Integrin targeting ligands and uses thereof

Abstract

To provide compounds having affinity for integrins, to provide the synthesis of these compounds, and to provide use of these compounds as ligands to facilitate the delivery of cargo molecules to cells expressing integrins.SOLUTION: Compounds having affinity for integrins, the synthesis of these compounds, and use of these compounds as ligands to facilitate the delivery of cargo molecules to cells expressing integrins are described. The described integrin targeting ligands have serum stability and affinity for αvβ3 integrin and / or αvβ5 integrin, and are suitable for conjugation to cargo molecules, such as oligonucleotide-based therapeutic agents (e.g., RNAi agents), to facilitate delivery of the cargo molecules to cells and tissues, such as tumor cells, that express integrin αvβ3, integrin αvβ5, or both integrin αvβ3 and integrin αvβ5. Compositions that include integrin targeting 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 / 663,763, filed April 27, 2018, and No. 62 / 790,372, filed January 9, 2019, both of which are incorporated by reference in their entireties.

[0002] FIELD OF THEINVENTION Disclosed herein are compounds having affinity for integrins, methods for the synthesis of such compounds, and the use of such compounds as ligands for delivering cargo molecules in vivo. [Background technology]

[0003] background Integrins are transmembrane glycoproteins that mediate cell-cell and cell-matrix interactions. Integrin αvβ-3 (αvβ3) and αvβ-5 (αvβ5) are known to be members of the integrin superfamily of adhesion molecules and receptors for the extracellular matrix (ECM) protein vitronectin (Horton, MA, 29(5) Int. J. Biochem. Cell Biol. 721-725 (1997)). It is believed that altered expression of certain integrins, including integrin αvβ3 and integrin αvβ5, is involved in cancer progression, invasiveness, and metastasis.

[0004] Indeed, overexpression of integrins, including integrins αvβ3 and αvβ5, has been reported in many tumor cells (Desgrosellier, JS et al., Nat Rev Cancer, 10(1):9-22 (2010)). Antagonists of αvβ3 (and to some extent αvβ5) have been considered for use in various diseases associated with altered integrin function. For example, attempts have been made to develop αvβ3 inhibitors as potential cancer treatments, since inhibition of the αvβ3 receptor has been shown to inhibit angiogenesis, thereby suppressing the formation of new blood vessels that are believed to be necessary for tumor growth (see, e.g., Brooks et al., 79 Cell 1157-1164 (1994); Mas-Moruno et al., Anticancer Agents Med Chem, 10(10):753-768). However, one leading example of an αvβ3 inhibitor, the antagonist Cilengitide, has been shown to be ineffective in clinical trials aimed at limiting tumor angiogenesis and progression in patients with glioblastoma (see, e.g., Ley et al., Integrin-based Therapeutics: Biological Basis, Clinical Use and New Drugs, 15(3) Nat. Rev. Drug Discov. 173-183 (2016)).

[0005] In general, the delivery of a cargo molecule, including a therapeutically effective pharmaceutical compound or active pharmaceutical ingredient, to a desired cell and / or tissue in vivo remains a common challenge in the development of a therapeutically viable pharmaceutical formulation. There remains a need for a stable and effective targeting compound that has affinity for and / or selectively binds to a specific cell or tissue, which can be used or utilized as a ligand to facilitate the delivery of a therapeutic cargo molecule to the specific cell or tissue. Moreover, there remains a unique need for a compound that can selectively target integrin αvβ-3 and is suitable for conjugation to a cargo molecule and delivers the cargo molecule to cells expressing such integrins, such as tumor cells, in vivo. With respect to oligonucleotides, particularly oligonucleotide-based therapeutic agents (e.g., oligonucleotide-based compounds such as antisense oligonucleotides or RNAi agents), there remains a need for a ligand that can target integrin αvβ-3 and / or integrin αvβ-5 and facilitate the delivery of these oligonucleotide-based compounds to cells expressing such integrins. Summary of the Invention

[0006] overview Described herein are compounds having affinity for certain integrins, including αvβ3 and αvβ5, which can be used as ligands to selectively target compounds or other molecules to cells or tissues expressing integrins αvβ3 and / or αvβ5 (referred to herein as "integrin targeting ligands", "αvβ3 integrin targeting ligands", "αvβ3 integrin ligands" or simply "integrin targeting ligands"). The integrin targeting ligands disclosed herein are stable in serum and can bind with affinity and specificity to these integrins. The integrin targeting ligands disclosed herein are conjugated to cargo molecule(s) to facilitate delivery of the cargo molecule(s) to cells or tissues expressing integrins αvβ3 and / or αvβ5.

[0007] In another embodiment, a method for delivering cargo molecules to tissues and / or cells expressing integrin αvβ3 and / or integrin αvβ5 in vivo is described herein, wherein the method comprises administering to a subject one or more integrin-targeting ligands disclosed herein conjugated to one or more cargo molecules. Further disclosed herein is a method for treating a subject suffering from a disease, condition or disorder that can be treated by delivery of a therapeutic cargo molecule (e.g., an active pharmaceutical ingredient) to cells expressing αvβ3 integrin and / or αvβ5 integrin, wherein the method comprises administering to a subject one or more integrin-targeting ligands disclosed herein conjugated to one or more therapeutic cargo molecules.

[0008] Further described herein is a method of inhibiting expression of a target gene in a cell in vitro or in vivo, the method comprising administering to the cell an effective amount of a conjugate comprising one or more integrin targeting ligands disclosed herein conjugated to one or more oligonucleotide-based therapeutics, such as RNAi agents, capable of inhibiting expression of a target gene in the cell. In some embodiments, described herein is a method of inhibiting expression of a target gene in a cell of a subject, the subject being administered an effective amount of one or more oligonucleotide-based therapeutics (such as RNAi agents) conjugated to one or more integrin targeting ligands disclosed herein.

[0009] In yet another embodiment, the composition described herein comprises the integrin targeting ligand disclosed herein.The composition described herein can be a pharmaceutical composition or drug that comprises one or more integrin targeting ligands disclosed herein that are conjugated with one or more therapeutic cargo molecules such as RNAi agents or other cargo molecules or therapeutic agents.

[0010] In some embodiments, described herein is a method for treating a subject suffering from a disease or disorder mediated at least in part by expression of a target gene in a cell expressing integrin αvβ3, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises one or more oligonucleotide-based therapeutics capable of inhibiting expression of the targeted gene, such as an RNAi agent conjugated to one or more integrin-targeting ligands disclosed herein. In some embodiments, described herein is a method for treating a subject suffering from a disease or disorder mediated at least in part by expression of a target gene in a tumor cell, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises one or more oligonucleotide-based therapeutics capable of inhibiting expression of the targeted gene, such as an RNAi agent conjugated to one or more integrin-targeting ligands disclosed herein. In some embodiments, described herein are methods for treating a subject suffering from a disease or disorder mediated at least in part by expression of a target gene in renal tumor cells, such as clear cell renal carcinoma tumor cells, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition, the pharmaceutical composition comprising one or more oligonucleotide-based therapeutic agents capable of inhibiting expression of a targeted gene, such as an RNAi agent conjugated to one or more integrin-targeting ligands disclosed herein.

[0011] In a first aspect, the disclosure provides synthetic integrin targeting ligands.

[0012] In some embodiments, the integrin targeting ligand disclosed herein has the following formula: [ka] {In the formula, X is -C(R 3 )2-, -NR3 -, [ka] and; Y is an optionally substituted C1-C8 alkylene; Z is O, NR 3 , or S; n is an integer from 1 to 8; R 1 is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted cycloalkyl, or R 1 comprises a cargo molecule; R 2 is H, optionally substituted alkyl, or R 2 comprises a cargo molecule; R 3 is independently selected from the group consisting of H and optionally substituted alkyl, or R 3 comprises a cargo molecule; R 4 is H or optionally substituted alkyl; and Here, Y, R 1 , R 2 , in either case R 3 , and R 4 at least one of which comprises a cargo molecule, or a pharma- ceutically acceptable salt thereof.

