisopropylsilane (3.877 g, 5.02 mL, 24.48 mmol, 2 equiv.) were dissolved in DCM (40 mL), the solution was cooled to 0 °C, and then TFA (8.5 equiv.) was added dropwise. The mixture was allowed to stand at RT for 1 h at RT. The reaction was monitored by TLC (hexane:ethyl acetate (8:2)). The solution was evaporated to a mixture of a white precipitate and a pale yellow oil. Hexane (40 mL) was added and gently heated with a heat gun until all of the white precipitate dissolved. The addition of hexane resulted in two layers (a clear upper layer and an oily layer). The hexane layer was poured off, leaving the oily layer. The addition of hexane was repeated and the mixture was poured off again. The oil was evaporated to a dry state. Aziridine (1.06 g, 10.5 mmol) was dissolved in a total of 60 mL of THF / HO (2 / 1). Fmoc-OSu (5.312 g, 15.75 mmol, 1.5 equiv.) and NaHCO3 (2.646 g, 31.5 mmol, 3 equiv., to maintain pH = 8.5) were added to the mixture at room temperature and allowed to react overnight. The reaction was monitored by TLC (hexane:ethyl acetate 8:2). The mixture was concentrated until all THF was removed and then diluted with ethyl acetate (350 mL) and H2O (25 mL). The layers were separated and the organics were washed with H2O (40 mL). The organics were then washed with water (2 x 40 mL) at pH 3-4, then H2O (40 mL), then saturated aqueous NaCl (40 mL). The organic phase was dried over Na2SO4, filtered, and concentrated. The product was purified on a silica column with 10%-20% ethyl acetate in hexane. [ka]

[0318] Compound 2 (Fmoc-aziridine) (1.46 g, 4.52 mmol) and HO-PEG-N (1.983 g, 9.04 mmol, 2 equiv.) were dissolved in DCM. The mixture was cooled to 0 °C. Boron trifluoride diethyl etherate (12 drops) was added dropwise. The mixture was stirred at room temperature for 48 h. The reaction was monitored by TLC (5% MeOH in DCM). The reaction was quenched with saturated NH Cl solution (5 mL), diluted with DCM (60 mL), washed with HO (3 × 20 mL), saturated aqueous NaCl (20 mL), dried over Na SO , filtered, and concentrated. The product was purified on a silica column (40%-60% ethyl acetate in hexane). [ka]

[0319] Compound 3 was dissolved in a DMF solution containing 20% ​​triethylamine. The reaction was monitored by TLC. The product was concentrated. [ka]

[0320] Compound 5 (tert-butyl(4-methylpyridin-2-yl)carbamate) (0.501 g, 2.406 mmol, 1.0 equiv.) was dissolved in DMF (17 mL). To this mixture, NaH (0.116 mg, 3.01 mmol, 1.25 equiv., 60% mineral oil dispersion) was added at room temperature. The mixture was stirred for 10 minutes, and then ethyl 5-bromovalerate (0.798 g, 3.82 mmol, 0.604 mL) was added. After 3 hours, the reaction was quenched with ethanol (18 mL) and concentrated. The product was dissolved in DCM (50 mL), washed with saturated aqueous NaCl (50 mL), dried over NaSO, filtered, and concentrated. The product was purified on a silica column (gradient of 0-5% methanol in DCM). [ka]

[0321] Compound 7 (0.80 g, 2.378 mmol) was dissolved in 100 mL of acetone:0.1 M NaOH (1:1) and the reaction was monitored by TLC (5% ethyl acetate in hexanes). The organics were concentrated and the mixture was acidified to pH 3-4 with 0.3 M citric acid (40 mL). The product was extracted with DCM (3 x 75 mL). The organics were pooled, dried over NaSO, filtered, and concentrated. The product was used without further purification. [ka]

[0322] Compound 4 (0.340 g, 1.104 mmol) was dissolved in DMF (10 mL). To this solution, TBTU (0.531 g, 1.655 mmol) and diisopropylethylamine (0.320 mL, 1.839 mmol) were added. Compound 8 (0.295 g, 0.9197 mmol) was then added. The reaction was monitored by LC-MS and TLC (5% MeOH in DCM). The reaction was complete in 2 h. The product was concentrated, dissolved in ethyl acetate (150 mL), and washed with pH 3-4 H2O (2 × 12 mL). The product was then washed with H2O (2 × 12 mL), saturated aqueous NaHCO3 (12 mL), and saturated aqueous NaCl (12 mL). The organic phase was dried over Na2SO4, filtered, and concentrated. The product was purified on a silica column (ethyl acetate with 20% hexane to 100% ethyl acetate). [ka]

[0323] Compound 9 (0.330 g, 0.540 mmol) was dissolved in 10 mL of MeOH:dioxane [1:1] and 1 M LiOH solution (10 mL). The mixture was stirred at room temperature for 2 h and monitored by LC-MS and TLC (EtOAc). The organics were concentrated to remove the material, and the mixture was diluted with HO (5 mL) and acidified to pH 4. The product was extracted with ethyl acetate (2 × 50 mL). The organics were pooled, washed with saturated aqueous NaCl (10 mL), dried over NaSO, filtered, and concentrated. The product was used without further purification. [ka]

[0324] Compound 11 ((S)-3-(4-bromophenyl)-3-((tert-butoxycarbonyl)amino)-propionic acid) (2.0 g, 5.81 mmol) was dissolved in DMF (40 mL). To this mixture was added KCO (1.2 g, 8.72 mmol). Then, iodomethane (1.65 g, 11.62 mmol, 0.72 mL) was added. The reaction was monitored by TLC (hexane:ethyl acetate (7:3)). Upon completion, the mixture was cooled to 0 °C, and HO (20 mL) and MTBE (40 mL) were added. The product was extracted with MTBE (4 × 40 mL). The combined organic phase was washed with saturated aqueous NaHCO (40 mL) and then HO (4 × 40 mL). The mixture was dried over NaSO, filtered, and concentrated.

[0325] To the dried product of compound 12 (1.0 g, 2.7915 mmol) was added compound 13 (1-naphthaleneboronic acid (0.960 g, 5.583 mmol, 2 equivalents)). To the mixture was added [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) or Pd(dppf)Cl2 (0.0817 g, 0.1117 mmol, 0.4 equiv.) along with Na2CO3 (0.888 g, 8.375 mmol, 3 equiv.). 1,4-Dioxane (5 mL) and HO (0.2 mL) were then added, and the mixture was stirred at 100 °C for 4 h. The reaction was monitored by TLC (hexane:ethyl acetate (7:3)). The product was purified by silica chromatography (gradient of 0% to 50% ethyl acetate in hexane).

[0326] Compound 14 (0.200 g, 0.493 mmol) was dissolved in DCM (2.5 mL) and then TFA (0.45 mL) was added. The reaction was monitored by TLC (DCM:methanol (9:1)). Upon completion, the reaction mixture was concentrated. The residue was dissolved in DCM (4 mL) and washed with saturated aqueous NaHCO (2 × 2 mL) and then saturated aqueous NaCl (2 × 2 mL). The organic phase was dried over NaSO, filtered, and concentrated. The product was used without further purification. [ka]

[0327] Compound 10 (0.3224 g, 0.54 mmol) was dissolved in DMF (7 mL). To this mixture, TBTU (0.236 g, 0.735 mmol) and diisopropylethylamine (0.170 mL, 0.98 mmol) were added. Compound 15 (0.1496 g, 0.49 mmol) was then added. The reaction was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The mixture was concentrated, and the residue was dissolved in ethyl acetate (90 mL) and washed with pH 3-4 H2O (3 × 10 mL). The product was washed with H2O (2 × 10 mL), saturated aqueous NaHCO3 (10 mL), and then saturated aqueous NaCl (1 × 10 mL). The organic phase was dried over Na2SO4, filtered, and concentrated. The product was purified by silica chromatography using a gradient of DCM to 5% MeOH. [ka]

[0328] Compound 16 (0.250 g, 0.2828 mmol) was dissolved in MeOH:dioxane [1:1] (4 mL) and 1 M LiOH (4 mL). The mixture was stirred at room temperature for 2 hours. The organics were concentrated to remove the residue, and the residue was diluted with HO (3 mL) and acidified to pH 4. The product was extracted with ethyl acetate (3 × 20 mL). The organics were pooled and washed with saturated aqueous NaCl (10 mL). The product was dried over NaSO. The product (0.200 g, 0.2299 mmol) was dissolved in 2 mL DCM:TFA [25:75] and stirred at room temperature for 2 hours. Toluene (4 mL) was added. The mixture was concentrated and then co-evaporated with acetonitrile (2 × 4 mL). The product was purified by HPLC (gradient 35% ACN to 50% over 30 minutes, 0.1% TFA buffer). =>C 41 H 51 [M+H]+ calculated for N7O8: 769.90, found: 770.45; [ka] Synthesis of structure 2b ((14S,17S)-1-azido-14-(4-((4-methylpyridin-2-yl)amino)butanamido)-17-(4-(naphthalen-1-yl)phenyl)-15-oxo-3,6,9,12-tetraoxa-16-azanonadecanoic acid). [ka]

[0329] Compound 5 (tert-butyl (4-methylpyridin-2-yl)carbamate) (0.501 g, 2.406 mmol, 1 eq) was dissolved in DMF (17 mL). To this mixture was added NaH (0.116 mg, 3.01 mmol, 1.25 eq, 60% oil dispersion). After stirring the mixture for 10 minutes, compound 20 (ethyl 4-bromobutyrate (0.745 g, 3.82 mmol, 0.547 mL)) (Sigma 167118) was added. After 3 hours, the reaction was quenched with ethanol (18 mL) and concentrated. The concentrate was dissolved in DCM (50 mL), washed with saturated aqueous NaCl (1 × 50 mL), dried over NaSO, filtered, and concentrated. The product was purified on a silica column (gradient of 0-5% methanol in DCM). [ka]

[0330] Compound 21 (0.80 g, 2.378 mmol) was dissolved in 100 mL of acetone:0.1 M The residue was acidified to pH 3-4 with 0.3 M citric acid (40 mL). The product was extracted with DCM (3 x 75 mL). The organics were pooled, dried over Na2SO4, filtered, and concentrated. The product was used without further purification. [ka]

[0331] Compound 22 (0.340 g, 1.104 mmol) was dissolved in DMF (10 mL). To this mixture, TBTU (0.531 g, 1.655 mmol) and diisopropylethylamine (0.320 mL, 1.839 mmol) were added. Compound 10 (0.295 g, 0.9197 mmol) was then added. The reaction was monitored by LC-MS and TLC (5% MeOH in DCM). The reaction was complete in 2 h. The mixture was concentrated, dissolved in ethyl acetate (150 mL), and washed with pH 3-4 H2O (2 × 12 mL). The mixture was then washed with H2O (2 × 12 mL), saturated aqueous NaHCO3 (12 mL), and saturated aqueous NaCl (12 mL). The organic phase was dried over Na2SO4, filtered, and concentrated. The product was purified on a silica column (ethyl acetate with 20% hexane to 100% ethyl acetate). [ka]

[0332] Compound 23 (0.330 g, 0.540 mmol) was dissolved in 10 mL of MeOH:dioxane [1:1] and 1 M LiOH (10 mL). The mixture was stirred at room temperature for 2 h and monitored by LC-MS and TLC (100% EtOAc). The organics were After concentration, the residue was diluted with H2O (5 mL) and acidified to pH 4. The product was extracted with ethyl acetate (2 x 50 mL). The combined organic phases were washed with saturated aqueous NaCl (1 x 10 mL). The organic phase was dried over Na2SO4, filtered, and concentrated. The product was used without further purification. [ka]

[0333] Compound 24 (0.3224 g, 0.54 mmol) was dissolved in DMF (7 mL). To this mixture, TBTU (0.236 g, 0.735 mmol) and diisopropylethylamine (0.170 mL, 0.98 mmol) were added. Compound 15 (0.1496 g, 0.49 mmol) was then added. The mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The mixture was concentrated, and the residue was dissolved in ethyl acetate (90 mL) and washed with pH 3-4 H2O (3 × 10 mL). The concentrate was washed with H2O (2 × 10 mL), saturated aqueous NaHCO3 (10 mL), and then saturated aqueous NaCl (10 mL). The organic phase was dried over Na2SO4, filtered, and concentrated. The product was purified on a silica column (5% DCM to MeOH gradient). [ka]

[0334] Compound 25 (0.250 g, 0.2828 mmol) was dissolved in MeOH:dioxane [1:1] (4 mL) and 1 M LiOH (4 mL). The mixture was stirred at room temperature for 2 h and monitored by LC-MS. The organics were concentrated, and the residue was diluted with H2O (3 mL) and acidified to pH 4. The product was extracted with ethyl acetate (3 x 20 mL). The organics were pooled and washed with saturated aqueous NaCl (1 x 10 mL). The organic phase was dried over Na2SO4 and concentrated. The residue (0.200 g, 0.2299 mmol) was dissolved in 2 mL DCM / T The mixture was dissolved in 25 / 75 ethanol and stirred at room temperature for 2 hours while being monitored by LC-MS. Toluene (4 mL) was added and the mixture was concentrated. Acetonitrile (2 x 4 mL) was then added and the mixture was concentrated. The product was purified by HPLC (gradient 35% ACN to 50% over 30 minutes, 0.1% TFA buffer). 40 H 49 [M+H]+ calculated for N7O8: 755.87, found: 756.32; [ka] Synthesis of structures 5b, 5.1b, and 5.2b. Structure 5b (3-(4-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-3,5-dichlorophenyl)-3-(2-(5-((4-methylpyridin-2-yl)amino)pentanamido)acetamido)propanoic acid) [ka]

[0335] To a solution of compound 5 (0.98 g, 4.70 mmol, 1 equiv.) in dry DMF (10 mL) was added NaH (0.226 g, 5.647 mmol, 1.2 equiv., 60% oil dispersion) in portions under a N atmosphere at 0 °C. The reaction mixture was kept at 0 °C for 30 min, and then compound 6 (1.18 mL, 5.647 mmol, 1.2 equiv.) was added at the same temperature. After further stirring at 0 °C for 30 min, the mixture was warmed to room temperature. After stirring at room temperature for 1 h, the reaction was quenched with saturated aqueous NH Cl solution. The aqueous phase was extracted with ethyl acetate (3 × 20 mL), and the organic layers were combined, dried over Na SO , and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase. LC-MS: [M+H] 337.20, found 337.39. [ka]

[0336] To a solution of compound 7 (1.347 g, 4.00 mmol, 1 equiv.) in THF (5 mL) and HO (5 mL) was added lithium hydroxide monohydrate (0.505 g, 12.01 mmol, 5 equiv.) in portions at 0 °C. The reaction mixture was warmed to room temperature. After stirring at room temperature for 1 h, the reaction mixture was acidified to pH 4.0 with HCl (6 N). The aqueous phase was extracted with ethyl acetate (3 × 20 mL), and the organic layers were combined, dried over NaSO, and concentrated. LC-MS: [M+H] 309.17, found 309.39. [ka]

[0337] To a solution of compound 8 (1.163 g, 3.77 mmol, 1 equiv.), compound 45 (568 mg, 4.52 mmol, 1.2 equiv.), and TBTU (1.453 g, 4.52 mmol, 1.2 equiv.) in anhydrous DMF (10 mL) was added diisopropylethylamine (1.97 mL, 11.31 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 3 h. The reaction was quenched with saturated NaHCO solution (20 mL). The aqueous layer was extracted with ethyl acetate (3 × 10 mL), and the organic phases were combined, dried over anhydrous NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase. LC-MS: [M+H] calculated 380.21, found 380.51. [ka]

[0338] To a solution of compound 47 (1.0 g, 5.23 mmol, 1 eq.) and malonic acid (1.09 g, 10.47 mmol, 2 eq.) in ethanol (10 mL) was added ammonium acetate (0.807 mg, 10.47 mmol, 2.0 eq.) at room temperature. The reaction mixture was stirred at reflux overnight. The solid was filtered and washed with cold ethanol. The product was used directly for the next step without further purification. LC-MS: [M+H]+ calculated 250.00, found 250.16. [ka]

[0339] To a solution of compound 46 (1.412 g, 3.72 mmol, 1 equiv.) in THF (5 mL) and HO (5 mL) was added lithium hydroxide monohydrate (0.469 g, 11.16 mmol, 3 equiv.) in portions at 0 °C. The reaction mixture was warmed to room temperature. After stirring at room temperature for 3 h, the reaction mixture was acidified to pH 4.0 with HCl (6 N). The aqueous phase was extracted with ethyl acetate (3 × 20 mL), and the organic layers were combined, dried over NaSO, and concentrated. LC-MS: [M+H] calculated 366.20, found 366.46. [ka]

[0340] To a suspension of compound 49 (0.531 g, 2.12 mmol, 1 equiv.) in anhydrous methanol (10 mL) was added thionyl chloride (308 μL, 4.24 mmol, 2.0 equiv.) on an ice bath. The reaction was allowed to warm to room temperature and stirred overnight. The solvent was removed under reduced pressure and the product was used directly without further purification. LC-MS: [M+H]+ calculated 264.01, found 264.20. [ka]

[0341] To a solution of compound 50 (150 mg, 0.410 mmol, 1 equiv.), compound 51 (148 mg, 0.492 mmol, 1.2 equiv.), and TBTU (158 mg, 0.492 mmol, 1.2 equiv.) in anhydrous DMF (5 mL) was added diisopropylethylamine (0.214 mL, 1.23 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 3 h. The reaction was quenched with saturated aqueous NaHCO (10 mL), and the product was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was purified by CombiFlash using silica gel as the stationary phase and eluted with 2-4% methanol in DCM. [ka]

[0342] To a solution of compound 52 (80 mg, 0.130 mmol, 1 equiv.) and azido-PEG3-OTs (86 mg, 0.262 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added K2CO3 (36 mg, 0.262 mmol, 2 equiv.) at 0 °C. The reaction mixture was stirred at 80 °C for 1 h. The solvent was removed by rotary evaporation. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 2-4% methanol. LC-MS: [M+H]+ calculated 768.28, found 769. [ka]

[0343] To a solution of compound 53 (58 mg, 0.0755 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide monohydrate (10 mg, 0.226 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another 2 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (0.25 mL) and DCM (0.75 mL) were added to the residue, and the mixture was stirred for another 1 h at room temperature. The solvent was removed by rotary evaporation. LC-MS: [M+H]+ calculated 654.21, found 655. Structure 5.1b (3-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3,5-dichlorophenyl)-3-(2-(5-((4-methylpyridin-2-yl)amino)pentanamido)acetamido)propanoic acid) [ka]

[0344] To a solution of compound 52 (100 mg, 0.163 mmol, 1 equiv.) and azido-PEG5-OTs (205 mg, 0.491 mmol, 3 equiv.) in anhydrous DMF (2 mL) was added K2CO3 (68 mg, 0.491 mmol, 2 equiv.) at 0 °C. The reaction mixture was stirred at 80 °C for 1 h. The solvent was removed by rotary evaporation. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 2-3% methanol. LC-MS: [M+H]+ calculated 856.33, found 857.07. [ka]

[0345] To a solution of compound 55 (119 mg, 0.139 mmol, 1.0 equiv.) in THF (4 mL) and water (4 mL) was added lithium hydroxide (10 mg, 0.417 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for an additional 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The combined organic phases were then washed with HCl (6 N). The residue was stirred at room temperature for another 3 hours. The solvent was removed by rotary evaporation. LC-MS: [M+H]+ calculated 742.27, found 743.02. Structure 5.2b (3-(4-((8-azidooctyl)oxy)-3,5-dichlorophenyl)-3-(2-(5-((4-methylpyridin-2-yl)amino)pentanamido)acetamido)propanoic acid) [ka]

[0346] To a solution of compound 52 (89 mg, 0.14 mmol, 1 equiv.) and 1,8-dibromooctane (80 uL, 0.436 mmol, 3 equiv.) in acetone (2 mL) was added K2CO3 (60 mg, 0.436 mmol, 3 equiv.) at room temperature. The reaction mixture was stirred at 55 °C for 6 h. The reaction was quenched with saturated NaHCO3 solution, and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. LC-MS: [M+H]+ calculated 801.23, found 801.98. [ka]

[0347] To a solution of compound 57 (97 mg, 0.114 mmol, 1 equiv.) in anhydrous DMF (2 mL) was added sodium azide (15 mg, 0.229 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 80° C. for 2 h. The reaction was quenched with water, and the aqueous layer was extracted with ethyl acetate (3×10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was used directly without further purification. LC-MS: [M+H]+ calculated 764.32, found 765.07. [ka]

[0348] To a solution of compound 58 (78 mg, 0.101 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (7 mg, 0.304 mmol, 3.0 equiv.). ) was added at room temperature. The mixture was stirred for another 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N) and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4 and concentrated. TFA (2 mL) and DCM (2 mL) were added to the residue and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation. LC-MS: [M+H]+ calculated 650.25, found 650.83. Synthesis of structures 6b, 6.1b, 6.2b, 6.3b, and 6.4b. Structure 6b ((S)-3-(4-(4-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)naphthalen-1-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid) [ka]

[0349] To a solution of compound 22 (1.1 g, 3.95 mmol, 1 equiv.), compound 45 (595 mg, 4.74 mmol, 1.2 equiv.), and TBTU (1.52 g, 4.74 mmol, 1.2 equiv.) in anhydrous DMF (10 mL) was added diisopropylethylamine (2.06 mL, 11.85 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 3 h. The reaction was quenched with saturated NaHCO solution (10 mL). The aqueous phase was extracted with ethyl acetate (3 × 10 mL), and the organic phases were combined, dried over anhydrous NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase. LC-MS: [M+H] calculated 366.20, found 367. [ka]

[0350] To a solution of compound 61 (2 g, 8.96 mmol, 1 eq.) and compound 62 (2.13 mL, 17.93 mmol, 2 eq.) in anhydrous DMF (10 mL) was added K2CO3 (2.48 g, 17.93 mmol, 2 eq.) at 0 °C. The reaction mixture was warmed to room temperature and stirred overnight. The reaction was quenched with water (10 mL). The aqueous phase was extracted with ethyl acetate (3 × 10 mL), and the organic phases were combined, dried over anhydrous Na2SO4, and concentrated. The products were separated by CombiFlash® using silica gel as the stationary phase. [ka]

[0351] To a solution of compound 60 (1.77 g, 4.84 mmol, 1 equiv.) in THF (5 mL) and HO (5 mL) was added lithium hydroxide monohydrate (0.61 g, 14.53 mmol, 3 equiv.) in portions at 0 °C. The reaction mixture was warmed to room temperature. After stirring at room temperature for 3 h, the reaction mixture was acidified to pH 3.0 with HCl (6 N). The aqueous phase was extracted with ethyl acetate (3 × 20 mL), and the organic layers were combined, dried over NaSO, and concentrated. LC-MS: calculated [M+H] 352.18, found 352. [ka]

[0352] To a solution of compound 63 (1.88 g, 6.0 mmol, 1.0 equiv) in anhydrous THF (20 mL) was added n-BuLi in hexane (3.6 mL, 9.0 mmol, 1.5 equiv) dropwise at −78° C. The reaction was maintained at −78° C. for an additional 1 h. Triisopropylborate (2.08 mL, 9.0 mmol, 1.5 equiv) was then added to the mixture at −78° C. The reaction was then warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NH4Cl solution (20 mL) and adjusted to pH 3. The aqueous phase was extracted with EtOAc (3×20 mL), and the organic phases were combined, dried over Na2SO4, and concentrated. [ka]

[0353] Compound 12 (300 mg, 0.837 mmol, 1.0 equiv.), compound 65 (349 mg, 1.256 mmol, 1.5 equiv.), XPhos Pd G2 (13 mg, 0.0167 mmol, 0.02 equiv.), and K3PO4 (355 mg, 1.675 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a septum-equipped screw cap, then evacuated and refilled with nitrogen (this process was repeated a total of three times). THF (8 mL) and water (2 mL) were then added via syringe. Nitrogen was bubbled through the mixture for 20 minutes, and the reaction was kept at room temperature overnight. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phase was dried over Na2SO4, concentrated and purified by CombiFlash® using silica gel as stationary phase and eluted with 15% EtOAc in hexane. LC-MS: [M+H]+ calculated 512.24, found 512.56. [ka]

[0354] Compound 66 (858 mg, 1.677 mmol, 1.0 equiv) was cooled in an ice bath. HCl in dioxane (8.4 mL, 33.54 mmol, 20 equiv) was added to the flask. The reaction was allowed to warm to room temperature and stirred for an additional hour. The solvent was removed by rotary evaporation and the product was used directly without further purification. LC-MS: [M+H]+ calculated 412.18, found 412.46. [ka]

[0355] To a solution of compound 64 (500 mg, 1.423 mmol, 1 equiv.), compound 67 (669 mg, 1.494 mmol, 1.05 equiv.), and TBTU (548 mg, 0.492 mmol, 1.2 equiv.) in anhydrous DMF (15 mL) was added diisopropylethylamine (0.744 mL, 4.268 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated aqueous NaHCO (10 mL), and the product was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with 3-4% methanol in DCM. The yield was 96.23%. LC-MS: [M+H]+ calculated 745.35, found 746.08. [ka]

[0356] To a solution of compound 68 (1.02 g, 1.369 mmol, 1 equiv.) in ethyl acetate (10 mL) was added 10% Pd / C (0.15 g, 50% HO) at room temperature. The reaction mixture was warmed to room temperature and the reaction was monitored by LC-MS. The reaction was kept at room temperature overnight. The solid was filtered through Celite® and the solvent was removed by rotary evaporation. The product was used directly without further purification. LC-MS: [M+H]+ 655.31, found 655.87. [ka]

[0357] To a solution of compound 69 (100 mg, 0.152 mmol, 1 equiv.) and azido-PEG3-OTs (100 mg, 0.305 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added K2CO3 (42 mg, 0.305 mmol, 2 equiv.) at 0 °C. The reaction mixture was stirred at 80 °C for 6 h. The reaction was quenched with saturated NaHCO3 solution, and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase. LC-MS: [M+H]+ calculated 812.39, found 813.14. [ka]

[0358] To a solution of compound 70 (77 mg, 0.0948 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (7 mg, 0.284 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another 2 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (0.5 mL) and DCM (0.5 mL) were added to the residue, and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation. LC-MS: [M+H]+ calculated 698.32, found 698.81. Structure 6.1b ((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid) [ka]

[0359] To a solution of compound 69 (100 mg, 0.152 mmol, 1 equiv.) and azido-PEG5-OTs (128 mg, 0.305 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added K2CO3 (42 mg, 0.305 mmol, 2 equiv.) at 0 °C. The reaction mixture was stirred at 80 °C for 6 h. The reaction was quenched with saturated NaHCO3 solution, and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. LC-MS: calculated [M+H]+ 900.40, found 901.46. [ka]

[0360] To a solution of compound 72 (59 mg, 0.0656 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (5 mg, 0.197 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (0.5 mL) and DCM (0.5 mL) were added to the residue, and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation. LC-MS: [M+H]+ calculated 786.37, found 786.95. Structure 6.2b ((S)-3-(4-(4-((8-azidooctyl)oxy)naphthalen-1-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid) [ka]

[0361] To a solution of compound 69 (150 mg, 0.229 mmol, 1 equiv.) and 1,8-dibromooctane (127 uL, 0.687 mmol, 3 equiv.) in acetone (2 mL) was added K2CO3 (95 mg, 0.687 mmol, 3 equiv.) at room temperature. The reaction mixture was stirred at 55 °C overnight. The reaction was quenched with saturated NaHCO3 solution, and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. LC-MS: [M+H]+ calculated 845.34, found 845.91. [ka]

[0362] To a solution of compound 74 (97 mg, 0.114 mmol, 1 equiv.) in anhydrous DMF (2 mL) was added sodium azide (15 mg, 0.229 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 80° C. for 2 h. The reaction was quenched with water, and the aqueous layer was extracted with ethyl acetate (3×10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. LC-MS: [M+H]+ calculated 808.43, found 809.00. [ka]

[0363] To a solution of compound 75 (92 mg, 0.114 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (8 mg, 0.342 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (0.5 mL) and DCM (0.5 mL) were added to the residue, and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation. LC-MS: [M+H]+ calculated 694.36, found 694.94. Structure 6.3b ((S)-3-(4-(4-((20-azido-3,6,9,12,15,18-hexaoxaicosyl)oxy)naphthalen-1-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid) [ka]

[0364] To a solution of compound 69 (100 mg, 0.152 mmol, 1 equiv.) and azido-PEG7-OTs (154 mg, 0.305 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (100 mg, 0.305 mmol, 2 equiv.) at 0 °C. The reaction mixture was stirred at 40 °C overnight. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase, and the product was eluted with DCM containing 2-3% methanol. LC-MS: [M+H]+ calculated 988.50, found 989.14. [ka]

[0365] To a solution of compound 21 (112 mg, 0.113 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (8 mg, 0.340 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another hour at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. TFA (4 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for another 3 hours at room temperature. The solvent was evaporated on a rotary evaporator. LC-MS: [M+H]+ calculated 874.43, found 875.08. Structure 6.4b ((S)-3-(4-(4-((35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)oxy)naphthalen-1-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid) [ka]

[0366] Compound 69 (80 mg, 0.122 mmol, 1 equiv.) and azide-PEG 12 To a solution of -OTs (184 mg, 0.244 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (80 mg, 0.244 mmol, 2 equiv.) at 0 °C. The reaction mixture was stirred at 40 °C for 5 h. The reaction was quenched with saturated NaHCO3 solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 2-3% methanol. LC-MS: [M+H]+ calculated 1208.63, found 1209.21. [ka]

[0367] To a solution of compound 82 (100 mg, 0.0972 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (7 mg, 0.292 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (4 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation. LC-MS: calculated [M+H]+ 1094.56, 1095.05. Structure 7b ((R)-3-(4-(4-(2-(2-azidoethoxy)ethoxy Synthesis of)ethoxy)naphthalen-1-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid). [ka]

[0368] To a solution of compound 84 (1.0 g, 2.90 mmol, 1 equiv.) and potassium carbonate (0.60 g, 4.36 mmol, 1.5 equiv.) in anhydrous DMF (10 mL) was added methyl iodide (362 μL, 5.81 mmol, 2.0 equiv.) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. LC-MS: [M+H]+ calculated 358.06, found 358.34. [ka]

[0369] Compound 85 (1.0 g, 2.791 mmol, 1.0 equiv) was cooled in an ice bath. HCl in dioxane (7.0 mL, 27.91 mmol, 10 equiv) was added to the flask. The reaction was warmed to room temperature and stirred for an additional 1 h. The solvent was removed by rotary evaporation and the product was used directly without further purification. LC-MS: [M+H]+ calculated 258.01, found 257.97. [ka]

[0370] To a solution of compound 64 (790 mg, 2.248 mmol, 1 equiv.), compound 86 (728 mg, 2.473 mmol, 1.10 equiv.), and TBTU (866 mg, 2.698 mmol, 1.20 equiv.) in anhydrous DMF (15 mL) was added diisopropylethylamine (1.175 mL, 6.744 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated aqueous NaHCO (10 mL), and the product was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 3-4% methanol. LC-MS: [M+H] calculated 591.17, found 591.49. [ka]

[0371] Compound 87 (200 mg, 0.338 mmol, 1.0 equiv.), compound 65 (141 mg, 0.507 mmol, 1.5 equiv.), XPhos Pd G2 (5.3 mg, 0.068 mmol, 0.02 equiv.), and K3PO4 (143 mg, 0.676 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a septum-equipped screw cap, then evacuated and refilled with nitrogen (this process was repeated a total of three times). THF (8 mL) and water (2 mL) were then added via syringe. Nitrogen was bubbled through the mixture for 20 minutes, and the reaction was kept at room temperature overnight. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phase was dried over Na2SO4 and concentrated. LC-MS: [M+H]+ calculated 745.35, found 746.08. [ka]

[0372] To a solution of compound 88 (0.247 g, 0.331 mmol, 1 equiv.) in ethyl acetate (10 mL) was added 10% Pd / C (100 mg) at room temperature. The reaction mixture was stirred overnight at room temperature. The catalyst was removed by filtration through Celite®, and the product was used directly without further purification. LC-MS: [M+H]+ calculated 655.31, found 655.96. [ka]

[0373] To a solution of compound 89 (50 mg, 0.076 mmol, 1 equiv.) and azido-PEG3-OTs (50 mg, 0.152 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (50 mg, 0.152 mmol, 2 equiv.) at 0 °C. The reaction mixture was stirred at room temperature for 72 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 4% MeOH in DCM. LC-MS: [M+H]+ calculated 812.39, found 813.14. [ka]

[0374] To a solution of compound 90 (36 mg, 0.0443 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (3 mg, 0.133 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (0.5 mL) and DCM (0.5 mL) were added to the residue, and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation. LC-MS: [M+H]+ calculated 698.32, found 698.90. Synthesis of structure 8b ((S)-3-(4-(7-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)naphthalen-1-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid). [ka]

[0375] To a solution of compound 92 (1.0 g, 4.48 mmol, 1 eq.) and compound 62 (1.06 mL, 8.96 mmol, 2 eq.) in anhydrous DMF (10 mL) was added K2CO3 (1.24 g, 8.96 mmol, 2 eq.) at room temperature. The reaction mixture was stirred at 80 °C overnight. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 5% ethyl acetate in hexane. [ka]

[0376] To a solution of compound 94 (0.5 g, 1.596 mmol, 1.0 equiv) in anhydrous THF (10 mL) was added n-BuLi in hexane (0.96 mL, 2.394 mmol, 1.5 equiv) dropwise at −78° C. The reaction was maintained at −78° C. for an additional 1 h. Triisopropylborate (0.553 mL, 2.394 mmol, 1.5 equiv) was then added to the mixture at −78° C. The reaction was then warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NH4Cl solution (20 mL) and adjusted to pH 3. The aqueous phase was extracted with EtOAc (3×20 mL), and the organic phases were combined, dried over Na2SO4, and concentrated. The solid was triturated with hexane and filtered. The product was used directly without further purification. LC-MS: [MH] − calculated 277.11, found 277.35. [ka]

[0377] Compound 96 (100 mg, 0.169 mmol, 1.0 equiv.), compound 95 (70 mg, 0.253 mmol, 1.5 equiv.), XPhos Pd G2 (2.7 mg, 0.0034 mmol, 0.02 equiv.), and K3PO4 (72 mg, 0.338 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a septum-equipped screw cap, then evacuated and backfilled with nitrogen (this process was repeated a total of three times). THF (8 mL) and water (2 mL) were then added via syringe. Nitrogen was bubbled through the mixture for 20 minutes, and the reaction was kept at room temperature overnight. The reaction was quenched with water (10 mL). The mixture was thawed and the aqueous phase was extracted with ethyl acetate (3 x 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The compounds were separated by CombiFlash® using silica gel as the stationary phase and eluted with 3% methanol in DCM. [ka]

[0378] To a solution of compound 97 (0.116 g, 0.157 mmol, 1 equiv.) in ethyl acetate (10 mL) was added 10% Pd / C (100 mg) at room temperature. The reaction mixture was stirred overnight at room temperature. The catalyst was removed by filtration through Celite®, and the product was used directly without further purification. LC-MS: [M+H]+ calculated 655.31, found 655.87. [ka]

[0379] To a solution of compound 98 (87 mg, 0.133 mmol, 1 equiv.) and azido-PEG3-OTs (87 mg, 0.266 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (87 mg, 0.266 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 40 °C for 6 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 3-4% MeOH in DCM. LC-MS: [M+H]+ calculated 812.39, found 813.05. [ka]

[0380] To a solution of compound 99 (65 mg, 0.0801 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (6 mg, 0.240 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (0.5 mL) and DCM (0.5 mL) were added to the residue, and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation. LC-MS: [M+H]+ calculated 698.32, found 698.99. Synthesis of structure 9b ((14S,17R)-1-azido-14-(4-((4-methylpyridin-2-yl)amino)butanamido)-17-(4-(naphthalen-1-yl)phenyl)-15-oxo-3,6,9,12-tetraoxa-16-azanonadecanoic acid). [ka]

[0381] Compound 102 (0.19 g, 0.468 mmol, 1.0 equiv) was cooled in an ice bath. HCl in dioxane (2.35 mL, 9.37 mmol, 20 equiv) was added to the flask. The reaction was allowed to warm to room temperature and stirred for an additional hour. The solvent was removed by rotary evaporation and the product was used directly without further purification. LC-MS: [M+H]+ calculated 306.14, found 306.51. [ka]