[0013] Any of the integrin targeting ligands disclosed herein can be linked to cargo molecules, reactive groups, and / or protected reactive groups.For example, linking to reactive groups can be used to facilitate the conjugation of integrin targeting ligands to cargo molecules.The integrin targeting ligands disclosed herein can enhance the targeting of cargo molecules to cells expressing integrins, including αvβ3 integrin and / or αvβ5 integrin.Cargo molecules can be, but are not limited to, medicamentously active ingredients or compounds, prodrugs, or other substances known for their therapeutic benefits. In some embodiments, the cargo molecule may 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, a 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, a polyethylene glycol, a hapten, digoxigenin, biotin, a radioactive atom or molecule, or a fluorophore. In some embodiments, the cargo molecule comprises a pharmaceutically active component or a prodrug. In some embodiments, the cargo molecule is or comprises an oligonucleotide-based therapeutic agent, such as an antisense compound or an RNAi agent. In some embodiments, the cargo molecule is or comprises an oligonucleotide-based compound that is a pharmaceutically active component. In some embodiments, the cargo molecule is or comprises an RNAi agent that is a pharmaceutically active component.

[0014] The use of the αvβ3 / 5 integrin targeting ligand described herein is described for targeting and delivering cargo molecules to cells expressing integrins. The cargo molecules can be delivered to cells in vitro, in situ, ex vivo, or in vivo.

[0015] In another aspect, this disclosure provides a composition comprising one or more of the integrin targeting ligands described herein.For example, in some embodiments, the composition comprising one or more integrin targeting ligands disclosed herein comprises one or more oligonucleotide-based compounds, such as one or more RNAi agents to be delivered to cells in vivo.In some embodiments, the composition described herein is for delivering RNAi agents to cells in vivo, where the RNAi agents are linked to one or more integrin targeting ligands.

[0016] The compositions comprising one or more integrin targeting ligands are described herein. In some embodiments, the compositions comprise a pharma- ceutically acceptable excipient. In some embodiments, the compositions comprising one or more integrin targeting ligands comprise one or more other medicinal substances or pharma- ceutical active ingredients or compounds. In some embodiments, the drugs comprising one or more integrin targeting ligands are described herein.

[0017] Compositions comprising one or more integrin targeting ligands disclosed herein can be used in vivo or in vitro to target, for example, clear cell renal carcinoma tumor cells (e.g., A498), other kidney cancer cells (e.g., ACHN, CAKI-2, 769-P, 786-O), melanoma cells (e.g., A375), glioblastoma cells (e.g., U87MG), pancreatic cancer cells (e.g., PANC-1), lung cancer cells (e.g., H460, H661, H1573, H2126), colon cancer cells (e.g., HT29, HCT 116), liver cancer cells (e.g., Hep2G, Hep3B), breast cancer cells (e.g., MCF7, SK-BR3), prostate cancer cells (e.g., DU145, PC3, LNCaP, MDA-PCa-2b), oral cancer cells (e.g., KB), tongue cancer cells (e.g., CAL27, SCC9), pharyngeal cancer cells (e.g., Detroit562), and / or ovarian cancer cells (e.g., OVCAR3, SKOV3, A2780) and / or xenografts derived from other patients.

[0018] In another aspect, the disclosure provides methods that include the use of one or more integrin targeting ligands and / or compositions described herein, and, if desired, forming the disclosed integrin targeting ligands and / or compositions into a form suitable for administration as a pharmaceutical product. In other embodiments, the disclosure provides methods of making the ligands and compositions, e.g., medicaments, described herein.

[0019] A composition comprising one or more integrin targeting ligands may be administered to a subject in vivo using routes of administration known in the art as appropriate for such administration in view of the cargo molecule to be administered, including, for example, subcutaneous, intravenous, intratumoral, inhalation (aerosol or dry powder formulation), intranasal, intraperitoneal, intradermal, transdermal, oral, sublingual, or topical administration. In some embodiments, a composition comprising one or more integrin targeting ligands may be administered for systemic delivery, for example, by intravenous or subcutaneous administration.

[0020] In some embodiments, disclosed herein is a method for delivering one or more desired cargo molecules to clear cell renal carcinoma tumor cells in vivo, the method comprising administering to a subject one or more integrin targeting ligands conjugated to one or more cargo molecules.

[0021] In some embodiments, disclosed herein is a method of delivering oligonucleotide-based compounds to tumor cells in vivo, comprising administering to the subject one or more integrin-targeting ligands conjugated to one or more oligonucleotide-based compounds. In some embodiments, disclosed herein is a method of delivering RNAi agents to tumor cells in vivo, comprising administering to the subject one or more integrin-targeting ligands conjugated to one or more RNAi agents. In some embodiments, disclosed herein is a method of inhibiting the expression of target genes in clear cell renal carcinoma tumor cells in vivo, comprising administering to the subject an RNAi agent conjugated to one or more ligands having affinity for αvβ3 integrin and / or αvβ5 integrin.

[0022] Other objects, features, aspects, and advantages of the present invention will become apparent from the following detailed description and claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description Integrin Targeting Ligands Described herein are compounds that have affinity for integrins, exhibit serum stability in vivo, and can be used as ligands to facilitate delivery of cargo molecules to cells and / or tissues expressing integrins, such as integrin αvβ3 and / or integrin αvβ5. The integrin targeting ligands can be used to target cells expressing integrins in vitro, in situ, ex vivo, and / or in vivo.

[0024] In some embodiments, the integrin targeting ligands disclosed herein are conjugated to one or more cargo molecules to preferentially direct and target the cargo molecule to cells or tissues expressing integrins, including integrin αvβ3 and / or integrin αvβ5. In some embodiments, the cargo molecule comprises or consists of a pharma- ceutically 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 integrin targeting ligands disclosed herein are conjugated to a cargo molecule to direct the cargo molecule to tumor cells in vivo. In some embodiments, the integrin targeting ligands disclosed herein are conjugated to a cargo molecule to direct the cargo molecule to clear cell renal carcinoma tumor cells in vivo.

[0025] Formula I In one aspect, the present invention provides a compound having the following structure: [ka] {In the formula, X is -C(R 3 )2-, -NR 3 -, [ka] and; Y is an optionally substituted alkylene; Z is O, NR 3 , or S; n is an integer from 1 to 8; R 1 is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted cycloalkyl, or R 1 comprises a cargo molecule; R 2 is H, optionally substituted alkyl, or R 2 comprises a cargo molecule; R3 is independently selected from the group consisting of H and optionally substituted alkyl, or R 3 comprises a cargo molecule; R 4 is H or optionally substituted alkyl; and Here, Y, R 1 , R 2 , in either case R 3 , and R 4 at least one of which comprises a cargo molecule.

[0026] In some embodiments of Formula I, R 1 below: [ka] {wherein: [ka] indicates the attachment point, and CM comprises the cargo molecule.

[0027] In some embodiments of Formula I, Y is a C1-C6 alkylene.

[0028] Formula II In some embodiments of Formula I, the integrin targeting ligand disclosed herein has the following formula: [ka] {In the formula, n is an integer from 1 to 8; R 1 is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted cycloalkyl, or R 1 comprises a cargo molecule; R 2 is H, optionally substituted alkyl, or R 2 comprises a cargo molecule; R4 is H or optionally substituted alkyl; and Here, R 1 or R 2 at least one of which comprises a cargo molecule, or a pharma- ceutically acceptable salt thereof.

[0029] Formula III In some embodiments of Formula I, the integrin targeting ligand disclosed herein has the following formula: [ka] {In the formula, n is an integer from 1 to 8; R 1 is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted cycloalkyl, or R 1 comprises a cargo molecule; R 2 is H, optionally substituted alkyl, or R 2 comprises a cargo molecule; R 3 is selected from the group consisting of H and optionally substituted alkyl, or R 3 comprises a cargo molecule; R 4 is H or optionally substituted alkyl; and Here, R 1 , R 2 and R 3 at least one of which comprises a cargo molecule, or a pharma- ceutically acceptable salt thereof.

[0030] Formula IV In some embodiments of Formula I, the integrin targeting ligand disclosed herein has the formula: [ka] {In the formula, n is an integer from 1 to 8; R 1is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted cycloalkyl, or R 1 comprises a cargo molecule; R 2 is H, optionally substituted alkyl, or R 2 comprises a cargo molecule; R 3 is selected from the group consisting of H and optionally substituted alkyl, or R 3 comprises a cargo molecule; R 4 is H or optionally substituted alkyl; and Here, R 1 , R 2 and R 3 at least one of which comprises a cargo molecule, or a pharma- ceutically acceptable salt thereof.

[0031] Formula V In some embodiments of Formula I, the integrin targeting ligand disclosed herein has the following formula: [ka] {In the formula, n is an integer from 1 to 8; R 1 is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted cycloalkyl, or R 1 comprises a cargo molecule; R 2 is H, optionally substituted alkyl, or R 2 comprises a cargo molecule; R 3 is independently selected from the group consisting of H and optionally substituted alkyl, or R 3 comprises a cargo molecule; R 4 is H or optionally substituted alkyl; and Here, R 1 , R 2and R 3 at least one of which comprises a cargo molecule, or a pharma- ceutically acceptable salt thereof.

[0032] Equation VI In some embodiments of Formula I, the integrin targeting ligand disclosed herein has the following formula: [ka] {In the formula, n is an integer from 1 to 8; R 1 is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted cycloalkyl, or R 1 comprises a cargo molecule; R 2 is H, optionally substituted alkyl, or R 2 comprises a cargo molecule; R 3 is independently selected from the group consisting of H and optionally substituted alkyl, or R 3 comprises a cargo molecule; R 4 is H or optionally substituted alkyl; and Here, R 1 , R 2 and R 3 at least one of which comprises a cargo molecule, or a pharma- ceutically acceptable salt thereof.

[0033] Formula VII In some embodiments of Formula I, the integrin targeting ligand disclosed herein has the following formula: [ka] {In the formula, R 1 is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted cycloalkyl, or R 1comprises a cargo molecule; R 2 is H, optionally substituted alkyl, or R 2 comprises a cargo molecule; R 4 is H or optionally substituted alkyl; and Here, R 1 , R 4 and R 5 at least one of which comprises a cargo molecule, or a pharma- ceutically acceptable salt thereof.

[0034] R 1 In an embodiment of Formula I, R 1 is R 1 is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted cycloalkyl, or R 1 comprises a cargo molecule. In some embodiments, R 1 below: [ka] {wherein: [ka] indicates an attachment point, and CM comprises a cargo molecule.

[0035] Integrin-targeting ligand precursors In some embodiments, the present invention provides integrin targeting ligand precursors that can be used to attach integrin targeting ligands to moieties that include cargo molecules, such as those of the following formula: [ka] {In the formula, X is -C(R 3 )2-, -NR 3 -, [ka] and; Y is an optionally substituted alkylene; Z is O, NR 3 , or S; n is an integer from 1 to 8; R 1 is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, optionally substituted cycloalkyl, or R 1 comprises a cargo molecule; R 2 is H, optionally substituted alkyl, or R 2 comprises a cargo molecule; R 3 is independently selected from the group consisting of H and optionally substituted alkyl, or R 3 comprises a cargo molecule; R 4 is H or optionally substituted alkyl; and Here, Y, R 1 , R 2 , in either case R 3 , and R 4 Provided herein are integrin targeting ligand precursors, wherein at least one of the following comprises a reactive group:

[0036] In some embodiments of the compound of Formula Ip, the reactive group comprises an azide.

[0037] Compounds of Formula I In some embodiments, the integrin targeting ligands disclosed herein have a structure that comprises, consists of, or consists essentially of any of the structures represented by the following: [ka] [ka] [ka] [ka] [ka] [ka]

[0038] In some embodiments, an integrin targeting ligand disclosed herein is conjugated to one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10; or 1-10, 2-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 3-10, 4-10, 5-10, 2-5, 2-4, or 3-5) cargo molecules (e.g., any of the cargo molecules described herein or known in the art).

[0039] In some embodiments, two or more of the integrin targeting ligands disclosed herein (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, 5-3 0, 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 integrin targeting ligands) are conjugated to one cargo molecule (e.g., any of the cargo molecules described herein or known in the art).

[0040] In some embodiments, the integrin targeting ligands disclosed herein are optionally conjugated to one or more cargo molecules via a linking group, such as, for example, a polyethylene glycol (PEG) group.

[0041] In some embodiments, the integrin targeting ligand disclosed herein is optionally conjugated to one or more cargo molecules via a scaffold that includes at least one binding point for each ligand and at least one binding point for each cargo molecule.In some embodiments, the integrin targeting ligand comprises, consists of, or essentially consists of an integrin targeting ligand conjugated to one cargo molecule.In some embodiments, the integrin targeting ligand comprises, consists of, or essentially consists of an integrin targeting ligand conjugated to two or more cargo molecules.

[0042] In some embodiments, the integrin targeting ligand comprises, consists of, or consists essentially of Structure 1a, Structure 2a, Structure 2.1a, Structure 2.2a, Structure 2.3a, Structure 2.4a, Structure 2.5a, Structure 2.6a, Structure 2.8a, Structure 2.9a, Structure 2.10a, Structure 2.11a, Structure 28a, Structure 29a, Structure 30a, Structure 31a, Structure 32a, Structure 33a, Structure 34a, Structure 36a, Structure 37a, Structure 38a, Structure 39a, Structure 40a, and Structure 41a, each of which is disclosed herein.

[0043] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0044] In some embodiments, the integrin targeting ligand of structure 1a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0045] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0046] In some embodiments, an integrin targeting ligand precursor may be synthesized to include an azide reactive group and have the following structure: [ka] Includes.

[0047] The reactive group (or a protected reactive group) can be used to facilitate conjugation (either directly or via one or more scaffolds and / or linkers) of an integrin targeting ligand to a molecule of interest, e.g., a cargo molecule.

[0048] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0049] In some embodiments, the integrin targeting ligand of structure 2a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0050] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0051] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0052] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0053] In some embodiments, the integrin targeting ligand of structure 2.1a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0054] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0055] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0056] In some embodiments, the integrin targeting ligand of structure 2.2a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0057] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0058] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0059] In some embodiments, the integrin targeting ligand of structure 2.3a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0060] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0061] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0062] In some embodiments, the integrin targeting ligand of structure 2.4a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0063] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0064] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0065] In some embodiments, the integrin targeting ligand of structure 2.5a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0066] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0067] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0068] In some embodiments, the integrin targeting ligand of structure 2.6a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0069] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0070] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0071] The reactive group (or a protected reactive group) can be used to facilitate conjugation (either directly or via one or more scaffolds and / or linkers) of an integrin targeting ligand to a molecule of interest, e.g., a cargo molecule.

[0072] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0073] In some embodiments, the integrin targeting ligand of structure 2.7a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0074] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0075] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0076] The reactive group (or a protected reactive group) can be used to facilitate conjugation (either directly or via one or more scaffolds and / or linkers) of an integrin targeting ligand to a molecule of interest, e.g., a cargo molecule.

[0077] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0078] In some embodiments, the integrin targeting ligand of structure 2.8a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0079] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0080] The reactive group (or a protected reactive group) can be used to facilitate conjugation (either directly or via one or more scaffolds and / or linkers) of an integrin targeting ligand to a molecule of interest, e.g., a cargo molecule.

[0081] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0082] In some embodiments, the integrin targeting ligand of structure 2.9a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0083] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0084] The reactive group (or a protected reactive group) can be used to facilitate conjugation (either directly or via one or more scaffolds and / or linkers) of an integrin targeting ligand to a molecule of interest, e.g., a cargo molecule.

[0085] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0086] In some embodiments, the integrin targeting ligand of structure 2.10a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0087] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0088] The reactive group (or a protected reactive group) can be used to facilitate conjugation (either directly or via one or more scaffolds and / or linkers) of an integrin targeting ligand to a molecule of interest, e.g., a cargo molecule.

[0089] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0090] In some embodiments, the integrin targeting ligand of structure 2.11a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0091] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0092] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure:

[0093] [ka] Includes.

[0094] The reactive group (or a protected reactive group) can be used to facilitate conjugation (either directly or via one or more scaffolds and / or linkers) of an integrin targeting ligand to a molecule of interest, e.g., a cargo molecule.

[0095] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0096] In some embodiments, the integrin targeting ligand of structure 28a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0097] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0098] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0099] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0100] In some embodiments, the integrin targeting ligand of structure 29a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0101] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0102] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0103] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0104] In some embodiments, the integrin targeting ligand of structure 30a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0105] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0106] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0107] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0108] In some embodiments, the integrin targeting ligand of structure 31a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0109] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0110] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0111] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0112] In some embodiments, the integrin targeting ligand of structure 32a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0113] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0114] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0115] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0116] In some embodiments, the integrin targeting ligand of structure 33a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0117] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0118] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0119] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0120] In some embodiments, the integrin targeting ligand of structure 34a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0121] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0122] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0123] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0124] In some embodiments, the integrin targeting ligand of structure 36a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0125] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0126] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0127] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0128] In some embodiments, the integrin targeting ligand of structure 37a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0129] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0130] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0131] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0132] In some embodiments, the integrin targeting ligand of structure 38a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0133] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0134] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0135] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0136] In some embodiments, the integrin targeting ligand of structure 39a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0137] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0138] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0139] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0140] In some embodiments, the integrin targeting ligand of structure 40a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0141] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0142] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0143] In some embodiments, the integrin targeting ligand disclosed herein has the following structure: [ka] Includes.