[0382] To a solution of compound 23 (110 mg, 0.188 mmol, 1 equiv.), compound 103 (71 mg, 0.207 mmol, 1.10 equiv.), and TBTU (72.7 mg, 0.226 mmol, 1.20 equiv.) in anhydrous DMF (2 mL) was added diisopropylethylamine (0.1 mL, 0.566 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated aqueous NaHCO (10 mL), and the product was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 3-4% methanol. LC-MS: [M+H] calculated 870.43, found 871.12. [ka]

[0383] To a solution of compound 104 (110 mg, 0.126 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (9 mg, 0.379 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another hour at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (4 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for another 3 hours at room temperature. The solvent was removed by rotary evaporation. LC-MS: [M+H]+ calculated 756.36, found 756.88. Synthesis of structure 10b ((S)-3-(4-(5-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid). [ka]

[0384] To a solution of compound 106 (1.0 g, 4.48 mmol, 1 equiv.) and compound 62 (1.06 mL, 8.96 mmol, 2 equiv.) in anhydrous DMF (10 mL) was added Cs2CO3 (2.92 g, 8.96 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred overnight at room temperature. The reaction was quenched with aqueous solution (20 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 5% ethyl acetate in hexane. [ka]

[0385] To a solution of compound 107 (1.188 g, 3.793 mmol, 1.0 equiv) in anhydrous THF (10 mL) was added n-BuLi in hexane (2.27 mL, 5.689 mmol, 1.5 equiv) dropwise at −78° C. The reaction was maintained at −78° C. for an additional 1 h. Triisopropylborate (1.31 mL, 5.689 mmol, 1.5 equiv) was then added to the mixture at −78° C. The reaction was then warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NH4Cl solution (20 mL) and adjusted to pH 3. The aqueous phase was extracted with EtOAc (3×20 mL), and the organic phases were combined, dried over Na2SO4, and concentrated. The solid was triturated with hexane and filtered. The product was used directly without further purification. LC-MS: [MH] − calculated 277.11, found 277.26. [ka]

[0386] Compound 96 (100mg, 0.169mmol, 1.0eq), Compound 108 (70m g, 0.253 mmol, 1.5 equiv.), XPhos Pd G2 (2.7 mg, 0.0034 mmol, 0.02 equiv.), and K3PO4 (72 mg, 0.338 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a septum-equipped screw cap, then evacuated and refilled with nitrogen (this process was repeated a total of three times). THF (8 mL) and water (2 mL) were then added via syringe. Nitrogen was bubbled through the mixture for 20 minutes, and the reaction was kept at room temperature overnight. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The compounds were separated by CombiFlash® using silica gel as the stationary phase and eluted with 3% methanol in DCM. LC-MS: [M+H]+ calculated 745.35, found 745.99. [ka]

[0387] To a solution of compound 109 (0.135 g, 0.181 mmol, 1 eq) in ethyl acetate (10 mL) was added 10% Pd / C (100 mg) at room temperature. The reaction mixture was stirred overnight at room temperature. The catalyst was removed by filtration through Celite®, and the product was used directly without further purification. LC-MS: [M+H]+ calculated 655.31, found 655.87. [ka]

[0388] To a solution of compound 110 (50 mg, 0.0764 mmol, 1 equiv.) and azido-PEG5-OTs (64 mg, 0.152 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (50 mg, 0.152 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 4% methanol. The yield is 62%. LC-MS: [M+H]+ calculated 900.44, found 901.19. [ka]

[0389] To a solution of compound 111 (43 mg, 0.0478 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (3.4 mg, 0.143 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (4 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation. LC-MS: [M+H]+ calculated 786.37, found 787.04. Synthesis of structure 11b ((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-((S)-1-(4-((4-methylpyridin-2-yl)amino)butanoyl)pyrrolidine-2-carboxamido)propanoic acid). [ka]

[0390] To a solution of compound 22 (500 mg, 1.698 mmol, 1 equiv.), compound 113 (295 mg, 1.783 mmol, 1.05 equiv.), and TBTU (654 mg, 2.038 mmol, 1.2 equiv.) in anhydrous DMF (10 mL) was added diisopropylethylamine (0.888 mL, 5.096 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated aqueous NaHCO (10 mL), and the product was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 2-3% methanol. The yield is 98.72%. LC-MS: [M+H]+ calculated 406.23, found 406.07. [ka]

[0391] To a solution of compound 114 (0.68 g, 1.676 mmol, 1 equiv.) in THF (5 mL) and HO (5 mL) was added lithium hydroxide (0.12 g, 5.030 mmol, 3 equiv.) in portions at 0 °C. The reaction mixture was warmed to room temperature. After stirring at room temperature for 1 h, the reaction mixture was acidified to pH 3.0 with HCl (6 N). The aqueous phase was extracted with ethyl acetate (3 × 10 mL), and the organic layers were combined, dried over NaSO, and concentrated. The product was used without further purification. LC-MS: [M+H] calculated 392.21, found 392.39. [ka]

[0392] To a solution of compound 115 (300 mg, 0.766 mmol, 1 equiv.), compound 116 (237 mg, 0.804 mmol, 1.05 equiv.), and TBTU (295 mg, 0.919 mmol, 1.2 equiv.) in anhydrous DMF (10 mL) was added diisopropylethylamine (0.400 mL, 2.299 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated aqueous NaHCO (10 mL), and the product was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with 3-4% methanol in DCM. The yield is 83%. LC-MS: [M+H]+ calculated 631.21, found 631.46. [ka]

[0393] Compound 118 (100 mg, 0.158 mmol, 1.0 equiv.), compound 65 (66 mg, 0.237 mmol, 1.5 equiv.), XPhos Pd G2 (2.5 mg, 0.0032 mmol, 0.02 equiv.), and K3PO4 (67 mg, 0.316 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a septum-equipped screw cap, then evacuated and refilled with nitrogen (this process was repeated a total of three times). THF (5 mL) and water (1 mL) were then added via syringe. Nitrogen was bubbled through the mixture for 20 minutes, and the reaction was held at 40 °C for 1 hour. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The compound was separated by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 3% methanol. The yield was 96%. LC-MS: [M+H]+ calculated 785.38, found 785.69. [ka]

[0394] To a solution of compound 119 (0.120 g, 0.153 mmol, 1 equiv.) in ethyl acetate (10 mL) was added 10% Pd / C (100 mg) at room temperature. The reaction mixture was stirred overnight at room temperature. The catalyst was removed by filtration through Celite®, and the product was used directly without further purification. LC-MS: [M+H]+ calculated 695.34, found 695.66. [ka]

[0395] To a solution of compound 120 (83 mg, 0.119 mmol, 1 equiv.) and azido-PEG5-OTs (100 mg, 0.239 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (78 mg, 0.239 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 4% methanol. The yield was 79%. LC-MS: calculated 940.47, found 941.16. [ka]

[0396] To a solution of compound 121 (89 mg, 0.0947 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (6.8 mg, 0.284 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another hour at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (4 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for another 3 hours at room temperature. The solvent was removed by rotary evaporation. LC-MS: [M+H]+ calculated 826.41, found 827.10. Synthesis of structure 12b ((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)benzo[d]oxazol-7-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid). [ka]

[0397] To a solution of compound 123 (1.0 g, 7.40 mmol, 1 equiv.) and compound 62 (1.32 mL, 11.10 mmol, 1.5 equiv.) in anhydrous DMF (10 mL) was added Cs2CO3 (3.62 g, 11.10 mmol, 1.5 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred overnight. The reaction was quenched with water (10 mL). The aqueous phase was extracted with ethyl acetate (3 × 10 mL), and the organic phases were combined, dried over anhydrous Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 5-7% ethyl acetate in hexane. Yield: 85%. [ka]

[0398] To a solution of compound 124 (1.425 g, 6.326 mmol, 1 equiv.) in anhydrous acetonitrile (20 mL) was added N-bromosuccinimide (1.216 g, 6.832 mmol, 1.08 equiv.) in portions at 0° C. The reaction mixture was kept at 0° C. for an additional 30 min. The mixture was then allowed to warm to room temperature and stirred overnight. The solvent was removed under reduced pressure and the residue was purified by CombiFlash® using silica gel as the stationary phase. The product was eluted with 4-5% ethyl acetate in hexane. Yield 65%. LC-MS: [M+H]+ calculated 303.99. Found 304.08. [ka]

[0399] A mixture of compound 125 (1.339 g, 4.402 mmol, 1 equiv.), bis(pinacolato)diboron (2.236 g, 8.805 mmol, 2 equiv.), potassium acetate (0.864 g, 8.805 mmol, 2 equiv.), and Pd(dppf)Cl (161 mg, 0.220 mmol, 0.05 equiv.) in 15 mL of anhydrous 1,4-dioxane was stirred at 100 °C under nitrogen for 8 h. After concentration, the residue was partitioned between HO and DCM, the aqueous phase was extracted with DCM, and the combined organic layers were washed with brine, dried over NaSO, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with 15-20% ethyl acetate in hexane. LC-MS: [M+H] calculated 352.16, found 352.06. [ka]

[0400] Compound 96 (200 mg, 0.338 mmol, 1.0 equiv.), compound 126 (178 mg, 0.507 mmol, 1.5 equiv.), XPhos Pd G2 (5.3 mg, 0.0068 mmol, 0.02 equiv.), and K3PO4 (143 mg, 0.676 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a septum-equipped screw cap, then evacuated and refilled with nitrogen (this process was repeated a total of three times). THF (5 mL) and water (1 mL) were then added via syringe. Nitrogen was bubbled through the mixture for 20 minutes, and the reaction was held at 40 °C for 1 hour. The reaction was quenched with saturated NaHCO3 (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The compounds were separated by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 2-3% methanol. LC-MS: [M+H]+ calculated 736.33, found 736.89. [ka]

[0401] To a solution of compound 127 (0.219 g, 0.297 mmol, 1 equiv.) in ethyl acetate (10 mL) was added 10% Pd / C (100 mg) at room temperature. The reaction mixture was stirred overnight at room temperature. The catalyst was removed by filtration through Celite®, and the product was used directly without further purification. LC-MS: [M+H]+ calculated 646.28, found 646.78. [ka]

[0402] To a solution of compound 128 (73 mg, 0.113 mmol, 1 eq.) and azido-PEG5-OTs (94 mg, 0.226 mmol, 2 eq.) in anhydrous DMF (2 mL) was added Cs2CO3 (74 mg, 0.226 mmol, 2 eq.) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 4% methanol. The yield is 80%. LC-MS: [M+H]+ calculated 891.42, found 892.00. [ka]

[0403] To a solution of compound 129 (43 mg, 0.0478 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (3.4 mg, 0.143 mmol, 3.0 equiv.) at room temperature. The mixture was stirred at room temperature for 1 h. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (4 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred at room temperature for an additional 3 h. The solvent was removed by rotary evaporation. LC-MS: [M+H]+ calculated 777.35, found 777.94. Synthesis of structure 13b ((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-5,6,7,8-tetrahydronaphthalen-1-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid). [ka]

[0404] A mixture of compound 1 (300 mg, 1.321 mmol, 1 equiv.), bis(pinacolato)diboron (671 mg, 2.642 mmol, 2 equiv.), potassium acetate (389 mg, 3.963 mmol, 2 equiv.), and Pd(dppf)Cl (48 mg, 0.066 mmol, 0.05 equiv.) in 10 mL of anhydrous 1,4-dioxane was stirred overnight at 80 °C under nitrogen. After concentration, the residue was partitioned between HO and DCM, the aqueous phase was extracted with DCM, and the combined organic layers were washed with brine, dried over NaSO, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with 10% ethyl acetate in hexane. LC-MS: [MH] calculated 273.17, found 273.29. [ka]

[0405] Compound 1 (100 mg, 0.169 mmol, 1.0 equiv.), compound 2 (70 mg, 0.253 mmol, 1.5 equiv.), XPhos Pd G2 (2.7 mg, 0.0034 mmol, 0.02 equiv.), and K3PO4 (72 mg, 0.338 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a septum-equipped screw cap, then evacuated and backfilled with nitrogen (this process was repeated a total of three times). THF (5 mL) and water (1 mL) were then added via syringe. Nitrogen was bubbled through the mixture for 20 minutes, and the reaction was held at 40 °C for 3 hours. The reaction was then cooled to room temperature and allowed to stand overnight. The reaction was quenched with saturated NaHCO3 (10 mL), and the aqueous phase was purified with ethyl acetate. The resulting mixture was extracted with hexane (3 x 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The compounds were separated by CombiFlash® using silica gel as the stationary phase and eluted with 4-5% methanol in DCM. LC-MS: [M+H]+ calculated 659.34, found 659.57. [ka]

[0406] To a solution of compound 1 (30 mg, 0.0455 mmol, 1 equiv.) and azido-PEG5-OTs (38 mg, 0.0911 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (30 mg, 0.0911 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 4% methanol. The yield is 70%. LC-MS: [M+H]+ calculated 904.47, found 904.88. [ka]

[0407] To a solution of compound 1 (29 mg, 0.0321 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (2.3 mg, 0.0962 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (4 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation. LC-MS: [M+H]+ calculated 790.41, found 790.64. Synthesis of structure 14b ((S)-3-(4'-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid). [ka]

[0408] Compound 1 (150 mg, 0.253 mmol, 1.0 equiv.), compound 2 (118 mg, 0.380 mmol, 1.5 equiv.), XPhos Pd G2 (4 mg, 0.0051 mmol, 0.02 equiv.), and K3PO4 (107 mg, 0.507 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a septum-equipped screw cap, then evacuated and refilled with nitrogen (this process was repeated a total of three times). THF (5 mL) and water (1 mL) were then added via syringe. Nitrogen was bubbled through the mixture for 10 minutes, and the reaction was maintained at 40 °C overnight. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The compounds were separated by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 2-4% methanol. LC-MS: [M+H]+ calculated 779.32, found 779.65. [ka]

[0409] To a solution of compound 1 (0.19 g, 0.244 mmol, 1 equiv.) in ethyl acetate (10 mL) was added 10% Pd / C (100 mg) at room temperature. The reaction was evacuated and recharged with hydrogen (this process was repeated three times). The reaction mixture was stirred overnight at room temperature. The catalyst was removed by filtration through Celite®, and the product was used directly without further purification. LC-MS: [M+H]+ calculated 689.27, found 689.54. [ka]

[0410] To a solution of compound 1 (80 mg, 0.116 mmol, 1 equiv.) and azido-PEG5-OTs (97 mg, 0.232 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (76 mg, 0.232 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 3-4% methanol. The yield was 82%. LC-MS: [M+H]+ calculated 934.41, found 935.04. [ka]

[0411] To a solution of compound 1 (90 mg, 0.0964 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (7 mg, 0.289 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (4 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation. LC-MS: [M+H]+ calculated 820.34, found 820.89. Synthesis of structure 15b ((S)-3-(3-(5-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid). [ka]

[0412] To a solution of compound 1 (1.0 g, 2.90 mmol, 1 equiv.) and potassium carbonate (0.60 g, 4.36 mmol, 1.5 equiv.) in anhydrous DMF (10 mL) was added methyl iodide (362 μL, 5.81 mmol, 2.0 equiv.) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The reaction was then quenched with water (20 mL), and the aqueous phase was extracted with ethyl acetate (3×10 mL). The organic phases were combined, dried over anhydrous NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 15% ethyl acetate in hexane. LC-MS: [M+H] calculated 358.06, found 358.18. [ka]

[0413] Compound 1 (858 mg, 1.677 mmol, 1.0 equiv) was cooled in an ice bath. HCl in dioxane (8.4 mL, 33.54 mmol, 20 equiv) was added to the flask. The reaction was allowed to warm to room temperature and stirred for an additional hour. The solvent was removed by rotary evaporation, and the product was used directly without further purification. LC-MS: [M+H]+ calculated 258.01, found 258.08. [ka]

[0414] To a solution of compound 1 (640 mg, 1.821 mmol, 1 equiv.), compound 2 (590 mg, 2.003 mmol, 1.10 equiv.), and TBTU (702 mg, 2.185 mmol, 1.20 equiv.) in anhydrous DMF (10 mL) was added diisopropylethylamine (0.952 mL, 5.464 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated aqueous NaHCO (10 mL), and the product was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 3-4% methanol. LC-MS: [M+H] calculated 591.17, found 591.40. [ka]

[0415] Compound 1 (150 mg, 0.253 mmol, 1.0 equiv.), compound 2 (106 mg, 0.380 mmol, 1.5 equiv.), XPhos Pd G2 (4 mg, 0.0051 mmol, 0.02 equiv.), and K3PO4 (107 mg, 0.507 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a septum-equipped screw cap, then evacuated and refilled with nitrogen (this process was repeated a total of three times). THF (5 mL) and water (1 mL) were then added via syringe. Nitrogen was bubbled through the mixture for 10 minutes, and the reaction was maintained at 40 °C for 2 hours. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The compounds were separated by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 3-4% methanol. LC-MS: [M+H]+ calculated 745.35, found 745.99. [ka]

[0416] To a solution of compound 1 (0.189 g, 0.253 mmol, 1 equiv.) in ethyl acetate (10 mL) was added 10% Pd / C (100 mg) at room temperature. The reaction was evacuated and recharged with hydrogen (this process was repeated three times). The reaction mixture was stirred overnight at room temperature. The catalyst was removed by filtration through Celite®, and the product was used directly without further purification. LC-MS: [M+H]+ calculated 655.31, found 655.42. [ka]

[0417] To a solution of compound 1 (80 mg, 0.122 mmol, 1 equiv.) and azido-PEG5-OTs (102 mg, 0.244 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (80 mg, 0.244 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 1-2% methanol. The yield is 90%. LC-MS: calculated 900.44, found 901.10. [ka]

[0418] To a solution of compound 1 (100 mg, 0.111 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (8 mg, 0.333 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (4 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation. LC-MS: [M+H]+ calculated 786.37, found 786.95. Synthesis of structure 16b ((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-((R)-1-(4-((4-methylpyridin-2-yl)amino)butanoyl)pyrrolidine-2-carboxamido)propanoic acid). [ka]

[0419] To a solution of compound 1 (500 mg, 1.698 mmol, 1 equiv.), compound 2 (295 mg, 1.783 mmol, 1.05 equiv.), and TBTU (654 mg, 2.038 mmol, 1.2 equiv.) in anhydrous DMF (10 mL) was added diisopropylethylamine (0.888 mL, 5.096 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated aqueous NaHCO (10 mL), and the product was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 2-3% methanol. The yield is 98.43%. LC-MS: [M+H]+ calculated 406.23, found 406.34. [ka]