[0144] In some embodiments, the integrin targeting ligand of structure 41a is linked to one or more cargo molecules (eg, RNAi agent(s)).

[0145] In some embodiments, the integrin targeting ligand may be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure: [ka] where X comprises a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent).

[0146] In some embodiments, the integrin targeting ligand may be synthesized to include an azide reactive group and has the following structure: [ka] Includes.

[0147] The azide reactive group disclosed in any of Structure 1c, Structure 2c, Structure 2.1c, Structure 2.2c, Structure 2.3c, Structure 2.4c, Structure 2.5c, Structure 2.6c, Structure 2.7c, Structure 2.8c, Structure 2.9c, Structure 2.10c, Structure 2.11c, Structure 28c, Structure 29c, Structure 30c, Structure 31c, Structure 32c, Structure 33c, Structure 34c, Structure 36c, Structure 37c, Structure 38c, Structure 39c, Structure 40c, and Structure 41c can be used to attach an integrin targeting ligand to a molecule of interest, i.e., a cargo molecule such as an RNAi agent. The cargo molecule can be any molecule that is desired to be targeted to a cell expressing an integrin.

[0148] As used herein, the term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms, unless otherwise specified. 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, mono-substituted, or di-substituted. Non-limiting examples of aminoalkyl groups include aminomethyl, dimethylaminomethyl, and 2-aminoprop-1-yl.

[0149] As used herein, the term "cycloalkyl" means a saturated or unsaturated non-aromatic hydrocarbon ring group having 3 to 14 carbon atoms, unless otherwise specified. Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, and cyclohexyl. Cycloalkyl groups may contain multiple spiro- or fused rings. Cycloalkyl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by normal valences.

[0150] As used herein, the term "alkenyl" refers to a straight or branched chain non-aromatic hydrocarbon group containing at least one carbon-carbon double bond and having 2 to 10 carbon atoms, unless otherwise specified. Up to 5 carbon-carbon double bonds may be present in such a group. For example, "C2-C6" alkenyl is defined as an alkenyl group having 2 to 6 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, and cyclohexenyl. The straight, branched, or cyclic portions of the alkenyl group may contain double bonds and are optionally mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted 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.

[0151] As used herein, the term "alkynyl" refers to a straight or branched chain hydrocarbon group containing 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond, unless otherwise specified. Up to five carbon-carbon triple bonds may be included. Thus, "C2-C6 alkynyl" means an alkynyl group 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 at any position as permitted by normal valences.

[0152] As used herein, "alkoxyl" or "alkoxy" refers to an -O-alkyl group having the indicated number of carbon atoms. For example, C1-C6 alkoxy is intended to include C1, C2, C3, C4, C5, and C6 alkoxy groups. For example, C1-C8 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.

[0153] 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., ethinoyl, propinoyl, butynoyl, pentinoyl, or hexinoyl), aryloyl (e.g., benzoyl), heteroaryloyl (e.g., pyrrolyl, imidazoloyl, quinolinyl, or pyridinoyl).

[0154] 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, but are not limited to, methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-propoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, or n-pentoxycarbonyl.

[0155] As used herein, "aryloxycarbonyl" refers to an aryl group as defined herein attached through an oxycarbonyl bridge (i.e., -C(O)O-aryl). Examples of aryloxycarbonyl groups include, but are not limited to, phenoxycarbonyl and naphthyloxycarbonyl.

[0156] 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.

[0157] As used herein, "aryl" or "aromatic" refers to any stable monocyclic or polycyclic carbocyclic ring of up to 6 atoms in each ring, with at least one ring being aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl. Anthracenyl, tetrahydronaphthyl, indanyl, and biphenyl. When the aryl group is bicyclic and one ring is non-aromatic, it is understood that the bond is via the aromatic ring. The aryl group is optionally mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by normal valences.

[0158] As used herein, the term "heteroaryl" refers to a stable monocyclic or polycyclic 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, benzimidazolonyl, benzoxazolyl, quinolinyl, isoquinolinyl, dihydroisoindolonyl, imidazopyridinyl, isoindolinyl, 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. When a heteroaryl group is bicyclic and one ring is non-aromatic or does not contain a heteroatom, attachment is understood to be through the aromatic ring or through the heteroatom-containing ring. Heteroaryl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by normal valences.

[0159] As used herein, the term "heterocycle," "heterocyclic," or "heterocyclyl" refers to a 3- to 14-membered aromatic or non-aromatic heterocycle containing 1 to 4 heteroatoms selected from the group consisting of O, N, and S, including polycyclic groups. As used herein, the term "heterocyclic" is also considered synonymous with the terms "heterocycle" and "heterocyclyl," and is understood to have the same definition as described herein. "Heterocyclyl" includes the heteroaryls described above, as well as dihydro and tetrahydro analogs thereof.Examples of heterocyclyl groups include, but are not limited to, azetidinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthopyridinyl, oxazolyl, and the like. Diazolyl, 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, tetrazo aryl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydroisophenyl ... Heterocyclyl groups include hydroxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dioxidothiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and N-oxides thereof. The attachment of heterocyclyl substituents can occur through a carbon atom or through a heteroatom. Heterocyclyl groups are optionally mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by normal valences.

[0160] As used herein, the terms "treat," "treatment," and the like refer to methods or steps taken to provide relief 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" may include prevention, management, prophylactic treatment, and / or inhibition of the number, severity, and / or frequency of one or more symptoms of a disease in a subject.

[0161] As used herein, phrases such as "delivery to cell" refer to cargo molecule and functionally deliver cargo molecule to cell.The phrase "functionally deliver" refers to deliver cargo molecule to cell in such a manner that cargo molecule can have expected biological activity.When specifically referring to cargo molecule that is RNAi agent, for example, expected biological activity is sequence-specific inhibition of gene expression.

[0162] Unless otherwise stated, as used herein, the following symbols: [ka] The use of means that any group or groups may be linked according to the scope of the invention described herein.

[0163] As used herein, the term "isomer" refers to compounds that have identical molecular formulae but differ in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are called "stereoisomers." Stereoisomers that are not mirror images of one another are called "diastereoisomers," and stereoisomers that are non-superimposable mirror images are also called "enantiomers" or sometimes optical isomers. A carbon atom bonded to four non-identical substituents is called a "chiral center."

[0164] As used herein, a linking group is one or more atoms that connect one molecule or a portion of a molecule to another second molecule or a second portion of a molecule. In the art, the terms linking group and spacer are sometimes used interchangeably. Similarly, when used in the art, the term scaffold is sometimes used interchangeably with linking group. In some embodiments. In some embodiments, linking group can include or consist of a PEG group or PEG moiety.

[0165] As used herein, the terms "linked" or "conjugated" when referring to a connection between two molecules means that the two molecules are joined by a covalent bond or that the two molecules are associated through a non-covalent bond (e.g., hydrogen bond or ionic bond). In some examples where the term "linked" refers to an association between two molecules through a non-covalent bond, the association between two different molecules may be at least 1x10 in a physiologically acceptable buffer (e.g., phosphate buffered saline). -4 Less than M (e.g. 1x10 -5 Less than M, 1x10 -6 Less than M or 1x10 -7 M or less). Unless stated otherwise, as used herein, the term linked may refer to a connection between a first compound and a second compound, with or without any intervening atoms or groups of atoms.

[0166] Those skilled in the art will readily understand and appreciate that the compounds and compositions disclosed herein may contain 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, the structures disclosed herein contemplate that certain functional groups, such as, for example, OH, SH, or NH, may be protonated or deprotonated. The disclosure herein is intended to cover the disclosed compounds and compositions regardless of their state of protonation based on the pH of the environment, as will be readily understood by those skilled in the art.

[0167] When used in the claims of this application, the phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. When used in the claims of this application, 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.