[0420] To a solution of compound 1 (0.678 g, 1.672 mmol, 1 equiv.) in THF (10 mL) and HO (10 mL) was added lithium hydroxide (0.12 g, 5.016 mmol, 3 equiv.) in portions at 0 °C. The reaction mixture was warmed to room temperature. After stirring at room temperature for 1 h, the reaction mixture was acidified to pH 3.0 with HCl (6 N). The aqueous phase was extracted with ethyl acetate (3 × 10 mL), and the organic layers were combined, dried over NaSO, and concentrated. The product was used without further purification. LC-MS: [M+H] calculated 392.21, found 392.39. [ka]

[0421] To a solution of compound 1 (130 mg, 0.332 mmol, 1 equiv.), compound 2 (125 mg, 0.348 mmol, 1.05 equiv.), and TBTU (128 mg, 0.398 mmol, 1.2 equiv.) in anhydrous DMF (5 mL) was added diisopropylethylamine (0.174 mL, 0.996 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined and diluted with Na2SO4. The mixture was dried at 4°C and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 3-4% methanol. The yield is 86%. LC-MS: [M+H]+ calculated 695.34, found 695.93. [ka]

[0422] To a solution of compound 1 (80 mg, 0.115 mmol, 1 equiv.) and azido-PEG5-OTs (96 mg, 0.230 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (75 mg, 0.230 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with 4-5% methanol in DCM. The yield is 60%. [ka]

[0423] To a solution of compound 1 (65 mg, 0.0691 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (5 mg, 0.207 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (4 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation. LC-MS: [M+H]+ calculated 826.41, found 827.01. Synthesis of structure 17b ((S)-3-(4-(7-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)benzo[b]thiophen-4-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid). [ka]

[0424] A solution of bromine (1.877 g, 11.745 mmol, 1.05 equiv) in dry tetrachloromethane (20 mL) was added dropwise over 1.5 h to a stirred solution of compound 1 (1.837 g, 11.186 mmol, 1 equiv) in tetrachloromethane (20 mL) at 0 °C. After an additional 1 h at 0 °C, the organic layer was washed with water and brine, dried over Na SO , and concentrated to give a residue that was purified by CombiFlash using silica gel as the stationary phase. The product, along with impurities, was eluted using pure hexane. [ka]

[0425] To a solution of compound 1 (2.70 g, 11.105 mmol, 1.0 equiv.) in dichloromethane (20 mL) was added boron trifluoride dimethylsulfide complex (3.5 mL, 33.317 mmol, 3.0 equiv.) under a nitrogen atmosphere at 0° C. and stirred at room temperature for 20 hours. The reaction mixture was cooled to 0° C. and quenched with saturated NH4Cl solution (20 mL). The aqueous phase was extracted with ethyl acetate (3×20 mL), and the combined organic phases were dried over Na2SO4 and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 5% ethyl acetate in hexane. LC-MS: [MH]- calculated 226.92, found 227.03. [ka]

[0426] To a solution of compound 1 (1.838 g, 8.023 mmol, 1 equiv.) and compound 2 (1.906 mL, 16.04 mmol, 2 equiv.) in anhydrous DMF (10 mL) was added CsCO (5.228 g, 16.04 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred overnight at room temperature. The reaction was quenched with water (20 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 2-3% ethyl acetate in hexane. [ka]

[0427] To a solution of compound 1 (2.22 g, 6.954 mmol, 1.0 equiv.) in anhydrous THF (20 mL) was added n-BuLi in hexane (4.17 mL, 10.43 mmol, 1.5 equiv.) dropwise at −78° C. The reaction was maintained at −78° C. for an additional 1 h. Triisopropylborate (2.40 mL, 10.43 mmol, 1.5 equiv.) was then added to the mixture at −78° C. The reaction was then warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NH4Cl solution (20 mL) and adjusted to pH 3. The aqueous phase was extracted with EtOAc (3×20 mL), and the organic phases were combined, dried over Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 4-6% methanol in DCM. LC-MS: [MH] − calculated 283.07, found 283.20. [ka]

[0428] Compound 1 (400 mg, 0.676 mmol, 1.0 equiv.), compound 2 (288 mg, 1.01 mmol, 1.5 equiv.), XPhos Pd G2 (10 mg, 0.0135 mmol, 0.02 equiv.), and K3PO4 (287 mg, 1.352 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a septum-equipped screw cap, then evacuated and refilled with nitrogen (this process was repeated a total of three times). THF (8 mL) and water (2 mL) were then added via syringe. Nitrogen was bubbled through the mixture for 10 minutes, and the reaction was maintained at 40 °C for 2 hours. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The compounds were separated by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 3-4% methanol. LC-MS: [M+H]+ calculated 751.31, found 751.84. [ka]

[0429] To a solution of compound 1 (0.50 g, 0.666 mmol, 1 equiv.) in ethyl acetate (10 mL) was added 10% Pd / C (100 mg) at room temperature. The reaction was evacuated and recharged with hydrogen (this process was repeated three times). The reaction mixture was stirred overnight at room temperature. The catalyst was removed by filtration through Celite®, and the product was separated by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 5% methanol. LC-MS: [M+H]+ calculated 661.26, found 661.73. [ka]

[0430] To a solution of compound 1 (130 mg, 0.196 mmol, 1 equiv.) and azido-PEG5-OTs (164 mg, 0.393 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (128 mg, 0.393 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 3-4% methanol. The yield is 82%. LC-MS: [M+H]+ calculated 906.40, found 906.95. [ka]

[0431] To a solution of compound 1 (147 mg, 0.162 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (12 mg, 0.486 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another hour at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (2 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for another 3 hours at room temperature. The solvent was removed by rotary evaporation, and the product was separated by CombiFlash® using silica gel as the stationary phase. LC-MS: [M+H]+ calculated 792.33, found 792.89. Synthesis of structure 18b ((S)-3-(4-(6-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-2-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid). [ka]

[0432] Compound 1 (150 mg, 0.253 mmol, 1.0 equiv.), compound 2 (71.5 mg, 0.380 mmol, 1.5 equiv.), XPhos Pd G2 (4 mg, 0.0051 mmol, 0.02 equiv.), and K3PO4 (107 mg, 0.507 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a septum-equipped screw cap, then evacuated and refilled with nitrogen (this process was repeated a total of three times). THF (5 mL) and water (1 mL) were then added via syringe. Nitrogen was bubbled through the mixture for 10 minutes, and the reaction was maintained at 40 °C for 2 hours. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The compounds were separated by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 2-3% methanol. LC-MS: [M+H]+ calculated 655.31, found 655.87. [ka]

[0433] To a solution of compound 1 (160 mg, 0.244 mmol, 1 equiv.) and azido-PEG5-OTs (204 mg, 0.488 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (160 mg, 0.488 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 60 °C for 3 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 3-4% methanol. The yield was 30%. LC-MS: [M+H]+ calculated 900.44, found 901.01. [ka]

[0434] To a solution of compound 1 (67 mg, 0.0744 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (5 mg, 0.223 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. TFA (2 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation, and the product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 10% methanol in DCM. LC-MS: [M+H] calculated 786.37, found 786.86. Synthesis of structure 19b ((S)-3-(3-(6-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-2-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid). [ka]

[0435] Compound 1 (150 mg, 0.253 mmol, 1.0 equiv.), compound 2 (71.5 mg, 0.380 mmol, 1.5 equiv.), XPhos Pd G2 (4 mg, 0.0051 mmol, 0.02 equiv.), and K3PO4 (107 mg, 0.507 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a septum-equipped screw cap, then evacuated and refilled with nitrogen (this process was repeated a total of three times). THF (5 mL) and water (1 mL) were then added via syringe. Nitrogen was bubbled through the mixture for 10 minutes, and the reaction was maintained at 40 °C for 2 hours. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The compounds were separated by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 2-3% methanol. LC-MS: [M+H]+ calculated 655.31, found 655.78. [ka]

[0436] To a solution of compound 1 (104 mg, 0.158 mmol, 1 equiv.) and azido-PEG5-OTs (132 mg, 0.317 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (103 mg, 0.317 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 60 °C for 3 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 3-4% methanol. LC-MS: [M+H]+ calculated 900.44, found 901.01. [ka]

[0437] To a solution of compound 1 (125 mg, 0.138 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (10 mg, 0.416 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. TFA (4 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation, and the product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 12% methanol in DCM. LC-MS: [M+H] calculated 786.37, found 786.86. Synthesis of structure 20b ((S)-3-(3-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid). [ka]

[0438] Compound 1 (150 mg, 0.253 mmol, 1.0 equiv.), compound 2 (102 mg, 0.380 mmol, 1.5 equiv.), XPhos Pd G2 (4 mg, 0.0051 mmol, 0.02 equiv.), and K3PO4 (107 mg, 0.507 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a septum-equipped screw cap, then evacuated and refilled with nitrogen (this process was repeated a total of three times). THF (5 mL) and water (1 mL) were then added via syringe. Nitrogen was bubbled through the mixture for 10 minutes, and the reaction was maintained at 40 °C for 2 hours. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The compounds were separated by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 2-3% methanol. LC-MS: [M+H]+ calculated 655.31, found 655.78. [ka]

[0439] To a solution of compound 1 (160 mg, 0.244 mmol, 1 equiv.) and azido-PEG5-OTs (204 mg, 0.488 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (159 mg, 0.488 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 60 °C for 3 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 3-4% methanol. LC-MS: [M+H]+ calculated 900.44, found 901.01. [ka]

[0440] To a solution of compound 1 (125 mg, 0.138 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (10 mg, 0.416 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for an additional 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. TFA (4 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for an additional 3 h at room temperature. The solvent was removed by rotary evaporation, and the product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 8-12% methanol in DCM. LC-MS: [M+H] calculated 786.37, found 786.86. Synthesis of structure 22b ((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-((S)-2-(4-((4-methylpyridin-2-yl)amino)butanamido)propanamido)propanoic acid). [ka]

[0441] To a solution of compound 1 (250 mg, 0.85 mmol), L-alanine methyl ester hydrochloride (130 mg, 0.93 mmol), and TBTU (327 mg, 1.02 mmol) in DMF (2 mL) was added DIPEA (329 mg, 444 μL, 2.55 mmol) at 0° C. The reaction mixture was warmed to room temperature and stirred for 1 h. The reaction was quenched with saturated NH4Cl(aq) solution (0.75 mL) and deionized water (1 mL) and then extracted with ethyl acetate (3 mL). The aqueous layer was further extracted with ethyl acetate (2 × 3 mL). The combined organic phase was washed with saturated NaHCO3(aq) solution (2 mL). The organic layer was dried over Na2SO4, filtered, and concentrated. The crude mixture was separated by CombiFlash® using silica gel as the stationary phase with 0-5% methanol in DCM. Yield of compound 2: 294 mg (91%). 19 H 29[M+H] calculated for N3O5: 380.46, found: 380.33. [ka]

[0442] To a solution of compound 2 (294 mg, 0.77 mmol) in THF (4.5 mL) and deionized water (3 mL) at 0 °C was added a solution of lithium hydroxide (56 mg, 2.32 mmol) in deionized water (1 mL). The reaction was warmed to room temperature and stirred for 40 minutes. The reaction mixture was acidified to pH = 3 with 6 M HCl (aq). The aqueous phase was extracted with ethyl acetate (3 x 10 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated. Compound 3 was used without further purification. Yield of compound 3: 267 mg (94%). C 18 H 27 [M+H] calculated for N3O5: 366.43, found: 366.19. [ka]

[0443] Compound 3 (267 mg, 0.73 mmol), Compound 3a (288 mg, 0.80 mmol) To a solution of 283 mg, 382 μL, 2.19 mmol) and TBTU (282 mg, 0.88 mmol) in DMF (3 mL) was added DIPEA (283 mg, 382 μL, 2.19 mmol) at 0° C. The reaction mixture was warmed to room temperature and stirred for 1 h. The reaction mixture was quenched with saturated NH4Cl(aq) solution (1.5 mL) and deionized water (1.5 mL) and then extracted with ethyl acetate (12 mL). The aqueous layer was further extracted with ethyl acetate (2 × 12 mL). The combined organic phases were washed with half-saturated NH4Cl(aq) solution (10 mL), half-saturated NaHCO3(aq) solution (10 mL), and saturated NaCl(aq) solution (10 mL). The organic layer was dried over Na2SO4, filtered, and concentrated. The crude mixture was separated by CombiFlash® using silica gel as the stationary phase with 0-5% methanol in DCM. Yield of compound 4: 342 mg (70%). 38 H44 [M+H] calculated for N4O7: 669.79, found: 669.74. [ka]

[0444] To a solution of compound 4 (150 mg, 0.22 mmol) and azido-PEG5-OTs (187 mg, 0.49 mmol) in anhydrous DMF (1.2 mL) was added Cs2CO3 (146 mg, 0.49 mmol). The reaction mixture was stirred at 60 °C for 3 h. The reaction mixture was quenched with saturated NaHCO3 (aqueous) solution (10 mL) and deionized water (5 mL) and then extracted with ethyl acetate (7.5 mL). The aqueous layer was further extracted with ethyl acetate (2 × 7.5 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated. The crude mixture was separated by CombiFlash® using silica gel as the stationary phase with 0-4% methanol in DCM. Yield of compound 5: 142 mg (69%). 48 H 63 N7O 11 [M+H] calculated for: 915.06, found: 914.96. [ka]

[0445] To a solution of compound 5 (142 mg, 0.16 mmol) in THF (2 mL) and deionized water (1.5 mL) at 0 °C was added a solution of lithium hydroxide (11 mg, 0.47 mmol) in deionized water (0.5 mL). The reaction was warmed to room temperature and stirred for 1 h. The reaction mixture was acidified to pH = 3 with 6 M HCl (aq). The aqueous phase was diluted with ethyl acetate (3 The crude mixture was extracted with 2×8 mL of acetonitrile and toluene (2×20 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated. To this crude residue, TFA (2.0 mL) and water (100 μL) were added. The reaction mixture was stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure, and the residue was co-evaporated with acetonitrile:toluene [1:1] (2×20 mL). The crude mixture was separated by CombiFlash® using silica gel as the stationary phase with 0-13% methanol in DCM. Yield of structure 22b: 100 mg (80%). C 42 H 53 [M+H] calculated for N7O9: 800.92, found: 800.81. Synthesis of structure 23b ((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-((S)-3-methyl-2-(4-((4-methylpyridin-2-yl)amino)butanamido)butanamido)propanoic acid). [ka]

[0446] To a solution of compound 1 (250 mg, 0.85 mmol), L-valine methyl ester hydrochloride (157 mg, 0.93 mmol), and TBTU (327 mg, 1.02 mmol) in DMF (2 mL) was added DIPEA (329 mg, 444 μL, 2.55 mmol) at 0° C. The reaction mixture was warmed to room temperature and stirred for 1 h. The reaction was quenched with saturated NH4Cl(aq) solution (0.75 mL) and deionized water (1 mL) and then extracted with ethyl acetate (3 mL). The aqueous layer was further extracted with ethyl acetate (2 × 3 mL). The combined organic phase was washed with saturated NaHCO3(aq) solution (2 mL). The organic layer was dried over Na2SO4, filtered, and concentrated. The crude mixture was separated by CombiFlash® using silica gel as the stationary phase with 0-5% methanol in DCM. Yield of compound 2: 297 mg (86%). 21 H 33 [M+H] calculated for N3O5: 408.51, found: 407.87. [ka]

[0447] To a solution of compound 2 (297 mg, 0.73 mmol) in THF (4.5 mL) and deionized water (3 mL) at 0 °C was added a solution of lithium hydroxide (52 mg, 2.19 mmol) in deionized water (1 mL). The reaction was warmed to room temperature and stirred for 40 minutes. The reaction mixture was acidified to pH = 3 with 6 M HCl (aq). The aqueous phase was extracted with ethyl acetate (3 × 10 mL). The combined organic phases were dried over NaSO, filtered, and concentrated. Compound 3 was used without further purification assuming 100% yield. 20 H 31 [M+H] calculated for N3O5: 394.49, found: 393.83. [ka]

[0448] To a solution of compound 3 (287 mg, 0.73 mmol), compound 3a (287 mg, 0.80 mmol), and TBTU (281 mg, 0.88 mmol) in DMF (3 mL) was added DIPEA (283 mg, 382 μL, 2.19 mmol) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 1 h. The reaction mixture was quenched with saturated NH4Cl(aqueous) solution (2.5 mL) and deionized water (2.5 mL) and then extracted with ethyl acetate (12 mL). The aqueous layer was further extracted with ethyl acetate (2 × 12 mL). The combined organic phases were washed with half-saturated NH4Cl(aqueous) solution (10 mL), half-saturated NaHCO3(aqueous) solution (10 mL), and saturated NaCl(aqueous) solution (10 mL). The organic layer was dried over Na2SO4, filtered, and concentrated. The crude mixture was separated by CombiFlash® using silica gel as the stationary phase with 0-5% methanol in DCM. Yield of compound 4: 374 mg (74%). 40 H 48 [M+H] calculated for N4O7: 697.84, found: 697.46. [ka]

[0449] To a solution of compound 4 (150 mg, 0.215 mmol) and azido-PEG5-OTs (180 mg, 0.43 mmol) in anhydrous DMF (1.2 mL) was added Cs2CO3 (140 mg, 0.43 mmol). The reaction mixture was stirred at 60 °C for 3 h. The reaction mixture was quenched with saturated NaHCO3 (aqueous) solution (10 mL) and deionized water (5 mL) and then extracted with ethyl acetate (7.5 mL). The aqueous layer was further extracted with ethyl acetate (2 × 7.5 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated. The crude mixture was separated by CombiFlash® using silica gel as the stationary phase with 0-4% methanol in DCM. Yield of compound 5: 134 mg (66%). C 50 H 67 N7O 11 [M+H] calculated for: 943.12, found: 942.96. [ka]

[0450] To a solution of compound 5 (134 mg, 0.14 mmol) in THF (2 mL) and deionized water (1.5 mL) at 0 °C was added a solution of lithium hydroxide (10 mg, 0.43 mmol) in deionized water (0.5 mL). The reaction was warmed to room temperature and stirred for 1 h. The reaction mixture was acidified to pH = 3 with 6 M HCl (aq). The aqueous phase was extracted with ethyl acetate (3 × 8 mL). The combined organic phases were dried over NaSO, filtered, and concentrated. To the crude residue was added TFA (1.9 mL) and water (95 μL). The reaction mixture was stirred at room temperature for 1.5 h. The solvent was removed under reduced pressure, and the residue was co-evaporated with acetonitrile:toluene [1:1] (2 × 20 mL). The crude mixture was separated by CombiFlash® using silica gel as the stationary phase with 0-10% methanol in DCM. Yield of structure 23b: 36 mg (30.5%). 44 H 57[M+H] calculated for N7O9: 828.97, found 828.90. Synthesis of structure 24b ((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-((S)-2-(4-((4-methylpyridin-2-yl)amino)butanamido)-3-phenylpropanamido)propanoic acid). [ka]