[0168] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Similar or equivalent methods and materials to those described herein can be used to carry out or test the present invention, but suitable methods and materials are described below. All publications, patent applications, patents, and other references described herein are incorporated by reference in their entirety. In case of discrepancy, the present specification, including definitions, will control. In addition, the materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0169] Multilocus αvβ3 integrin ligands and scaffolds As disclosed herein, in some embodiments, one or more αvβ3 / 5 integrin ligands may be linked to one or more cargo molecules. In some embodiments, only one integrin ligand is conjugated to a cargo molecule (referred to herein as a "monodentate form" or "monovalent" ligand). In some embodiments, two integrin ligands are conjugated to a cargo molecule (referred to herein as a "bidentate" or "bivalent" targeting group). In some embodiments, three integrin ligands are conjugated to a cargo molecule (referred to herein as a "tridentate" or "trivalent" targeting group). In some embodiments, four integrin ligands are conjugated to a cargo molecule (referred to herein as a "tetradentate" or "tetravalent" targeting group). In some embodiments, five or more integrin ligands are conjugated to a cargo molecule.

[0170] In some embodiments, when only one integrin ligand is conjugated to a cargo molecule (referred to herein as a "monodentate" ligand), the integrin ligand may be directly conjugated to the cargo molecule. In some embodiments, the integrin ligands disclosed herein are conjugated to the cargo molecule via a scaffold or other linker structure.

[0171] In some embodiments, the integrin ligands disclosed herein include 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 integrin ligands disclosed herein. Useful scaffolds compatible with the ligands disclosed herein are widely known in the art. Non-limiting examples of scaffolds that can be used with the αvβ3 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 includes a cysteine ​​linker or group, DBCO-PEG, 1-24 -NHS, propargyl-PEG 1-24 -NHS, and / or multidentate DBCO and / or propargyl moieties may also be mentioned.

[0172] In some embodiments, the scaffold used to link one or more integrin ligands disclosed herein to one or more cargo molecules has the following structure: [ka] has.

[0173] For example, the use of scaffold 1 facilitates effective conjugation of both integrin ligand monomers and one or more cargo molecules. Scaffold 1 contains an amine-reactive p-nitrophenol (also called 4-nitrophenol) ester, an amide linkage, and three PEG2 unit arms, and a terminal alkyne. The 4-nitrophenol ester can be conjugated by amide formation to a primary amine on a cargo molecule, such as a primary amine on an RNA trigger constructed with a terminal amine group (e.g., (NH2-(CH2)6). The terminal alkyne is conjugated to an azide-modified ligand (both peptides and small molecules) by copper-catalyzed click chemistry.

[0174] In some embodiments, the cargo molecule is an RNAi agent. In some embodiments, scaffold 1 may be attached to an end of an RNAi agent, for example, the 5' end of the sense strand of the RNAi agent. For example, the 5' end of the sense strand of an RNAi agent may be modified to include a C6 amine (-(CH2)6-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 resulting in a terminal amine) has the following structure: [ka] {wherein: [ka] can be readily conjugated to scaffold 1, as indicated by the designations in {denoting the RNAi agent}.

[0175] The alkyne group of structure 380 above can then be conjugated to an integrin ligand disclosed herein to form a tridentate integrin targeting group.

[0176] In some embodiments, the scaffold may be synthesized using DBCO (dibenzocyclooctyne), which has the following structure: [ka] {wherein: [ka] indicates attachment to a moiety that contains a reactive group or cargo molecule.

[0177] In some embodiments, the triazole group has the following general structure: [ka] {wherein: [ka] shows any suitable scaffold or linker that can be used to attach a ligand to an RNAi agent, and includes the following: [ka] represents the RNAi agent} are formed between an RNAi agent and an integrin ligand disclosed herein.

[0178] In some embodiments, the scaffold may be synthesized as a phosphoramidite compound, an example of which is shown in the following structure: [ka] As shown in.

[0179] The trialkyne compound of structure 400 allows the tridentate ligand to be readily attached to the 5' end of the sense strand of an RNAi agent by click reaction of an azide-containing targeting ligand with an alkyne.

[0180] In some embodiments, integrin targeting groups disclosed herein include structure 1a, structure 2a, structure 2.1a, structure 2.2a, structure 2.3a, structure 2.4a, structure 2.5a, structure 2.6a, structure 2.7a, structure 2.8a, structure 2.9a, structure 2.10a, structure 2.11a, structure 28a, structure 29a, structure 30a, structure 31a, structure 32a, structure 33a, structure 34a, structure 36a, structure 37a, structure 38a, structure 39a, structure 40a, and structure 41a, wherein the αvβ3 integrin targeting group is a tridentate targeting group and comprises three ligands.

[0181] In some embodiments, the αβ triangular targeting group disclosed herein comprises three ligands of structure 2a and has the following structure: [ka] It is represented by:

[0182] In some embodiments, a tridentate targeting group disclosed herein comprises three ligands of structure 2a and has the following structure: [ka] It is represented by:

[0183] In some embodiments, a tridentate targeting group disclosed herein comprises three ligands of structure 2a and has the following structure: [ka] It is represented by:

[0184] In some embodiments, the tridentate targeting group comprising a glutaric acid linker comprises three ligands of structure 2a and has the following structure: [ka] It is represented by:

[0185] In some embodiments, a tridentate targeting group disclosed herein comprises three ligands of structure 2a and has the following structure: [ka] {wherein: [ka] indicates an RNAi agent, and X=O or S}.

[0186] In some embodiments, a tridentate targeting group disclosed herein comprises three ligands of structure 2a and has the following structure: [ka] {wherein: [ka] represents any suitable scaffold or linker that can be used to connect the ligand and cargo molecule.

[0187] In some embodiments, the αvβ3 triangular targeting group conjugated to the RNAi agent comprises three ligands of structure 2a and has the following structure: [ka] {wherein: [ka] shows any suitable scaffold or linker that can be used to connect the ligand and the RNAi agent, and the following: [ka] indicates the RNAi agent}.

[0188] Reactive Groups and Protected Reactive Groups Reactive groups are well known in the art and provide for the formation of a covalent bond between two molecules or reactants. Reactive groups suitable for use within the scope of the invention herein include, but are not limited to: amino groups, amide groups, carboxylic acid groups, azides, alkynes, propargyl groups, BCN (bicyclo[6.1.0]nonyne, DBCO (dibenzocyclooctyne)thiol, maleimide groups, aminooxy groups, N-hydroxysuccinimide (NHS) or other activated esters (e.g., PNP, TFP, PFP), bromo groups, aldehydes, carbonates, tosylates, tetrazines, trans-cyclooctene (TCO), hydrazides, hydroxyl groups, disulfides, and orthopyridyl disulfide groups.

[0189] The incorporation of reactive groups can facilitate the conjugation of the integrin ligands disclosed herein to cargo molecules.Conjugation reactions are well known in the art and provide for the formation of a covalent bond between two molecules or reactants.Conjugation reactions suitable for use within the scope of the invention herein include, but are not limited to, amide coupling reactions, Michael addition reactions, hydrazone formation reactions, and click chemistry cycloaddition reactions.

[0190] In some embodiments, the integrin targeting ligands disclosed herein are synthesized as tetrafluorophenyl (TFP) esters, which can be substituted with reactive amino groups to conjugate to cargo molecules. In some embodiments, the integrin targeting ligands disclosed herein are synthesized as azides, which can be coupled to propargyl or DBCO, for example, by click chemistry cycloaddition to attach cargo molecules.

[0191] Protected reactive groups are also commonly used in the art. Protecting groups provide a temporary chemical transformation of a reactive group into a group that does not react under the conditions in which the unprotected group reacts, for example, to provide chemoselectivity in a subsequent chemical reaction. Protected reactive groups suitable for use within the scope of the invention herein 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-pentamethylpentamethyldihydrobenzofuran-5-sulfonyl).

[0192] Cargo molecules (including RNAi agents) A cargo molecule is any molecule that, when removed from the integrin ligand described herein, will have a desired effect on cells that contain integrin receptors. A cargo molecule can be, but is not limited to, a pharmaceutical compound, a pharmaceutical product, a prodrug, a substance with a therapeutic effect, 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 integrin ligands to target the cargo molecule to cells expressing integrin αvβ3 and / or integrin αvβ5.

[0193] In some embodiments, the one or more cargo molecules are pharmaceutical ingredients or pharmaceutical compositions. In some embodiments, the one or more cargo molecules are oligonucleotide-based compounds. As used herein, an "oligonucleotide-based compound" refers to an oligonucleotide-based 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 5 ... 28, 12-26, 12-24, 12-22, 12-20, 12-18, 12-16, 12-14, 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, 2 2~46, 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, 34~3 8, 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, 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 (e.g., a gene transcript or mRNA of the target gene) in a cell. In some embodiments, the oligonucleotide-based compounds can inhibit expression of basal genes upon delivery to cells expressing the gene, and are referred to herein as "expression-inhibiting oligonucleotide-based compounds." Gene expression can be inhibited in vitro or in vivo.

[0194] "Oligonucleotide-based compounds" include, but are not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short or small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and dicer substrates. In some embodiments, the oligonucleotide-based compounds are single-stranded oligonucleotide-based compounds, such as antisense oligonucleotides. In some embodiments, the oligonucleotide-based compounds are double-stranded oligonucleotide-based compounds. In some embodiments, the oligonucleotide-based compounds are double-stranded oligonucleotides that are RNAi agents.

[0195] In some embodiments, one or more cargo molecules are "RNAi agents", and an RNAi agent as defined herein is a chemical composition comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule, which can inhibit the degradation of messenger RNA (mRNA) transcripts of target mRNA or their translation in a sequence-specific manner. As used herein, an RNAi agent can operate via an RNA interference mechanism (i.e., induce RNA interference via interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells) or by any alternative mechanism or pathway. As the term is used herein, an RNAi agent is believed to operate primarily via an RNA interference mechanism, but the disclosed RNAi agents are not constrained or limited to any particular pathway or mechanism of action. The RNAi agents disclosed herein are comprised of a sense strand and an antisense strand, and include, but are not limited to: short or small interfering RNA (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.

[0196] Typically, an RNAi agent may be comprised of at least a sense strand (also referred to as a passenger strand) that includes a first sequence, and an antisense strand (also referred to as a guide strand) that includes a second sequence. The length of the sense and antisense strands of an RNAi agent may each be 16-49 nucleotides long. In some embodiments, the sense and antisense strands of an RNAi agent are independently 17-26 nucleotides long. In some embodiments, the sense and antisense strands are independently 19-26 nucleotides long. In some embodiments, the sense and antisense strands are independently 21-26 nucleotides long. In some embodiments, the sense and antisense strands are independently 21-24 nucleotides long. In some embodiments, the sense and antisense strands are each 21 nucleotides long. The sense and antisense strands may be the same length or different lengths. An RNAi agent comprises an antisense strand sequence that is at least partially complementary to a sequence in a target gene, and upon delivery to a cell expressing the target, the RNAi agent can inhibit expression of one or more target genes in vivo or in vitro.

[0197] Oligonucleotide-based compounds in general, and RNAi agents in particular, 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 ribonucleotide (2'-hydroxyl nucleotide).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 nucleotides are modified nucleotides. As used herein, modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimetics, abasic nucleotides, 2'-modified nucleotides, 3' to 3' linked (inverted) nucleotides, non-natural base-containing nucleotides, bridged nucleotides, peptide nucleic acids, 2',3'-seconucleotide mimetics (non-locked nucleobase analogues, locked nucleotides, 3'-O-methoxy (2' internucleoside linkage) nucleotides), 2'-F-arabinonucleotides, 5'-Me,2'-fluoro nucleotides, morpholino nucleotides, vinyl phosphonate deoxyribonucleotides, vinyl phosphonate-containing nucleotides, and cyclopropyl phosphonate-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.

[0198] Also, one or more nucleotides of an oligonucleotide-based compound, such as an RNAi agent, may be linked by a non-standard bond or backbone (i.e., a modified internucleotide bond or a modified backbone). The modified internucleotide bond may be a non-phosphate-containing covalent internucleotide bond. Modified internucleotide linkages or backbones include, but are not limited to, 5'-phosphorothioate groups, chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g., methyl phosphonates or 3'-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-amino phosphoramidates, aminoalkyl phosphoramidates, or thionophosphoramidates), thionoalkyl-phosphonates, thionoalkyl phosphotriesters, morpholino linkages, boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates with inverted polarity in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.

[0199] It is not necessary for all positions in a given compound to be uniformly modified, rather, more than one modification can be incorporated into a single oligonucleotide-based compound or even into a single nucleotide thereof.

[0200] In some embodiments, the cargo molecule is an RNAi agent for inhibiting HIF-2α (EPAS1) gene expression.The cargo molecule may be the RNAi agent described in International Publication No. WO2016 / 196239 and International Publication No. WO2014 / 134255 (incorporated herein in its entirety by reference).

[0201] 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 RNAi agent for inhibiting HIF-2α expression can be found in, for example, International Publication No. WO2016 / 196239 (its entirety is incorporated herein by reference).

[0202] In some embodiments, the cargo molecule or molecules may comprise or consist of a PEG moiety that can act as a pharmacokinetic (PK) enhancer or modulator. In some embodiments, the cargo molecule or molecules may comprise a PEG moiety, which comprises 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, 100-80 ... 00~750, 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, 250~900, 250~850, 250~80 0, 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, 350~850, 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, 450~600, 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, 600~750, 600~700, 600~650, 650~90 0, 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, the one or more cargo molecules are comprised of a PEG moiety having about 455 ethylene oxide units (molecular weight of about 20 kilodaltons (kDa)). In some embodiments, the PEG moiety has a molecular weight of about 2 kilodaltons. In some embodiments, the PEG moiety has a molecular weight of about 20 kilodaltons. In some embodiments, the PEG moiety has a molecular weight of about 40 kilodaltons. The PEG moieties described herein may be linear or branched. The PEG moieties may be discrete (monodisperse) or non-disperse (polydisperse). PEG moieties for use as PK enhancing cargo molecules can be purchased commercially. In some embodiments, one or more cargo molecules include a PEG moiety that can act as a PK modulator or enhancer, as well as a different cargo molecule, e.g., a pharma- ceutically active ingredient or compound.

[0203] The integrin ligands described include salts or solvates. Solvates of the integrin ligands are understood to mean the addition of inert solvent molecules to the integrin ligands which form due to their mutual attractive force. Solvates are, for example, mono- or dihydrates or addition compounds with alcohols, such as, for example, methanol or ethanol.

[0204] Free amino or hydroxyl groups can be provided as substituents on the integrin ligand with corresponding protecting groups.

[0205] αvβ3 integrin ligand also includes, for example, derivatives, ie, integrin ligands that are modified, for example, with alkyl or acyl groups, sugars or oligopeptides, which are cleaved either in vitro or in vivo.

[0206] In some embodiments, the integrin ligands disclosed herein facilitate delivery of cargo molecules to the cytosol of cells that present integrin αvβ3 and / or integrin αvβ5 on their surface, either via ligand-mediated endocytosis, pinocytosis, or by other means. In some embodiments, the integrin ligands disclosed herein facilitate delivery of cargo molecules to the plasma membrane of cells that present integrin αvβ3 and / or integrin αvβ5.

[0207] Pharmaceutical Compositions In some embodiments, the present disclosure provides pharmaceutical compositions comprising, consisting of, or consisting essentially of one or more of the integrin ligands disclosed herein.

[0208] As used herein, a "pharmaceutical composition" contains a pharmacologically effective amount of an active pharmaceutical ingredient (API), and optionally one or more pharma- ceutical acceptable excipients. A pharma-ceutical acceptable excipient (vehicle) is a substance other than an active pharmaceutical ingredient (API, therapeutic product) 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 dose. An excipient may act to a) aid in the processing of the drug delivery system during manufacture, b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API, c) aid in product identification, and / or d) enhance any other attribute of the overall safety, efficacy, or delivery of the API during storage or use. A pharma-ceutical acceptable excipient may or may not be an inert substance.

[0209] Excipients include, but are not limited to: absorption enhancers, anti-adherents, antifoaming agents, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water repellents, and wetting agents.

[0210] The pharmaceutical compositions described herein may contain other additional components commonly found in pharmaceutical compositions. In some embodiments, the additional components are pharma- ceutical active substances. Pharmaceutically active substances include, but are not limited to: antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.), small molecule drugs, antibodies, antibody fragments, aptamers, and / or vaccines.

[0211] Pharmaceutical compositions may also contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for varying osmotic pressure, buffers, coating agents, or antioxidants. They may also contain other agents with known therapeutic effects.