[0451] To a solution of compound 1 (200 mg, 0.679 mmol, 1 equiv.), compound 2 (161 mg, 0.747 mmol, 1.2 equiv.), and TBTU (261 mg, 0.815 mmol, 1.2 equiv.) in anhydrous DMF (4 mL) was added diisopropylethylamine (0.355 mL, 2.038 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NaHCO solution (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 2-3% methanol. LC-MS: [M+H] calculated 456.24, found 456.12. [ka]

[0452] To a solution of compound 1 (300 mg, 0.658 mmol, 1 equiv) in THF (5 mL) and HO (5 mL) was added lithium hydroxide (47 mg, 1.975 mmol, 3 equiv) in portions at 0 °C. The reaction mixture was warmed to room temperature. After stirring at room temperature for 1 h, the reaction mixture was acidified to pH 3.0 with HCl (6 N). The aqueous phase was extracted with ethyl acetate (3 × 10 mL) and the organic layers were combined, dried over NaSO and concentrated. The product was used without further purification. LC-MS: [M+H] calculated 442.23, found 442.08. [ka]

[0453] To a solution of compound 1 (290 mg, 0.656 mmol, 1 equiv.), compound 2 (258 mg, 0.722 mmol, 1.1 equiv.), and TBTU (253 mg, 0.788 mmol, 1.2 equiv.) in anhydrous DMF (5 mL) was added diisopropylethylamine (0.343 mL, 1.970 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NaHCO solution (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 3-4% methanol in DCM. LC-MS: [M+H] calculated 745.35, found 745.63. [ka]

[0454] To a solution of compound 1 (113 mg, 0.151 mmol, 1 equiv.) and azido-PEG5-OTs (126 mg, 0.303 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (99 mg, 0.303 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 3-4% methanol. LC-MS: [M+H]+ calculated 990.49, found 990.87. [ka]

[0455] To a solution of compound 1 (140 mg, 0.141 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (10 mg, 0.424 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for an additional 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. TFA (4 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for an additional 3 h at room temperature. The solvent was removed by rotary evaporation, and the product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 6-10% methanol in DCM. LC-MS: [M+H] calculated 876.42, found 876.88. Synthesis of structure 25b ((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-((S)-3-(benzyloxy)-2-(4-((4-methylpyridin-2-yl)amino)butanamido)propanamido)propanoic acid). [ka]

[0456] To a solution of compound 1 (100 mg, 0.339 mmol, 1 equiv.), compound 2 (92 mg, 0.373 mmol, 1.1 equiv.), and TBTU (131 mg, 0.407 mmol, 1.2 equiv.) in anhydrous DMF (4 mL) was added diisopropylethylamine (0.178 mL, 1.019 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NaHCO solution (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 2-4% methanol. LC-MS: [M+H] calculated 486.25, found 486.37. [ka]

[0457] To a solution of compound 1 (160 mg, 0.329 mmol, 1 equiv) in THF (5 mL) and HO (5 mL) was added lithium hydroxide (23 mg, 0.988 mmol, 3 equiv) in portions at 0 °C. The reaction mixture was warmed to room temperature. After stirring at room temperature for 1 h, the reaction mixture was acidified to pH 3.0 with HCl (6 N). The aqueous phase was extracted with ethyl acetate (3 × 10 mL) and the organic layers were combined, dried over NaSO and concentrated. The product was used without further purification. LC-MS: [M+H] calculated 472.24, found 472.32. [ka]

[0458] To a solution of compound 1 (1600 mg, 0.339 mmol, 1 equiv.), compound 2 (133 mg, 0.373 mmol, 1.1 equiv.), and TBTU (130 mg, 0.815 mmol, 1.2 equiv.) in anhydrous DMF (3 mL) was added diisopropylethylamine (0.177 mL, 1.018 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NaHCO solution (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 2-3% methanol. LC-MS: [M+H] calculated 775.36, found 775.87. [ka]

[0459] To a solution of compound 1 (140 mg, 0.180 mmol, 1 equiv.) and azido-PEG5-OTs (150 mg, 0.361 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (117 mg, 0.361 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 3-4% methanol. LC-MS: [M+H]+ calculated 1020.50, found 1020.88. [ka]

[0460] To a solution of compound 1 (170 mg, 0.166 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (12 mg, 0.499 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for an additional 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. TFA (4 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for an additional 3 h at room temperature. The solvent was removed by rotary evaporation, and the product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 6-10% methanol in DCM. LC-MS: [M+H] calculated 906.43, found 906.95. Synthesis of structure 27b ((S)-3-(3-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid). [ka]

[0461] To a solution of compound 1 (3.0 g, 8.71 mmol, 1 equiv.) and potassium carbonate (1.806 g, 13.073 mmol, 1.5 equiv.) in anhydrous DMF (10 mL) was added methyl iodide (1.085 mL, 17.431 mmol, 2.0 equiv.) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. The reaction was then quenched with water (20 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over anhydrous NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 15% ethyl acetate in hexane. LC-MS: [M+H] calculated 358.06, found 358.15. [ka]

[0462] A mixture of compound 1 (200 mg, 0.558 mmol, 1 equiv.), compound 2 (169 mg, 0.837 mmol, 1.5 equiv.), copper(I) iodide (106 mg, 0.558 mmol, 1 equiv.), potassium carbonate (154 mg, 1.116 mmol, 2 equiv.), and trans-N,N'-dimethylcyclohexane-1,2-diamine (88 μL, 0.558 mmol, 1 equiv.) in anhydrous DMF (5 mL) was refilled with nitrogen three times. The mixture was stirred at 120 °C for 24 h. The mixture was cooled to room temperature and concentrated. The product was separated by CombiFlash® chromatography using silica gel as the stationary phase and eluted with 30-40% ethyl acetate in hexane. LC-MS: [M+H]+ calculated 480.24, found 480.43. [ka]

[0463] Compound 1 (30 mg, 0.0626 mmol, 1.0 equiv) was cooled in an ice bath. HCl in dioxane (0.313 mL, 1.25 mmol, 20 equiv) was added to the flask. The reaction was allowed to warm to room temperature and stirred for an additional hour. The solvent was removed by rotary evaporation, and the product was used directly without further purification. LC-MS: [M+H]+ calculated 380.19, found 380.33. [ka]

[0464] To a solution of compound 1 (10 mg, 0.0571 mmol, 1 equiv.), compound 2 (26 mg, 0.0628 mmol, 1.1 equiv.), and TBTU (22 mg, 0.0685 mmol, 1.2 equiv.) in anhydrous DMF (1 mL) was added diisopropylethylamine (0.030 mL, 0.171 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NaHCO3 solution (5 mL), and the aqueous phase was extracted with ethyl acetate (3 x 5 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 3-4% methanol in DCM. LC-MS: [M+H]+ calculated 537.26, found 537.41. [ka]

[0465] Compound 1 (30 mg, 0.0626 mmol, 1.0 equiv) was cooled in an ice bath. HCl in dioxane (0.313 mL, 1.25 mmol, 20 equiv) was added to the flask. The reaction was allowed to warm to room temperature and stirred for an additional hour. The solvent was removed by rotary evaporation, and the product was used directly without further purification. LC-MS: [M+H]+ calculated 437.21, found 437.31. [ka]

[0466] To a solution of compound 1 (20 mg, 0.0569 mmol, 1 equiv.), compound 2 (26 mg, 0.0626 mmol, 1.1 equiv.), and TBTU (22 mg, 0.0683 mmol, 1.2 equiv.) in anhydrous DMF (2 mL) was added diisopropylethylamine (0.03 mL, 0.170 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NaHCO solution (5 mL), and the aqueous phase was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 4-5% methanol in DCM. LC-MS: [M+H] calculated 713.36, found 713.85. [ka]

[0467] To a solution of compound 1 (0.033 g, 0.0463 mmol, 1 equiv.) in ethyl acetate (10 mL) was added 10% Pd / C (20 mg) at room temperature. The reaction mixture was stirred with hydrogen gas at room temperature overnight. The catalyst was removed by filtration through Celite®, and the product was used directly without further purification. LC-MS: [M+H]+ calculated 623.31, found 623.56. [ka]

[0468] To a solution of compound 1 (16 mg, 0.0257 mmol, 1 equiv.) and azido-PEG5-OTs (22 mg, 0.0514 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (17 mg, 0.0514 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 3-4% methanol. LC-MS: [M+H]+ calculated 868.45, found 868.96. [ka]

[0469] Compound 1 (5 mg, 0.0058 mmol, 1.0 equiv.) in THF (1 mL) and To a solution of 1 mL of HCl in water (1 mL) was added lithium hydroxide (1 mg, 0.0346 mmol, 6.0 equiv.) at room temperature. The mixture was stirred for another 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N) and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (1 mL) and DCM (1 mL) were added to the residue and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation. LC-MS: [M+H]+ calculated 754.38, found 755. Synthesis of structure 29b ((S)-3-(4-(3-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid). [ka]

[0470] Compound 1 (100 mg, 0.169 mmol, 1.0 equiv.), compound 2 (68 mg, 0.253 mmol, 1.5 equiv.), XPhos Pd G2 (3 mg, 0.0034 mmol, 0.02 equiv.), and K3PO4 (72 mg, 0.338 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a septum-equipped screw cap, then evacuated and refilled with nitrogen (this process was repeated a total of three times). THF (5 mL) and water (1 mL) were then added via syringe. Nitrogen was bubbled through the mixture for 10 minutes, and the reaction was maintained at 40 °C for 2 hours. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The compounds were separated by CombiFlash® using silica gel as stationary phase and eluted with DCM containing 4% methanol. LC-MS: [M+H]+ calculated 655.31, found 656. [ka]

[0471] To a solution of compound 1 (100 mg, 0.152 mmol, 1 equiv.) and azido-PEG5-OTs (127 mg, 0.305 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (100 mg, 0.305 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with 3-4% methanol in DCM. LC-M S: [M+H]+ calculated value 900.44, found value 901. [ka]

[0472] To a solution of compound 1 (125 mg, 0.138 mmol, 1.0 equiv.) in THF (1 mL) and water (1 mL) was added lithium hydroxide (10 mg, 0.416 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (3 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation. The product was used directly without further purification. LC-MS: [M+H]+ calculated 786.37, found 787. Synthesis of structure 30b ((S)—N-(1-azido-21-(4-(naphthalen-1-yl)phenyl)-19,23-dioxo-3,6,9,12,15-pentaoxa-18,22-diazatetracosan-24-yl)-4-((4-methylpyridin-2-yl)amino)butanamide). [ka]

[0473] Compound 1 (100 mg, 0.169 mmol, 1.0 equiv.), compound 2 (43 mg, 0.253 mmol, 1.5 equiv.), XPhos Pd G2 (3 mg, 0.0034 mmol, 0.02 equiv.), and K3PO4 (72 mg, 0.338 mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a septum-equipped screw cap, then evacuated and refilled with nitrogen (this process was repeated a total of three times). THF (5 mL) and water (1 mL) were then added via syringe. Nitrogen was bubbled through the mixture for 10 minutes, and the reaction was maintained at 40 °C for 2 hours. The reaction was quenched with water (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The compounds were separated by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 3-4% methanol. LC-MS: [M+H]+ calculated 639.31, found 640. [ka]

[0474] To a solution of compound 1 (90 mg, 0.140 mmol, 1 equiv) in THF (5 mL) and HO (5 mL) was added lithium hydroxide (10 mg, 0.422 mmol, 3 equiv) in portions at 0 °C. The reaction mixture was warmed to room temperature. After stirring at room temperature for 1 h, the reaction mixture was acidified to pH 3.0 with HCl (6 N). The aqueous phase was extracted with ethyl acetate (3 × 10 mL) and the organic layers were combined, dried over NaSO and concentrated. The product was used without further purification. LC-MS: [M+H] calculated 625.29, found 625.36. [ka]

[0475] To a solution of compound 1 (88 mg, 0.140 mmol, 1 equiv.), compound 2 (48 mg, 0.154 mmol, 1.1 equiv.), and TBTU (54 mg, 0.169 mmol, 1.2 equiv.) in anhydrous DMF (3 mL) was added diisopropylethylamine (0.074 mL, 0.422 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NaHCO solution (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 4-6% methanol in DCM. LC-MS: [M+H] calculated 913.47, found 913.70. [ka]

[0476] To a solution of compound 1 (93 mg, 0.101 mmol, 1.0 equiv.) in DCM (2 mL) was added TFA (3 mL), and the mixture was stirred at room temperature for an additional 3 h. The solvent was removed by rotary evaporation, and the product was separated by CombiFlash® using silica gel as the stationary phase. The product was eluted with 10-12% methanol in dichloromethane. LC-MS: [M+H]+ calculated 813.42, found 813.68. Synthesis of structure 31b ((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-((S)-3-hydroxy-2-(4-((4-methylpyridin-2-yl)amino)butanamido)propanamido)propanoic acid). [ka]

[0477] To a solution of compound 1 (150 mg, 0.509 mmol, 1 equiv.), compound 2 (87 mg, 0.560 mmol, 1.1 equiv.), and TBTU (196 mg, 0.196 mmol, 1.2 equiv.) in anhydrous DMF (3 mL) was added diisopropylethylamine (0.074 mL, 0.422 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NaHCO solution (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 4-6% methanol in DCM. LC-MS: [M+H] calculated 396.21, found 396.17. [ka]

[0478] To a solution of compound 1 (196 mg, 0.495 mmol, 1 equiv) in THF (5 mL) and HO (5 mL) was added lithium hydroxide (35 mg, 1.486 mmol, 3 equiv) in portions at 0 °C. The reaction mixture was warmed to room temperature. After stirring at room temperature for 1 h, the reaction mixture was acidified to pH 3.0 with HCl (6 N). The aqueous phase was extracted with ethyl acetate (3 × 10 mL) and the organic layers were combined, dried over NaSO and concentrated. The product was used without further purification. LC-MS: [M+H] calculated 382.19, found 382.13. [ka]

[0479] To a solution of compound 1 (189 mg, 0.495 mmol, 1 equiv.), compound 2 (195 mg, 0.545 mmol, 1.1 equiv.), and TBTU (190 mg, 0.595 mmol, 1.2 equiv.) in anhydrous DMF (5 mL) was added diisopropylethylamine (0.259 mL, 1.486 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NaHCO solution (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 4-6% methanol in DCM. LC-MS: [M+H] calculated 685.32, found 685.58. [ka]

[0480] A solution of compound 1 (75 mg, 0.109 mmol, 1 equiv.) and azido-PEG5-OTs (91 mg, 0.219 mmol, 2 equiv.) in anhydrous DMF (2 mL) was treated with Cs 2CO3 (71 mg, 0.219 mmol, 2 equiv.) was added at room temperature. The reaction mixture was stirred at 40°C overnight. The reaction was quenched with saturated NaHCO3 solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3 x 10 mL). The organic phases were combined, dried over Na2SO4 and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 4% methanol. The yield is 29%. LC-MS: [M+H]+ calculated 930.45, found 930.90. [ka]

[0481] To a solution of compound 1 (30 mg, 0.0323 mmol, 1.0 equiv.) in THF (1 mL) and water (1 mL) was added lithium hydroxide (2.3 mg, 0.0968 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (2 mL) and DCM (1 mL) were added to the residue, and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation, and the product was separated by CombiFlash® using silica gel as the stationary phase. The product was eluted with 12-15% methanol in dichloromethane. LC-MS: [M+H]+ calculated 816.39, found 816.92. Synthesis of structure 32b ((S)-4-(((S)-1-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-2-carboxyethyl)amino)-3-(4-((4-methylpyridin-2-yl)amino)butanamide)-4-oxobutanoic acid). [ka]

[0482] To a solution of compound 1 (100 mg, 0.404 mmol, 1 equiv.), compound 2 (160 mg, 0.444 mmol, 1.1 equiv.), and TBTU (155 mg, 0.485 mmol, 1.2 equiv.) in anhydrous DMF (2 mL) was added diisopropylethylamine (0.211 mL, 1.213 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® chromatography using silica gel as the stationary phase. ) and eluted with DCM containing 2-3% methanol. LC-MS: [M+H]+ calculated 551.23, found 551.45. [ka]

[0483] Compound 1 (0.164 g, 0.297 mmol, 1.0 equiv) was cooled in an ice bath. HCl in dioxane (0.745 mL, 2.978 mmol, 10 equiv) was added to the flask. The reaction was allowed to warm to room temperature and stirred for an additional hour. The solvent was removed by rotary evaporation, and the product was used directly without further purification. LC-MS: [M+H]+ calculated 451.18, found 451.35. [ka]

[0484] To a solution of compound 1 (100 mg, 0.404 mmol, 1 equiv.), compound 2 (160 mg, 0.444 mmol, 1.1 equiv.), and TBTU (155 mg, 0.485 mmol, 1.2 equiv.) in anhydrous DMF (2 mL) was added diisopropylethylamine (0.211 mL, 1.213 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NaHCO solution (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 3-5% methanol in DCM. LC-MS: [M+H] calculated 727.33, found 727.53. [ka]

[0485] To a solution of compound 1 (150 mg, 0.206 mmol, 1 equiv.) and azido-PEG5-OTs (172 mg, 0.412 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (134 mg, 0.412 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred overnight at room temperature. The reaction was quenched with saturated NaHCO3 solution (10 mL), and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 4% methanol. The yield is 29%. LC-MS: [M+H]+ calculated 940.45, found 940.71. [ka]

[0486] To a solution of compound 1 (30 mg, 0.0344 mmol, 1.0 equiv.) in THF (1 mL) and water (1 mL) was added lithium hydroxide (2.5 mg, 0.103 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another hour at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (2 mL) and DCM (1 mL) were added to the residue, and the mixture was stirred for another 3 hours at room temperature. The solvent was removed by rotary evaporation, and the product was separated by CombiFlash® using silica gel as the stationary phase. The product was eluted with 20% methanol in dichloromethane. LC-MS: [M+H]+ calculated 844.38, found 844.56. Synthesis of structure 33b ((S)-3-((S)-6-amino-2-(4-((4-methylpyridin-2-yl)amino)butanamido)hexanamido)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)propanoic acid). [ka]

[0487] To a solution of compound 1 (150 mg, 0.509 mmol, 1 equiv.), compound 2 (166 mg, 0.560 mmol, 1.1 equiv.), and TBTU (196 mg, 0.611 mmol, 1.2 equiv.) in anhydrous DMF (3 mL) was added diisopropylethylamine (0.266 mL, 1.528 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NaHCO solution (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 3-5% methanol in DCM. LC-MS: [M+H] calculated 537.32, found 537.23. [ka]

[0488] To a solution of compound 1 (230 mg, 0.428 mmol, 1 equiv) in THF (5 mL) and HO (5 mL) was added lithium hydroxide (31 mg, 1.285 mmol, 3 equiv) in portions at 0 °C. The reaction mixture was warmed to room temperature. After stirring at room temperature for 1 h, the reaction mixture was acidified to pH 3.0 with HCl (6 N). The aqueous phase was extracted with ethyl acetate (3 × 10 mL) and the organic layers were combined, dried over NaSO and concentrated. The product was used without further purification. LC-MS: [M+H] calculated 523.31, found 523.55. [ka]

[0489] To a solution of compound 1 (230 mg, 0.440 mmol, 1 equiv.), compound 2 (173 mg, 0.484 mmol, 1.1 equiv.), and TBTU (170 mg, 0.528 mmol, 1.2 equiv.) in anhydrous DMF (2 mL) was added diisopropylethylamine (0.230 mL, 1.320 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NaHCO solution (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 4-6% methanol in DCM. LC-MS: [M+H] calculated 826.43, found 826.65. [ka]

[0490] To a solution of compound 1 (150 mg, 0.181 mmol, 1 equiv.) and azido-PEG5-OTs (113 mg, 0.272 mmol, 1.5 equiv.) in anhydrous DMF (2 mL) was added Cs2CO3 (118 mg, 0.363 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. The reaction was quenched with saturated NaHCO3 solution (5 mL), and the aqueous layer was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFla column chromatography using silica gel as the stationary phase. Purified by sh® eluting with DCM containing 4% methanol. Yield: 66%. LC-MS: [M+H]+ calculated 1071.57, found 1071.89. [ka]

[0491] To a solution of compound 1 (130 mg, 0.121 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (8.7 mg, 0.364 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another hour at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. TFA (3 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for another 3 hours at room temperature. The solvent was removed by rotary evaporation, and the product was separated by CombiFlash® using silica gel as the stationary phase. The product was eluted with 20% methanol in dichloromethane. LC-MS: [M+H]+ calculated 857.45, found 857.64. Synthesis of structure 34b ((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-((S)-4-methyl-2-(4-((4-methylpyridin-2-yl)amino)butanamido)pentanamido)propanoic acid). [ka]

[0492] To a solution of compound 1 (150 mg, 0.509 mmol, 1 equiv.), compound 2 (101 mg, 0.560 mmol, 1.1 equiv.), and TBTU (196 mg, 0.611 mmol, 1.2 equiv.) in anhydrous DMF (3 mL) was added diisopropylethylamine (0.266 mL, 1.528 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NaHCO solution (5 mL), and the aqueous phase was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with 3-5% methanol in DCM. LC-MS: [M+H] calculated 422.26, found 422.36. [ka]

[0493] To a solution of compound 1 (186 mg, 0.441 mmol, 1 equiv) in THF (3 mL) and HO (3 mL) was added lithium hydroxide (31 mg, 1.323 mmol, 3 equiv) in portions at 0 °C. The reaction mixture was warmed to room temperature. After stirring at room temperature for 1 h, the reaction mixture was acidified to pH 3.0 with HCl (6 N). The aqueous phase was extracted with ethyl acetate (3 × 10 mL) and the organic layers were combined, dried over NaSO and concentrated. The product was used without further purification. LC-MS: [M+H] calculated 408.24, found 408.23. [ka]

[0494] To a solution of compound 1 (168 mg, 0.412 mmol, 1 equiv.), compound 2 (162 mg, 0.453 mmol, 1.1 equiv.), and TBTU (159 mg, 0.494 mmol, 1.2 equiv.) in anhydrous DMF (2 mL) was added diisopropylethylamine (0.215 mL, 1.237 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NaHCO solution (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 2-4% methanol. LC-MS: [M+H] calculated 711.37, found 711.69. [ka]

[0495] A solution of compound 1 (150 mg, 0.206 mmol, 1 equiv.) and azido-PEG5-OTs (132 mg, 0.317 mmol, 1.5 equiv.) in anhydrous DMF (2 mL) To this was added Cs2CO3 (137 mg, 0.422 mmol, 2 equiv.) at room temperature. The reaction mixture was stirred at 40 °C for 3 h. The reaction was quenched with saturated NaHCO3 solution (10 mL) and the aqueous layer was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and eluted with DCM containing 3-4% methanol. The yield is 82%. LC-MS: [M+H]+ calculated 956.51, found 956.64. [ka]

[0496] To a solution of compound 1 (160 mg, 0.167 mmol, 1.0 equiv.) in THF (2 mL) and water (2 mL) was added lithium hydroxide (12 mg, 0.502 mmol, 3.0 equiv.) at room temperature. The mixture was stirred for another 1 h at room temperature. The pH was adjusted to 3.0 with HCl (6 N), and the aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined, dried over NaSO, and concentrated. TFA (3 mL) and DCM (2 mL) were added to the residue, and the mixture was stirred for another 3 h at room temperature. The solvent was removed by rotary evaporation, and the product was separated by CombiFlash® using silica gel as the stationary phase. The product was eluted with 8-10% methanol in dichloromethane. LC-MS: [M+H] calculated 842.44, found 842.67. Synthesis of structure 35b ((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-((2S,3R)-3-hydroxy-2-(4-((4-methylpyridin-2-yl)amino)butanamido)butanamido)propanoic acid). [ka]

[0497] To a vial containing L-Threonine-OMe HCl (1.000 g, 5.896 mmol, 1.3 equiv.), compound 1 (1.335 g, 4.535 mmol, 1 equiv.), dimethylaminopyridine (0.277 g, 2.268 mmol, 0.5 equiv.), and CHCl (13.3 mL) were added. To this mixture, diisopropylamine (2.054 mL, 11.792 mmol, 2.6 equiv.) was added, and the resulting solution was cooled to 0 °C. EDC·HCl (1.130 g, 5.896 mmol, 1.3 equiv.) was added, and the reaction was stirred at 0 °C for 30 min before warming to room temperature. The reaction was judged complete by HPLC after 16 h and was transferred to a separatory funnel and washed with 66% saturated NH4Cl (4 × 20 mL) and saturated NH4Cl (20 mL). The organic layer was dried over Na2SO4 and concentrated to give a viscous oil (1.7588 g, 94.7%), which was used directly in the next step. LC-MS :[M+H] + Calculated value: 410.22, measured value: 410.03 [ka]

[0498] Compound 1 was dissolved in MeOH (4.5 mL), and to this mixture was added 2.0 M LiOH (9.1 mL). The reaction was stirred for 1.5 hours and concentrated to remove MeOH. The mixture was then acidified with 20% KHSO to pH = 4 and extracted with EtOAc (3 x 15 mL). The combined organics were washed with brine (20 mL), dried over NaSO, and concentrated to give 3 as a solid (1.5095 g, 88.9% yield). LC-MS: [M-H] - Calculated value: 394.21, measured value: 394.37. [ka] [ka]

[0499] A vial was charged with compound 1 (0.200 g, 0.506 mmol, 1 equiv.), TBTU (0.195 g, 0.607 mmol, 1.2 equiv.), DMF (2.0 mL), and DIEA (0.264 mL, 1.517 mmol, 3.0 equiv.). The reaction was stirred for 2 minutes, after which 2 (0.253 g, 0.708 mmol, 1.4 equiv.) was added. Upon completion, the reaction was diluted with saturated aqueous NaHCO3 (10 mL) and extracted with EtOAc (3 x 5 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, and concentrated. The crude material was purified by column chromatography eluting with 0-20% MeOH in CHCl2 to give the product (150.8 mg, 42.7% yield). LC-MS: [M+H] + Calculated value: 699.33, measured value: 699.53 [ka]

[0500] To a vial containing compound 1 (0.151 g, 0.216 mmol, 1 equiv.) was added Cs2CO3 (0.106 g, 0.324 mmol, 1.5 equiv.) and DMF (1.9 mL). N3-PEG5-OTs (0.135 g, 0.324 mmol, 1.5 equiv.) was added to the mixture, and the reaction was stirred at 40 °C. Upon completion, the reaction was diluted with EtOAc (10 mL), saturated aqueous NaHCO3 (5 mL), and water (5 mL). The layers were separated and aqueous extracted with EtOAc for a total of 3 × 10 mL. The combined organic layers were dried over Na2SO4 and concentrated. The crude material was purified by column chromatography eluting with 0-20% MeOH in CHCl2 to give the product (103 mg, 50.4% yield). LC-MS: [M+H] + Calculated value: 944.47, measured value: 944.56 [ka]

[0501] To a vial containing compound 1 (0.103 g, 0.109 mmol, 1 equiv.) was added MeOH (1.5 mL) and 2.0 M LiOH (2.0 mL). The reaction was stirred at room temperature, then concentrated to remove MeOH and acidified to pH=2 with 20% KHSO4. To this mixture was added EtOAc (5 mL) and water (4 mL). The aqueous layer was extracted with EtOAc (3×5 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, and concentrated to give the product (0.0879 g, 86.9%). LC-MS: [M+H] + Calculated value: 930.45, measured value: 930.56. [ka]

[0502] To a vial containing compound 1 (0.0879 g, 0.0945 mmol, 1 equiv.) was added CHCl (0.3 mL) and trifluoroacetic acid (0.64 mL). The solution was stirred at room temperature. Upon completion (>97% product), the reaction was concentrated and coevaporated with toluene (3 mL) followed by acetonitrile (2 × 3 mL). The product was obtained (115.6 mg) with residual TFA. Synthesis of structure 36b ((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-((2S,3S)-3-methyl-2-(4-((4-methylpyridin-2-yl)amino)butanamido)pentanamido)propanoic acid). [ka]

[0503] To a vial containing L-isoleucine-OMe HCl (1.000 g, 5.505 mmol, 1.3 equiv.), compound 1 (1.246 g, 4.234 mmol, 1 equiv.), dimethylaminopyridine (0.259 g, 2.117 mmol, 0.5 equiv.), and CHCl (12.5 mL) were added. To this mixture, diisopropylamine (2.054 mL, 11.792 mmol, 2.6 equiv.) was added, and the resulting solution was cooled to 0 °C. EDC·HCl (1.055 g, 5.505 mmol, 1.3 equiv.) was added, and the reaction was stirred at 0 °C for 30 min before warming to room temperature. The reaction was judged complete by HPLC after 16 h and was transferred to a separatory funnel and washed with 66% saturated NH4Cl (4 × 20 mL) and saturated NH4Cl (1 × 20 mL). The organic layer was dried over Na2SO4 and concentrated to give a viscous oil (1.8634 g, wet with CH2Cl2) which was used directly in the next step. LC-MS: [M+H] + Calculated value: 422.26, actual value: 422.00. [ka]

[0504] Compound 1 was dissolved in MeOH (4.2 mL), and to this mixture was added 2.0 mL of LiOH (8.5 mL). The reaction was stirred for 1.5 hours and concentrated to remove MeOH. The mixture was then acidified with 20% KHSO to pH=4 and extracted with EtOAc (3×15 mL). The combined organics were washed with brine (20 mL), dried over NaSO, and concentrated to give the product as a viscous oil (1.6123 g, 93.4% yield for two steps). LC-MS: [M-H] - Calculated value: 406.24, measured value: 406.43. [ka] [ka]

[0505] A vial was charged with compound 1 (0.200 g, 0.491 mmol, 1 equiv.), TBTU (0.189 g, 0.589 mmol, 1.2 equiv.), DMF (2.0 mL), and DIEA (0.256 mL, 1.472 mmol, 3.0 equiv.). The reaction was stirred for 2 minutes, after which 2 (0.246 g, 0.687 mmol, 1.4 equiv.) was added. Upon completion, the reaction was diluted with saturated aqueous NaHCO3 (10 mL) and extracted with EtOAc (3 x 5 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, and concentrated. The crude material was purified by column chromatography eluting with 0-20% MeOH in CHCl2 to give the product (0.3024 mg, 86.7% yield). LC-MS: [M+H] + Calculated value: 711.37, measured value 711.51. [ka]

[0506] To a vial containing compound 1 (0.170 g, 0.238 mmol, 1 equiv.) was added Cs2CO3 (0.116 g, 0.358 mmol, 1.5 equiv.) and DMF (2.1 mL). N3-PEG5-OTs (0.149 g, 0.358 mmol, 1.5 equiv.) was added to the mixture, and the reaction was stirred at 40 °C. Upon completion, the reaction was diluted with EtOAc (10 mL), saturated aqueous NaHCO3 (5 mL), and water (5 mL). The layers were separated and aqueous extracted with EtOAc for a total of 3 × 10 mL. The combined organic layers were dried over Na2SO4 and concentrated. The crude material was purified by column chromatography eluting with 0-20% MeOH in CHCl2 to give the product (0.1645 g, 72.1% yield). LC-MS: [M+H] + Calculated value: 956.51, measured value: 956.78. [ka]

[0507] To a vial containing compound 1 (0.164 g, 0.172 mmol, 1 equiv.) was added MeOH (2.0 mL) and 2.0 M LiOH (3.0 mL). The reaction was stirred at room temperature and monitored by HPLC. Additional LiOH (33 mg, 1.38 mmol, 8 equiv.), water (5 mL), and MeOH (4 mL) were required to dissolve the material and drive the reaction forward. HPLC revealed the formation of two new peaks, believed to be diastereomers. Upon reaching >94% conversion, the reaction was concentrated to remove MeOH and acidified to pH = 2 with 20% KHSO4. To this mixture was added EtOAc (5 mL) and water (4 mL). The aqueous layer was extracted with EtOAc (4 x 5 mL). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, and concentrated to give the product (0.1417 g, 87.4%). LC-MS: [M+H] + Calculated value: 942.49, measured value: 942.56. [ka]

[0508] To a vial containing compound 1 (0.1417 g, 0.1504 mmol, 1 equiv.) was added CHCl (0.5 mL) and trifluoroacetic acid (1.0 mL). The solution was stirred at room temperature. Upon completion (>97% product), the reaction was concentrated and co-evaporated with toluene (3 mL) followed by acetonitrile (2×3 mL). The product was obtained (150.3 mg) with residual TFA. Two peaks were present, representing both the starting material and the product. LC-MS: [M+H] + Calculated: 842.44, Found: 842.56. Both product peaks were found to be identical in mass, indicating the presence of diastereomers. Synthesis of structure 37b ((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-((R)-3-methyl-2-(4-((4-methylpyridin-2-yl)amino)butanamido)butanamido)propanoic acid). [ka]

[0509] To a solution of compound 1 (150 mg, 0.509 mmol, 1 equiv.), compound 2 (94 mg, 0.560 mmol, 1.1 equiv.), and TBTU (196 mg, 0.611 mmol, 1.2 equiv.) in anhydrous DMF (3 mL) was added diisopropylethylamine (0.266 mL, 1.528 mmol, 3 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NaHCO solution (10 mL), and the aqueous phase was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over NaSO, and concentrated. The product was isolated by CombiFlash® and eluted with 2-3% methanol in DCM. Yield: 205 mg (99%). [ka]

[0510] To a solution of compound 1 (207 mg, 0.508 mmol, 1 equiv.) in THF (5 mL) and HO (5 mL) was added lithium hydroxide (36 mg, 1.523 mmol, 3 equiv.) in portions at 0 °C. The reaction mixture was warmed to room temperature. After stirring at room temperature for 1 h, the reaction mixture was acidified to pH 3.0 with HCl (6 N). The aqueous phase was extracted with ethyl acetate (3 × 10 mL), and the organic layers were combined, dried over NaSO, and concentrated. The product was used without further purification. Yield: 180 mg (91%). [ka]

[0511] To a solution of compound 3 (180 mg, 0.46 mmol), compound 3a (180 mg, 0.50 mmol), and TBTU (176 mg, 0.55 mmol) in DMF (2.5 mL) was added DIPEA (177 mg, 239 μL, 1.37 mmol) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 1 h. The reaction mixture was quenched with saturated NH4Cl(aqueous) solution (1.75 mL) and deionized water (1.75 mL) and then extracted with ethyl acetate (8 mL). The aqueous layer was further extracted with ethyl acetate (2 × 8 mL). The combined organic phases were washed with half-saturated NH4Cl(aqueous) solution (6 mL) and half-saturated NaHCO3(aqueous) solution (6 mL). The organic layer was dried over Na2SO4, filtered, and concentrated. The crude mixture was separated by CombiFlash® using silica gel as the stationary phase with 0-5% methanol in DCM. Yield of compound 4: 295 mg (92%). 40 H 48 Calculated [M+H]+ for N4O7: 697.84, found: 697.82.

[0512] To a solution of compound 4 (200 mg, 0.29 mmol) and azido-PEG5-OTs (240 mg, 0.57 mmol) in anhydrous DMF (2.5 mL) was added Cs2CO3 (187 mg, 0.57 mmol). The reaction mixture was stirred at 60°C for 2 hours. The reaction mixture was quenched with saturated NaHCO3 (aqueous) solution (15 mL) and deionized water (7.5 mL) and then extracted with ethyl acetate (10 mL). The aqueous layer was further extracted with ethyl acetate (2 x 10 mL). The combined organic phases were dried over Na2SO4, filtered, and concentrated. The crude mixture was separated by CombiFlash® using silica gel as the stationary phase with 0-5% methanol in DCM. Yield of compound 5: 97 mg (36%). C 50 H 67 N7O 11 Calculated [M+H]+ for: 943.15, Found: 942.96.