[0212] The pharmaceutical composition can be administered in many ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be by any method commonly known in the art, including, but not limited to, topical (e.g., by transdermal patch), intrapulmonary (e.g., by inhalation or insufflation of powder or aerosol, including by 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; subcutaneous (e.g., via implantable device), intracranial, intraparenchymal, intrathecal, and intraventricular administration. In some embodiments, the pharmaceutical composition described herein is administered by intravenous injection, infusion or subcutaneous injection. The pharmaceutical composition can be administered orally, for example, in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, liquids, emulsions or suspensions. Administration can also be rectally, for example, using a suppository; topically or transdermally, for example, using ointments, creams, gels, or solutions; or parenterally, for example, using injectable solutions.

[0213] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents in the composition, for example, sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride. Prolonged absorption of injectable compositions is brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0214] Sterile injectable solution can be prepared by incorporating the active compound in the required amount in suitable solvent with one or combination of the above-listed components, and then optionally by sterilization by filtration.Generally, dispersion is prepared by incorporating active compound in a sterile vehicle that contains basic dispersion medium and other necessary components from above-listed.For the preparation of sterile powder for sterile injectable solution, the preparation method includes vacuum drying and freeze-drying, which obtains the powder of active ingredient plus any other desired components from the solution that has been previously sterilized and filtered.

[0215] 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, for example in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems may also be used to present any of the ligands described herein for both intra-articular and ophthalmic administration.

[0216] The active compound can be prepared with a carrier that will protect the compound from rapid excretion from the body, such as a controlled release formulation, 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 of preparing such formulations will be clear to those skilled in the art.Liposomal suspensions can also be used as pharma-ceutically acceptable carriers.These can be prepared according to the method known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0217] The pharmaceutical composition may contain other additional ingredients commonly found in pharmaceutical compositions. Such additional ingredients include, but are not limited to: antipruritic agents, 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 an amount of a pharma- ceutical active agent to produce a pharmacological, therapeutic, or prophylactic result.

[0218] Pharmaceuticals containing an αvβ3 integrin ligand are also an object of the present invention, as are methods for producing such pharmaceuticals, which methods comprise bringing one or more compounds comprising an αvβ3 integrin ligand and, if desired, one or more substances having known therapeutic activity into a pharma- ceutically acceptable form.

[0219] The integrin ligands described herein, and pharmaceutical compositions containing the disclosed integrin ligands, may be packaged or contained in a kit, container, pack, or dispenser. The integrin ligands and pharmaceutical compositions containing the integrin ligands may also be packaged in pre-filled syringes or vials.

[0220] Linking Groups, Pharmacokinetic (PK) Enhancers, Pharmacodynamic (PD) Modulators, Delivery Vehicles, and Targeting Groups In some embodiments, the αvβ3 ligand is conjugated to one or more non-nucleotide groups, including but not limited to a linking group, a pharmacokinetic (PK) enhancer (also called a PK modulator), a pharmacodynamic (PD) modulator, a delivery polymer, or a delivery vehicle. The non-nucleotide group can facilitate targeting, delivery, or attachment of the cargo molecule. Examples of scaffolds for targeting groups and linking groups are disclosed herein. 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 RNAi agent sense strand. The integrin ligands disclosed herein can be directly or indirectly linked to the cargo molecule via a linker / linking group. In some embodiments, the integrin ligand is linked to the cargo molecule via a labile, cleavable, or reversible bond or linker.

[0221] In some embodiments, the non-nucleotide group enhances the pharmacokinetics or biodistribution properties of the RNAi agent or conjugate to which it is attached, so as to improve cell- or tissue-specific distribution and cell-specific uptake of the RNAi agent or conjugate.In some embodiments, the non-nucleotide group enhances the endocytosis of the RNAi agent.In some embodiments, the non-nucleotide group enhances or modulates the pharmacodynamic properties of the RNAi agent or conjugate to which it is attached, so as to improve cell- or tissue-specific distribution and cell-specific uptake of the RNAi agent or conjugate.

[0222] Targeting groups or targeting moieties enhance the pharmacokinetic or biodistribution properties of the cargo molecule to which they are attached to improve 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 αvβ3 ligand as described herein. In some embodiments, the targeting group comprises a linker. In some embodiments, the targeting group comprises a PK enhancer. In some embodiments, the αvβ3 integrin 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, in some cases, serve as a linker). The targeting group may comprise one or more targeting ligands. In some embodiments, the targeting group may comprise one to four integrin ligands as disclosed herein. In some embodiments, the targeting group is a tridentate targeting group and comprises three integrin ligands as disclosed herein.

[0223] Cargo molecules can be synthesized with reactive groups such as amino groups (also referred to herein as amines). In embodiments where the cargo molecule is an RNAi agent, the reactive groups can be linked at the 5'-end and / or 3'-end. The reactive groups can then be used to attach αvβ3 integrin ligands using methods typical in the art.

[0224] For example, in some embodiments, an RNAi agent can be 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 a group that includes, for example, an integrin targeting ligand. In some embodiments, an RNAi agent can be 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 a group that includes, for example, an αvβ3 integrin targeting ligand.

[0225] In some embodiments, a linking group is conjugated to the αvβ3 ligand. The linking group facilitates the covalent attachment of the αvβ3 ligand to a cargo molecule, a PK enhancer, 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 amines and alkynes, alkyl groups, abasic residues / nucleotides, amino acids, trialkyne functional groups, ribitol, and / or PEG groups.

[0226] A linker or linking group is a connection between two atoms that connects one chemical group (such as an RNAi agent) or segment of interest to another chemical group (such as an αvβ3 integrin ligand, a PK enhancer, a PD modulator, or a delivery polymer) or segment of interest through one or more covalent bonds. A labile linkage contains a labile bond. The linkage optionally includes a spacer that increases the distance between the two joined atoms. The spacer may further add flexibility and / or length to the linkage. 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 can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides. Spacer groups are well known in the art, so the above list is not meant to limit the scope of the description.

[0227] In some embodiments, αvβ3 ligand is linked to cargo molecule without using additional linker.In some embodiments, αvβ3 ligand is designed with linker that presents simple and easy to link to cargo molecule.In some embodiments, when two or more RNAi agents are included in the composition, two or more RNAi agents are linked to their respective targeting groups using the same linker.In some embodiments, when two or more RNAi agents are included in the composition, two or more RNAi agents are linked to their respective targeting groups using different linkers.

[0228] Examples of specific linking groups and scaffolds are provided in Table A.

[0229] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12]

[0230] wherein: [ka] indicates the point of attachment to the cargo molecule.

[0231] Alternatively, other linking groups known in the art may be used. Examples of suitable linking groups are provided in PCT Application No. PCT / US19 / 18232, which is incorporated herein by reference in its entirety.

[0232] The embodiments and articles provided above are illustrated herein by the following non-limiting examples.

[0233] Internally linked targeting ligands In some embodiments, when an integrin targeting ligand described herein is bound or linked to an RNAi molecule, the integrin targeting ligand may be bound to an internal nucleotide of the sense strand or antisense strand. In some embodiments, up to 15 targeting ligands may be conjugated to internal nucleotides on the sense strand of the RNAi agent. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 targeting ligands may be conjugated to internal nucleotides on the sense strand of the HIF-2α RNAi agent. In some embodiments, 1-5 (e.g., 1, 2, 3, 4, or 5) targeting ligands are conjugated to internal nucleotides on the sense strand of the RNAi agent. In some embodiments, 3-4 targeting ligands are conjugated to internal nucleotides on the sense strand of the RNAi agent.

[0234] In some embodiments, the displacement of internal targeting ligand can affect the effectiveness or potency of RNAi agent.In some embodiments of the αvβ3 integrin targeting ligand that is bound to RNAi agent, the targeting group is conjugated to the 5'-end of the sense strand, and at least 10 nucleotides are arranged between the tridentate targeting group located at the 5'-end of the sense strand and the next closest targeting ligand located on the sense strand.In some embodiments, at least 5 nucleotides are arranged between the tridentate targeting group located at the 5'-end of the sense strand and the next closest targeting ligand located on the sense strand.

[0235] In some embodiments, where two or more targeting ligands are conjugated to the internal nucleotide located in the sense strand of the RNAi agent, there is at least one gap of nucleotide that is not conjugated to the targeting ligand located between two internal nucleotides that are conjugated to the targeting ligand.In some embodiments, where two or more targeting ligands are conjugated to the sense strand of the RNAi agent, at least two nucleotides that are not conjugated to the targeting ligand are located between two internal nucleotides that are conjugated to the targeting ligand.