[0513] To a solution of compound 5 (94 mg, 0.10 mmol) in THF (1.5 mL) and deionized water (1 mL) was added a solution of lithium hydroxide (7.2 mg, 0.30 mmol) in deionized water (0.5 mL). The reaction mixture was stirred for 1 h and then acidified to pH = 3 with 6 M HCl (aq). The aqueous phase was extracted with ethyl acetate (3 × 5 mL). The combined organic phase was dried over Na2SO4, filtered, and concentrated. To this crude residue, TFA (1.34 mL) and water (67 μL) were added. The reaction mixture was stirred at room temperature for 1.5 hours. The solvent was removed under reduced pressure, and the residue was co-evaporated with acetonitrile:toluene [1:1] (2 × 20 mL). The crude mixture was separated by CombiFlash® using silica gel as the stationary phase with 0-10% methanol in DCM. Yield of structure 37b: 44 mg (53%). C 44 H 57 [M+H]+ calculated for N7O9: 828.97, found: 828.63. Example 2. Synthesis of tridentate αvβ6 integrin ligands and conjugation of αvβ6 integrin ligands to cargo molecules (RNAi agents).

[0514] The αvβ6 integrin ligand can be linked to one or more RNAi agents, which are useful for inhibiting the expression of one or more targeted genes. The αvβ6 integrin ligand facilitates the delivery of the RNAi agent to targeted cells and / or tissues. Above Example 1 describes the synthesis of certain αvβ6 integrin ligands disclosed herein. The following describes the general synthesis procedure of certain αvβ6 integrin ligand-RNAi agent conjugates, which are shown in the non-limiting examples described herein.

[0515] A. Synthesis of RNAi Agents. RNAi agents can be synthesized using methods commonly known in the art. For the synthesis of the RNAi agents shown in the examples described herein, the sense and antisense strands of the RNAi agents were synthesized according to the solid-phase phosphoramidite technology used in oligonucleotide synthesis. Depending on the scale, a MerMade 96E® (Bioautomation), a MerMade 12® (Bioautomation), or an OP Pilot 100 (GE Healthcare) was used. Synthesis was performed on a solid support made of controlled pore glass (CPG, 500 Å or 600 Å, obtained from Prime Synthesis, Aston, PA, USA). All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the following 2'-O-methyl phosphoramidite was used: (5'-O-dimethoxytrityl-N 6 -(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, 5'-O-dimethoxy-trityl-N 4 -(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino)phosphoramidite, (5'-O-dimethoxytrityl-N 2-(Isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, and 5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite. The 2'-deoxy-2'-fluoro-phosphoramidite possessed the same protecting groups as the 2'-O-methyl-RNA amidite. 5'-Dimethoxytrityl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was purchased from Glen Research (Virginia). Inverted abasic (3'-O-dimethoxytrityl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was purchased from ChemGenes (Wilmington, MA, USA). The following UNA phosphoramidites were used: 5'-(4,4'-dimethoxytrityl)-N6-(benzoyl)-2',3'-seco-adenosine, 2'-benzoyl 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-acetyl-2',3'-seco-cytosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-isobutyryl-2',3'-seco-guano ... 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-(4,4'-dimethoxy-trityl)-2',3'-seco-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. TFA amino-linked phosphoramidites were also purchased (ThermoFisher).

[0516] In some examples, the αβ integrin ligands disclosed herein are attached to RNAi agents by linking the building blocks to a scaffold that includes a trialkyne group. In some examples, the trialkyne group is added using a trialkyne-containing phosphoramidite that can be added to the 5' end of the sense strand of the RNAi agent. When used in conjunction with the RNAi agents described herein, trialkyne-containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other amidites were dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3 Å) were added. 5-benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used as the activator solution. Coupling times were 10 minutes (RNA), 90 seconds (2'O-Me), and 60 seconds (2'F). To introduce phosphorothioate linkages, a solution of 100 mM 3-phenyl-1,2,4-dithiazolin-5-one (POS, available from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was used.

[0517] Alternatively, when linking an αvβ6 integrin ligand to an RNAi agent via a trialkyne scaffold, trialkyne-containing compounds can be introduced post-synthetically instead of using a phosphoramidite approach (see, e.g., Section E below). When used in conjunction with an RNAi agent as illustrated in certain embodiments herein, when a trialkyne group is attached to the 5' end of the sense strand post-synthetically, the 5'-terminal nucleotide of the sense strand was functionalized with a nucleotide containing a primary amine at the 5' end to facilitate attachment to the trialkyne-containing scaffold. The TFA amino-linked phosphoramidite was dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3 Å) were added. 5-benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used as the activator solution. Coupling times were 10 minutes (RNA), 90 seconds (2'O-Me), and 60 seconds (2'F). To introduce phosphorothioate linkages, a solution of 100 mM 3-phenyl-1,2,4-dithiazolin-5-one (POS, available from PolyOrg, Inc., Leominster, Mass., USA) in anhydrous acetonitrile was used.

[0518] B. Cleavage and deprotection of support-bound oligomers. After completion of solid-phase synthesis, the dry solid support was treated with a 1:1 volume solution of 40 wt% aqueous methylamine and 28%-31% ammonium hydroxide solution (Aldrich) for 1.5 hours at 30°C. The solution was evaporated, and the solid residue was reconstituted in water (see below).

[0519] C. Purification. The crude oligomer was purified by anion-exchange HPLC using a TSKgel SuperQ-5PW 13 μm column and a Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA (pH 9.0) containing 20% ​​acetonitrile, and buffer B was the same as buffer A but with the addition of 1.5 M sodium chloride. UV traces at 260 nm were recorded. Appropriate fractions were pooled and then purified by size-exclusion HPLC using a GE Healthcare XK 16 / 40 column packed with Sephadex G-25 fine, with a running buffer of 100 mM ammonium bicarbonate (pH 6.7) and 20% acetonitrile or filtrate. I used it to drive.

[0520] D. Annealing. Complementary strands were mixed to form RNAi agents by combining equimolar RNA solutions (sense and antisense) in 1x PBS (phosphate-buffered saline, 1x, Corning, Cellgro). Some RNAi agents were lyophilized and stored at -15 to -25°C. The concentration of the duplex was determined by measuring the absorbance of the solution in 1x PBS with a UV-Vis spectrometer. The absorbance of the solution at 260 nm was then multiplied by the conversion factor and dilution factor to determine the duplex concentration. The conversion factor was 0.037 mg / (mL·cm), or, depending on the experiment, the conversion factor was calculated from the experimentally determined extinction coefficient.

[0521] E. Trialkyne Scaffold Attachment. Either before or after annealing, the 5' or 3' amine-functionalized sense strand of the RNAi agent can be attached to the trialkyne scaffold. Exemplary trialkyne scaffold structures that can be used in forming the constructs disclosed herein include: [ka] [ka]

[0522] The conjugation of the trialkyne scaffold to the annealed duplex is described below: The amine-functionalized duplex was dissolved in 90% DMSO / 10% HO at approximately 50-70 mg / mL. 40 equivalents of triethylamine were added, followed by 3 equivalents of trialkyne-PNP. Upon completion, the conjugate was precipitated twice in a solvent system of 1x phosphate-buffered saline / acetonitrile (1:14 ratio) and dried.

[0523] F. Attachment of αvβ6 integrin ligand. A 5' or 3' tridentate alkyne-functionalized sense strand was attached to the αvβ6 integrin ligand either before or after annealing. The following example describes the attachment of the αvβ6 integrin ligand to the annealed duplex: Stock solutions of 0.5 M Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 0.5 M copper(II) sulfate pentahydrate (Cu(II)SO4·5 HO), and 2 M sodium ascorbate solution were prepared in deionized water. A 75 mg / mL DMSO solution of the αvβ6 integrin ligand was made. To a 1.5 mL centrifuge tube containing the trialkyne-functionalized duplex (3 mg, 75 μL, 40 mg / mL in deionized water, approximately 15,000 g / mol), 25 μL of 1 M Hepes (pH 8.5) buffer was added. After vortexing, 35 μL of DMSO was added and the solution was vortexed. αvβ6 integrin ligand was added to the reaction (6 equivalents / duplex, 2 equivalents / alkyne, approximately 15 μL) and the solution was vortexed. The pH was checked using pH paper and confirmed to be approximately pH 8. In a separate 1.5 mL centrifuge tube, 50 μL of 0.5 M THPTA was mixed with 10 μL of 0.5 M Cu(II)SO4·5H2O, vortexed, and incubated at room temperature for 5 minutes. After 5 minutes, THPTA / Cu solution (7.2 μL, 6 equivalents 5:1 THPTA:Cu) was added to the reaction vial and vortexed. Immediately after, 2 M ascorbate (5 μL, 50 equivalents / duplex, 16.7 equivalents / alkyne) was added. The reaction mixture was added to the reaction vial and vortexed. Upon completion of the reaction (typically within 0.5-1 h), the reaction mixture was immediately purified by native anion exchange chromatography.

[0524] G. Functionalization of Thiol Groups on Cysteine ​​Linkers. In some examples, a cysteine ​​linker can be used to facilitate attachment of an αvβ6 integrin ligand to an RNAi agent. Either before or after annealing, the 5' or 3' tridentate alkyne-Cys(Stbu)-PEG2-functionalized sense strand can be functionalized with a maleimide-containing moiety or reduced to release a free thiol, as shown in the following structure: [ka]

[0525] The following describes an example of modification of trialkyne-Cys(Stbu)-PEG2-duplex with N-ethylmaleimide: Trialkyne-Cys(Stbu)-PEG2-duplex (35 mg) was dissolved in 500 μL of deionized water. HEPES buffer (1 M, pH 8.5, 82 μL) was added to the reaction mixture, and the solution was vortexed. 1 M dithiothreitol solution (DTT, 100 equivalents, 236 μL) was added, and the solution was placed on a vortex shaker for 3 hours. After confirming disulfide reduction by denaturing RP-HPLC, the conjugate was precipitated three times with a solvent system of 1× phosphate-buffered saline / acetonitrile (1:14 ratio). The precipitated pellet was reconstituted in 0.5 mL of 0.1 M HEPES (pH 6.5), and N-ethylmaleimide (3 mg, 10 equivalents) was added to the solution, which was then placed on a vortex mixer for approximately 15 minutes. After the reaction was completed, the conjugate was precipitated three times with a solvent system of 1× phosphate buffered saline / acetonitrile (1:14 ratio), desalted, and dried. Example 3. αvβ6 integrin ligand binding activity.

[0526] IC50 binding data was obtained for the αvβ6 integrin ligands of structures 1 and 2, as reported in Table 1 below: [Table 1]

[0527] The azide-functionalized constructs (i.e., constructs 1b and 2b) were tested for IC50 under conditions typically used and known in the art. As shown in Table 1 above, constructs 1 and 2 demonstrated selective binding to αβ integrin. Example 4. In vivo intratracheal administration of an RNAi agent targeting α-ENaC bound to αvβ6 integrin ligand in rats.

[0528] RNAi agents, including sense and antisense strands, were synthesized by solid-phase phosphoramidite technology according to the general procedures commonly used in oligonucleotide synthesis, as described in Example 2 herein. The RNAi agents included antisense strands with nucleobase sequences at least partially complementary to the gene expressing the α subunit of the amiloride-sensitive epithelial sodium channel (commonly referred to as α-ENaC or SCNN1A). The α-ENaC RNAi agents were designed to degrade or inhibit the translation of α-ENaC messenger RNA (mRNA) transcripts in a sequence-specific manner, thereby inhibiting the expression of the α-ENaC gene. The RNAi agent (AD04835) used in this example was composed of modified nucleotides and more than one non-phosphodiester bond and contained the following nucleotide sequence: Sense strand sequence (5'→3'): (NH2-C6)sgscugugcaAfCfCfagaacaaauas(invAb) (SEQ ID NO: 1) Antisense strand sequence (5'→3') cPrpusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsc (SEQ ID NO: 2) where (invAb) represents an inverted (3'-3' linked) abasic deoxyribonucleotide; s represents a phosphorothioate linkage; a, c, g, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, or 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, or 2'-fluoro uridine, respectively; cPrpu represents 5'-cyclopropylphosphonate-2'-O-methyl uridine (see, e.g., Table A); and (NH2-C6) represents a C6-terminal amine to facilitate targeting ligand attachment, if desired (see, e.g., Table A).

[0529] As one of ordinary skill in the art will clearly understand, the nucleotide monomers are linked by standard phosphodiester bonds, except for the inclusion of phosphorothioate linkages shown in the modified nucleotide sequences disclosed herein in place of the phosphodiester linkages typically present in oligonucleotides.

[0530] On study days 1 and 2, male Sprague-Dawley rats received 200 microliter doses administered intratracheally via a microspray device (Penn Century, Philadelphia, PA), and the doses included the following dosing groups: (1) aqueous vehicle containing 5% dextrose (D5W); (2) 3.0 mg / kg of a ligand-free α-ENaC RNAi agent (AD04835) formulated in 5% dextrose in water (d5w) (“naked RNAi agent”); or (3) 3.0 mg / kg of α-ENaC RNAi agent (AD04835) conjugated to a tridentate αvβ6 integrin ligand of structure 1 formulated in d5w.

[0531] The same α-ENaC RNAi agent was used in Groups 2 and 3. For Group 3, the terminal amine (NH2-C6) present on the 5' end of the sense strand of the RNAi agent was then coupled to a scaffold containing three terminal alkyne groups. The alkyne groups were then coupled to the azide functional groups present on Structure 1b, thereby forming the tridentate αvβ6 integrin ligand of Structure 1. The general synthetic procedure is described in Example 2 above.

[0532] Four (4) rats were dosed per group. Rats were euthanized on day 5, and total RNA was isolated from both lungs after harvesting and homogenization. α-ENaC mRNA abundance was quantified by probe-based quantitative PCR, normalized to GAPDH expression, and expressed as a percentage of the vehicle control group (geometric mean, + / - 95% confidence interval). [Table 2]

[0533] As shown in Table 2 above, the tridentate form of construct 1 αvβ6 integrin ligand bound to the α-ENaC RNAi agent (i.e., Group 3) showed increased relative knockdown of α-ENaC mRNA (approximately 81% knockdown) compared to the naked RNAi agent without any ligand (64% knockdown) (i.e., Group 2) and the vehicle control in vivo. Example 5. In vivo oropharyngeal aspiration administration of an RNAi agent targeting α-ENaC bound to αvβ6 integrin ligand in rats.

[0534] In the following examples, various RNAi agents are used as cargo molecules to test the delivery of cargo molecules to cells of interest via αβ integrin. Certain RNAi agents used herein are described in U.S. Patent Application No. 62 / 679,549 (the entire contents of which are incorporated herein by reference).

[0535] On study day 1, male Sprague Dawley rats were dosed by oropharyngeal ("OP") aspiration using a pipette with 200 microliters according to the following dosing groups: [Table 3]

[0536] RNAi agents were synthesized with nucleotide sequences directed to target the human α-ENaC gene, which contained a functionalized amine-reactive group (NH2-C6) at the 5' end of the sense strand to facilitate conjugation to αvβ6 integrin ligands. Each αvβ6 integrin ligand was then conjugated to the RNAi agent via a tridentate scaffold containing a cysteine-n-ethyl-maleimide linker. For the RNAi agent-αvβ6 integrin ligand conjugates of Example 5, the RNAi agent and scaffold / linker structures were consistent across groups 2-7. Thus, the only difference between groups 2-7 was the specific αvβ6 integrin ligand used (each tridentate). The RNAi agent-αvβ6 integrin ligand conjugates of Example 5 had the structures shown below: [ka] (In the formula, [ka] represents the RNAi agent, and "avb6 ligand" represents each ligand structure.) The structure of the RNAi agent (AD04835) used in this example is described in Example 4 above.

[0537] Five (5) rats in each group were dosed (n=5). On study day 9, rats were sacrificed, and total RNA was isolated from both lungs after collection and homogenization. α-ENaC (SCNN1A) mRNA expression was quantified by probe-based quantitative PCR, normalized to GAPDH expression, and expressed as a percentage of the vehicle control group (geometric mean, + / - 95% confidence interval). [Table 4-1] [Table 4-2]

[0538] As shown in Table 4 above, each of the α-ENaC RNAi agents reduced mRNA expression in rats compared to the control. For example, Group 6 (AD04835-triloctate-construct 6.1) showed approximately a 65% reduction (0.351) in mean rENaC mRNA expression compared to the control; Group 2 (AD04835-triloctate-construct 2) showed approximately a 65% reduction (0.351) in mean rENaC mRNA expression compared to the control. showed an approximately 46% decrease in mRNA expression (0.543); Group 4 (AD04835-trilocate-construct 5.2) showed an approximately 48% decrease in mean rENaC mRNA expression (0.522) compared to controls. Example 6. In vivo oropharyngeal aspiration administration of an RNAi agent targeting α-ENaC bound to an αvβ6 integrin ligand in rats.

[0539] On study day 1, male Sprague Dawley rats were dosed by oropharyngeal ("OP") aspiration using a pipette with 200 microliters according to the following dosing groups: [Table 5-1] [Table 5-2]

[0540] An RNAi agent was synthesized having a nucleotide sequence directed to target the human α-ENaC gene, which contained a functionalized amine-reactive group (NH2-C6) at the 5' end of the sense strand to facilitate binding to the αββ integrin ligand. The RNAi agent used in this example was composed of modified nucleotides and more than one non-phosphodiester linkage, and contained the following nucleotide sequence: AD05347: Sense strand sequence (5'→3'): (NH2-C6)cscugugcaAfCfCfagaacaaauas(invAb) (SEQ ID NO: 3) Antisense strand sequence (5'→3') cPrpusAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsc (SEQ ID NO: 2), and AD05453: Sense strand sequence (5'→3'): (NH2-C6)cscugugcaAfCfCfagaacaaauas(invAb) (SEQ ID NO: 3) Antisense strand sequence (5'→3') usAfsusUfuGfuUfcUfgGfuUfgCfaCfaGfsg(sequence number No. 4), where (invAb) represents an inverted (3'-3' linked) abasic deoxyribonucleotide; s represents a phosphorothioate linkage; a, c, g, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, or 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, or 2'-fluoro uridine, respectively; cPrpu represents 5'-cyclopropylphosphonate-2'-O-methyl uridine (see, e.g., Table A); and (NH2-C6) represents a C6-terminal amine to facilitate targeting ligand attachment, if desired (see, e.g., Table A).

[0541] For groups 2, 3, 4, 5, and 6, each αβ integrin ligand was conjugated to the RNAi agent via a tridentate scaffold / linker structure that includes a glutaric acid linker (via the addition of glutaric acid), as shown in structure 300a below: [ka] (In the formula, [ka] represents the RNAi agent, and "avb6 ligand" represents each ligand structure).

[0542] For groups 7 and 8, each αβ integrin ligand was linked to the RNAi agent via a tridentate scaffold / linker structure having the structure shown in structure 330a: [ka] (In the formula, [ka] represents the RNAi agent, and "avb6 ligand" represents each ligand structure).

[0543] Four (4) rats were dosed (n=4) in groups 1, 3, 4, 6, and 7; five (5) rats were dosed (n=5) in groups 5 and 8; and three (3) rats were dosed (n=3) in group 2. Rats were sacrificed on study day 9, and total RNA was isolated from both lungs after harvesting and homogenization. α-ENaC (SCNN1A) mRNA expression was quantified by probe-based quantitative PCR, normalized to GAPDH expression, and presented as a percentage of the vehicle control group (geometric mean, + / - 95% confidence interval). [Table 6-1] [Table 6-2]

[0544] As shown in Table 6 above, each α-ENaC RNAi agent reduced mRNA expression in rats compared to the control. For example, Group 5 (AD05453-tridentate αvβ6 integrin ligand construct 6.1) showed an approximately 51% reduction in mean rENaC mRNA expression (0.494) compared to the control, and Group 3 (AD05453-tridentate αvβ6 integrin ligand construct 2) showed an approximately 38% reduction in mean rENaC mRNA expression (0.615) compared to the control. Furthermore, Group 5 (which contained αvβ6 integrin ligand construct 6.1) was improved over Group 6 (which contained αvβ6 integrin ligand construct 7), indicating the chirality dependence of the (s) observed with construct 6.1 over the (r) observed with construct 7 for the αvβ6 integrin ligand. Example 7. In vivo oropharyngeal aspiration administration of an RNAi agent targeting α-ENaC bound to αvβ6 integrin ligand in rats.

[0545] On study day 1, male Sprague Dawley rats were dosed by oropharyngeal ("OP") aspiration using a pipette with 200 microliters according to the following dosing groups: [Table 7-1] [Table 7-2]

[0546] RNAi agents were synthesized with nucleotide sequences directed to target the human α-ENaC gene, which contained a functionalized amine-reactive group (NH2-C6) at the 5' end of the sense strand to facilitate conjugation to αvβ6 integrin ligands. The nucleotide sequences of the RNAi agents used in this example are described in Example 6 above. For groups 2, 3, 4, 5, 6, 7, 8, 9, and 10, each αvβ6 integrin ligand was conjugated to the RNAi agent via a tridentate scaffold / linker structure containing a glutaric acid linker, as shown in construct 300a in Example 6 above. For group 11, the epithelial cell-targeting ligand was composed of an RGD-mimetic peptide known to bind to αvβ6 integrin and contained a 20 kDa PEG moiety as a pharmacokinetic (PK) modulator.

[0547] Four (4) rats in each group were dosed (n=4). On study day 9, rats were sacrificed, and total RNA was isolated from both lungs after collection and homogenization. α-ENaC (SCNN1A) mRNA expression was quantified by probe-based quantitative PCR, normalized to GAPDH expression, and expressed as a percentage of the vehicle control group (geometric mean, + / - 95% confidence interval). [Table 8]

[0548] As shown in Table 8 above, each α-ENaC RNAi agent reduced mRNA expression in rats compared to the control. For example, Group 3 (AD05347-glutaric acid-tridentate αvβ6 integrin ligand construct 6.1) showed an approximately 64% reduction in mean rENaC mRNA expression (0.358) compared to the control, and Group 8 (AD05453-glutaric acid-tridentate αvβ6 integrin ligand construct 6.1) showed an approximately 55% reduction in mean rENaC mRNA expression (0.454) compared to the control. Furthermore, all of the αvβ6 integrin ligands in Example 7 (i.e., construct 2, construct 6, construct 6.1, construct 8, construct 9, construct 10, and construct 11) showed knockdown levels comparable to those of Group 11, a tridentate peptide-based epithelial cell targeting ligand that further contained a relatively bulky 20-kilodalton PEG moiety to enhance its pharmacokinetic effect. Example 8. In vivo intratracheal administration of an RNAi agent targeting α-ENaC bound to αvβ6 integrin ligand in rats

[0549] On study days 1 and 2, male Sprague Dawley rats were administered a 200 microliter dose intratracheally via a micronebulizer device (Penn Century, Philadelphia, PA), with the following dose groups: [Table 9]

[0550] An RNAi agent was synthesized having a nucleotide sequence directed to target the human α-ENaC gene, which contained a functionalized amine-reactive group (NH2-C6) at the 5' end of the sense strand to facilitate binding to the αββ integrin ligand. The nucleotide sequence of the RNAi agent used in this example is described in Example 4 above.

[0551] For groups 3 and 4, the αβ integrin ligand of structure 1 was conjugated to the RNAi agent via a tridentate scaffold and linker structure containing a cysteine ​​linker, as shown in structure 331a below: [ka] (In the formula, [ka] represents the RNAi agent, and "avb6 ligand" represents each ligand structure).

[0552] For Group 5, the αvβ6 integrin ligand was conjugated to the RNAi agent via a linker structure comprising a tridentate scaffold and a cysteine-n-ethyl-maleimide linker, as shown in structure 330a in Example 6 above. For Group 7, the αvβ6 integrin ligand was conjugated to the RNAi agent via a linker structure comprising a tridentate scaffold and a glutaric acid linker, as shown in structure 300a in Example 6 above. For Groups 2 and 6, the peptide-based epithelial cell targeting ligand was composed of an RGD mimetic peptide and included a 20 kDa PEG moiety as a pharmacokinetic (PK) modulator.

[0553] The same α-ENaC RNAi agent was used in each of groups 2-7.

[0554] Five (5) rats were dosed (n=5) in each of groups 1, 2, 3, 4, 5, and 6, and four (4) rats were dosed (n=4) in group 7. Rats were sacrificed on study day 8, and total RNA was isolated from both lungs after collection and homogenization. α-ENaC (SCNN1A) mRNA expression was quantified by probe-based quantitative PCR, normalized to GAPDH expression, and presented as a percentage of the vehicle control group (geometric mean, + / - 95% confidence interval). [Table 10]

[0555] As shown in Table 10 above, each of the α-ENaC RNAi agents reduced mRNA expression in rats compared to controls. For example, Group 5 (containing AD04835-Cys-(n-ethyl-Mal)-tridentate αvβ6 integrin ligand construct 1) showed an approximately 65% ​​reduction (0.358) in mean rENaC mRNA expression compared to controls, which was comparable to the knockdown level achieved in Group 2, which contained a peptide-based epithelial cell-targeting ligand that also contained a 20 kDa PEG moiety as a pharmacokinetic modulator. Example 9. In vivo intratracheal administration of an RNAi agent targeting α-ENaC bound to αvβ6 integrin ligand in rats

[0556] On study days 1 and 2, male Sprague Dawley rats were administered a 200 microliter dose intratracheally via a micronebulizer device (Penn Century, Philadelphia, PA), with the following dose groups: [Table 11-1] [Table 11-2]

[0557] RNA with a nucleotide sequence directed to target the human α-ENaC gene Ai agents were synthesized that contained a functionalized amine-reactive group (NH2-C6) at the 5' end of the sense strand to facilitate conjugation to the αvβ6 integrin ligand. The nucleotide sequences of the RNAi agents used in this example are described in Example 4 above. For groups 2 and 3, the respective αvβ6 integrin ligands were conjugated to the RNAi agents via a tridentate scaffold / linker structure containing a glutaric acid linker, as shown in structure 300a in Example 6 above. For group 6, the respective αvβ6 integrin ligands were conjugated to the RNAi agents via a tridentate scaffold / linker structure containing a cysteine ​​linker, as shown in structure 331a in Example 8 above.

[0558] The same α-ENaC RNAi agent was used in each of groups 2-8. Five (5) rats were dosed in Group 1 (n=5), and four (4) rats were dosed in each of Groups 2 and 3 (n=4). Rats were sacrificed on study day 9, and total RNA was isolated from both lungs after collection and homogenization. α-ENaC (SCNN1A) mRNA expression was quantified by probe-based quantitative PCR, normalized to GAPDH expression, and presented as a percentage of the vehicle control group (geometric mean, + / - 95% confidence interval). [Table 12-1] [Table 12-2]

[0559] As shown in Table 12 above, each α-ENaC RNAi agent reduced mRNA expression in rats compared to controls. For example, Group 3 (RNAi agent-glutarate-tridentate αvβ6 integrin ligand construct 2) showed an approximately 52% reduction in mean rENaC mRNA expression (0.483) compared to controls, and Group 6 (RNAi agent-Cys-tridentate αvβ6 integrin ligand construct 2) showed an approximately 76% reduction in mean rENaC mRNA expression (0.237) compared to controls. Example 10. In vivo intratracheal administration of an RNAi agent targeting α-ENaC bound to αvβ6 integrin ligand in rats.

[0560] On study days 1 and 2, male Sprague Dawley rats were administered a 200 microliter dose intratracheally via a micronebulizer device (Penn Century, Philadelphia, PA), with the following dose groups:

[0561] [Table 13-1] [Table 13-2]

[0562] RNAi agents were synthesized with nucleotide sequences directed to target the human α-ENaC gene, which contained a functionalized amine-reactive group (NH2-C6) at the 5' end of the sense strand to facilitate conjugation to αvβ6 integrin ligands. The nucleotide sequences of the RNAi agents used in this example are described in Example 4 above. For groups 3, 4, 5, and 6, the respective αvβ6 integrin ligands were conjugated to the RNAi agents via a tridentate scaffold / linker structure containing a cysteine ​​linker, as shown in construct 331a in Example 8 above. For group 2, the targeting ligand was composed of an RGD-mimetic peptide and included a 20 kDa PEG moiety as a pharmacokinetic (PK) modulator and an FCFP peptide linker.

[0563] The same α-ENaC RNAi agent was used in each of groups 2-7.

[0564] Five (5) rats in each group were dosed (n=5). On study day 9, rats were sacrificed, and total RNA was isolated from both lungs after collection and homogenization. α-ENaC (SCNN1A) mRNA expression was quantified by probe-based quantitative PCR, normalized to GAPDH expression, and expressed as a percentage of the vehicle control group (geometric mean, + / - 95% confidence interval). [Table 14] As shown in Table 14 above, each of the α-ENaC RNAi agents reduced mRNA expression in rats compared to the control. Notably, Group 5 (1.0 mg / kg AD04835-Cys-tridentate αvβ6 integrin ligand construct 2) did not contain the large 20 kilodalton PEG moiety as a pharmacokinetic modulator, yet Group 2 (1.0 mg / kg AD04835-Cys-PEG20kDa-FCFP-PEG 20-peptide-based epithelial cell targeting ligands), the level of inhibition of α-ENaC expression was numerically superior (Group 5 = approximately 56% knockdown (0.436); Group 2 = approximately 47% knockdown (0.531)). Example 11. In vivo oropharyngeal aspiration administration of an RNAi agent targeting α-ENaC bound to αvβ6 integrin ligand in rats.

[0565] On study days 1, 2, and 3, male Sprague Dawley rats were dosed by oropharyngeal ("OP") aspiration using a pipette with 200 microliters according to the following dosing groups: [Table 15]

[0566] RNAi agents were synthesized with nucleotide sequences directed to target the human α-ENaC gene, which contained a functionalized amine-reactive group (NH2-C6) at the 5' end of the sense strand to facilitate conjugation to αvβ6 integrin ligands. The nucleotide sequences of the RNAi agents used in this example are described in Example 6 above. Each αvβ6 integrin ligand was conjugated to the RNAi agent via a tridentate scaffold / linker structure containing a glutaric acid linker, as shown in structure 300a in Example 6 above.

[0567] Five (5) rats were dosed (n=5) in each of groups 1, 3, 4, 5, 8, and 9, and six (6) rats (n=6) were dosed in groups 2, 6, and 7. Rats were sacrificed on study day 9, and total RNA was isolated from both lungs after collection and homogenization. α-ENaC (SCNN1A) mRNA expression was quantified by probe-based quantitative PCR, normalized to GAPDH expression, and presented as a percentage of the vehicle control group (geometric mean, + / - 95% confidence interval). [Table 16]

[0568] As shown in Table 16 above, each α-ENaC RNAi agent conjugated to the avb6 integrin ligand with structure 6.1 (tridentate form) reduced mRNA expression in rats compared to controls. Furthermore, at each dosage level, the α-ENaC RNAi agent conjugated to the avb6 integrin ligand with structure 6.1 was superior to the naked α-ENaC RNAi agent, demonstrating the effect of the ligand on delivery of the RNAi agent (e.g., compare groups 2 and 6; groups 3 and 7; groups 4 and 8; and groups 5 and 9). Example 12. Additional αvβ6 integrin ligand binding activity.

[0569] Further IC50 binding data was obtained for the αvβ6 integrin ligands of structures 2, 6.1, 7, and 23 used in certain examples herein, as reported in Table 17 below: [Table 17]

[0570] The azide-functionalized structures (i.e., structures 2b and 6.1b, 7b, and 23b) were tested for IC50 under conditions typically used and known in the art. As shown in Table 17 above, structure 6.1 exhibited potent binding activity to αβ integrin (IC50=1.6 nM). Example 13. In vivo oropharyngeal aspiration administration of an RNAi agent targeting α-ENaC bound to an αvβ6 integrin ligand in rats.

[0571] On study day 1, male Sprague Dawley rats were dosed according to the following dosing groups: Pets were administered 200 microliters by oropharyngeal ("OP") aspiration: [Table 18]

[0572] RNAi agents were synthesized with nucleotide sequences directed to target the human α-ENaC gene. The RNAi agents, including the AD05347 duplex, contained a functionalized amine-reactive group (NH2-C6) at the 5' end of the sense strand to facilitate conjugation to αvβ6...

Claims

1. The following structures: 【Chemistry 1-1】 or a pharmaceutically acceptable salt thereof, where: n is 3 or 4; J is N; Z is OR １３ and R １ is H or optionally substituted C １ ~C ６ is alkyl; R ２ is an optionally substituted cycloalkylene, optionally substituted arylene, optionally substituted heterocycloalkylene, or optionally substituted heteroarylene; R Ｐ１ and R Ｐ２ are each independently H or halo; R １０ is H or optionally substituted alkyl; R １１ is H or optionally substituted alkyl; R １２ is H or optionally substituted alkyl; Each R １３ are independently H, optionally substituted alkyl; R １４ is optionally substituted alkyl; and Here, R ２ comprises an RNAi agent, provided that the αvβ6 integrin ligand has the following structure: 【Chemistry 1-2】 wherein X is not an αvβ6 integrin ligand or a pharmaceutically acceptable salt thereof, comprising an RNAi agent. An αvβ6 integrin ligand or a pharmaceutically acceptable salt thereof.

2. The following structures: 【Chemistry 2-1】 or a pharmaceutically acceptable salt thereof, where: n is 3 or 4; J is N; R １ is H or C １ ~C ６ is alkyl; R ２ is optionally substituted cycloalkylene, optionally substituted arylene, optionally substituted heterocycloalkylene, optionally substituted heteroarylene; R １０ is H or optionally substituted alkyl; R １１ is H or optionally substituted alkyl; R １２ is H or optionally substituted alkyl; R １３ is H or optionally substituted alkyl; R １４ is optionally substituted alkyl; Here, R ２ comprises an RNAi agent, provided that the αvβ6 integrin ligand has the following structure: 【Chemistry 2-2】 wherein X is not an αvβ6 integrin ligand or a pharmaceutically acceptable salt thereof, comprising an RNAi agent. An αvβ6 integrin ligand or a pharmaceutically acceptable salt thereof.

3. The following structures: 【Chemistry 4】 (In the formula, n is 3 or 4; and R ９ 2. The αvβ6 integrin ligand of claim 1, or a pharmaceutically acceptable salt thereof, comprising one or more RNAi agents.

4. below: 【Chemistry 5-1】 【Chemistry 5-2】 【Chemistry 5-3】 【Chemistry 5-4】 【Chemistry 5-5】 【Chemistry 5-6】 【Chemistry 5-7】 【Chemistry 5-8】 or a pharmaceutically acceptable salt thereof, wherein X comprises an RNAi agent.

5. below: 【Chemistry 6-1】 【Chemistry 6-2】 【Chemistry 6-3】 【Chemistry 6-4】 【Chemistry 6-5】 【Chemistry 6-6】 【Chemistry 6-7】 【Chemistry 6-8】 【Chemistry 6-9】 【Chemistry 6-10】 【Chemistry 6-11】 (In the formula, 【Chemistry 6-12】 indicates the point of attachment to the moiety that comprises the RNAi agent), or a pharmaceutically acceptable salt thereof.

6. A structure, wherein said structure has the following formula: 【Chemistry 7-1】 (In the formula, 【Chemistry 7-2】 represents the RNAi agent, and "avb ６ The structure is a structure of the αvβ6 integrin ligand described in claim 5.

7. A composition comprising the αvβ6 integrin ligand according to any one of claims 1 to 5 or the structure according to claim 6, and a pharmaceutically acceptable excipient.

8. The composition of claim 7 , wherein the RNAi agent is capable of inhibiting expression of a target gene in an epithelial cell.

9. The composition of claim 7 , wherein the RNAi agent is capable of inhibiting expression of a target gene in bronchiolar epithelial cells.

10. A composition comprising an αvβ6 integrin ligand described in any one of claims 1 to 5 or a structure described in claim 6 for use in a method for delivering one or more RNAi agents to a cell, the method comprising administering the αvβ6 integrin ligand or the structure to the cell.

11. 11. A composition comprising an αvβ6 integrin ligand according to any one of claims 1 to 5, a structure according to claim 6, or a composition according to any one of claims 7 to 10, for use in delivering one or more RNAi agents to cells or tissues of a subject in vivo.

12. 12. The composition of claim 10 or 11, wherein the cells are selected from the group consisting of type I and type II alveolar epithelial cells, goblet cells, secretory epithelial cells, ciliated epithelial cells, corneal and conjunctival epithelial cells, dermal epithelial cells, bile duct cells, intestinal cells, ductal epithelial cells, glandular epithelial cells, and epithelial tumors (carcinomas).

13. A composition for inhibiting the expression of a target gene in a cell in vivo, wherein the composition comprises an αvβ6 integrin ligand described in any one of claims 1 to 5.

14. 14. The composition of claim 13, wherein the cells are selected from the group consisting of type I and type II alveolar epithelial cells, goblet cells, secretory epithelial cells, ciliated epithelial cells, corneal and conjunctival epithelial cells, dermal epithelial cells, bile duct cells, intestinal cells, ductal epithelial cells, glandular epithelial cells, and epithelial tumors (carcinomas).

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