[0236] In some embodiments, the targeting ligand is conjugated to the second, fourth, and sixth nucleotides on the sense strand, numbered from 3' to 5', starting from the 3' furthest nucleotide that base pairs with the nucleotide on the antisense strand. In some embodiments, the targeting ligand is conjugated to the second, fourth, sixth, and eighth nucleotides (3'→5') from the 3' terminal nucleotide on the sense strand that base pairs with the antisense strand.

[0237] Examples of modified nucleotides for attaching internal targeting ligands are shown in Table B below:

[0238] [Table 2-1] [Table 2-2] EXAMPLES

[0239] The following examples are intended to be non-limiting and to illustrate certain embodiments disclosed herein.

[0240] Example 1. Synthesis of integrin targeting ligands. Some of the abbreviations used in the Experimental Details of the Synthesis of the Examples below are defined as follows: h or hr = hour(s); min = minute; mol = mole(s); mmol = millimole(s); M = mole; μM = micromole; g = gram(s); μg = microgram(s); rt or RT = room temperature; L = liter(s); mL = milliliter(s); wt = weight; Et2O = diethyl ether; THF = tetrahydrofuran; DMSO = dimethylsulfoxide; EtOAc = ethyl acetate; Et3N or TEA = triethylamine; i-Pr2NEt, DIPEA or DIEA = diisopropylethylamine; CH2Cl2 or DCM = methylene chloride; CHCl3 = chloroform; CDCl3 = deuterated chloroform; CCl4 = carbon tetrachloride; MeOH = methanol; EtOH = ethanol; DMF = dimethylformamide. d;BOC=t-butoxycarbonyl;CBZ=benzyloxycarbonyl;TBS=t-butyldimethylsilyl;TBSC1 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=hydroxyl potassium fluoride;NH4OH=ammonium hydroxide;NH4Cl=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;MeOH=methanol;Ar=argon;N2=nitrogen;SiO2=silica;R T = retention time; PTSA = para-toluenesulfonic acid; PPTS = pyridinium para-toluenesulfonate.

[0241] Synthesis of Structure 1c ((S)-3-(6-((1-azido-15-oxo-3,6,9,12-tetraoxa-16-azanonadecane-19-yl)oxy)pyridin-3-yl)-3-(2-oxo-3-(3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl)imidazolidin-1-yl)propanoic acid) [ka]

[0242] A mixture containing compound 1 (1.03 g, 8.23 ​​mmol), compound 2 (0.92 g, 14.8 mol), and PTSA hydrate (156 mg, 0.82 mmol) in benzene (25 mL) was refluxed overnight in a Dean-Stark apparatus. The next morning, the reaction mixture was poured into saturated sodium bicarbonate and ethyl acetate was subsequently added. The organic phase was separated, filtered through sodium sulfate, and concentrated to give compound 3 in 95% yield, which was subsequently used without further purification. [ka]

[0243] To a solution containing compound 4 (5.39 g, 53.3 mmol) and 3 Å molecular sieves in DMF (100 mL) was added sodium hydride (60 wt%, 2.13 g, 53.3 mmol) and the reaction was stirred for 1 h. A solution of compound 3 (7.52 g, 7.52 g) in DMF (20 mL) was subsequently added and the suspension was heated at 80° C. overnight. Upon completion, the suspension was filtered through a cotton plug and concentrated under reduced pressure. The residue was partitioned between diethyl ether and water and the organic phase was separated, filtered through sodium sulfate and concentrated under reduced pressure. The residue was treated with 20 ml of 10% H2O in TFA and stirred for 30 min. Upon completion, the solution was cooled to 0° C. and the pH was adjusted to 11 with 6 M NaOH whereupon the product precipitated as an oil. Compound 5 was extracted from the oily suspension three times with diethyl ether. The organic phases were combined, filtered through sodium sulfate, and concentrated. Compound 5 was then isolated in 26% yield by separation on silica eluted with a gradient of ethyl acetate in hexane. [ka]

[0244] A mixture containing compound 5 (2.29 g, 9.94 mmol), compound 6 (4.82 g, 39.8 mmol), PPTS (125 mg, 0.50 mmol), magnesium sulfate (3 g, 24.9 mmol), copper sulfate (3.97 g, 24.9 mmol), and 3 Å molecular sieves in DCM (22 mL) was heated at reflux overnight. Upon completion, the mixture was filtered and concentrated under reduced pressure. Compound 7 was then isolated in 76% yield by separation on silica eluting with a gradient of ethyl acetate in hexanes. [ka]

[0245] A flame-dried flask was charged with THF (40 mL) and diisopropylamine (2.29 g, 22.6 mmol). It was cooled to -20 °C and n-BuLi (2.5 M, 8.64 mL, 21.6 mmol) was added via cannula. The solution was stirred at -20 °C for 10 min and then cooled to -78 °C. Compound 8 (2.02 mL, 20.6 mmol) was added dropwise with vigorous stirring. After the addition, the solution was stirred at -78 °C for 30 min. ClTi(iPrO)3 ​​(11.26 g, 43.2 mmol) as a solution in THF (10 mL) was then added via addition funnel over approximately 10 min with vigorous stirring. The reaction was stirred at -78 °C for 30 min. Finally, compound 7 (2.29 g, 6.86 mmol) was added dropwise as a suspension in THF and stirred at -78°C for 1.25 hours until the reaction was complete. To the reaction at -78°C was added saturated aqueous ammonium chloride solution. The reaction was then removed from cooling and quenched by slowly thawing the aqueous phase (yellow-orange color disappeared). The mixture was partitioned between EtOAc and saturated aqueous ammonium chloride solution. The organic phase was separated and the aqueous phase was extracted twice with EtOAc. The organic phases were combined, washed with brine, then dried over sodium sulfate, then filtered and concentrated. The residue was purified by silica eluting with a gradient of ethyl acetate in hexanes. After purification, compound 9 was obtained as a single diastereomer in 75% yield. [ka]

[0246] Compound 9 (1.28 g, 3.21 mmol) in MeOH (3.2 mL) was treated with HCl in dioxane (4 M, 3.2 mL, 12.9 mmol) and stirred at room temperature for 30 min. Upon completion, the reaction mixture was diluted with water and washed with diethyl ether. Subsequently, the pH was adjusted to 11 using 2N aqueous NaOH solution and the product was extracted with ethyl acetate. The organic phase was dried over sodium sulfate, filtered and concentrated to give compound 10 in 92% yield, which was subsequently used without further purification. [ka]

[0247] To a mixture of compound 10 (0.78 g, 2.67 mmol) and compound 11 (0.60 g, 3.46 mmol) in THF (6 mL) at 15° C., STAB-H (1.29 g, 6.12 mmol) was added portionwise as a solid. After the addition, the cooling was removed and the mixture was stirred for about 2.5 hours until completion. The reaction was quenched by the addition of a saturated aqueous solution of sodium bicarbonate and the pH was brought to 9. The product was extracted three times with EtOAc, and the organic phases were combined, dried with brine, then filtered through sodium sulfate and concentrated. Compound 12 was isolated in 85% yield by separation on silica eluting with a gradient of ethyl acetate in hexane. [ka]

[0248] To DIPEA (7.53 mL, 53.75 mmol) in THF (35 mL) was added n-BuLi (2.5 M, 19.9 mL, 49.8 mmol) via an oven-dried gas-tight syringe at -10 °C over 2 min. The mixture was stirred at -10 °C for 10 min, then cooled to -60 °C, and a solution of dimethyl methylphosphonate (6.42 g, 51.8 mmol) in THF (8 mL) was added dropwise over 5-10 min. After aging at -60 °C for approximately 1 h, compound 13 (7.37 g, 39.82 mmol) was added dropwise over 5 min at -60 °C as a solution in THF (15 mL). The reaction mixture was stirred at approximately -60 °C for 1 h and then at -41 °C for 1.5 h. The reaction was quenched by the addition of 2.6 equivalents of H2SO4 (2.0M) and extracted three times with ethyl acetate (~50 mL). The organic phases were combined, dried with brine, filtered through sodium sulfate, and briefly concentrated to weigh the crude product and obtain a sample for NMR. Once the dry weight was determined, compound 14 was dissolved in MeOH for use in the next reaction without further purification. A yield of 75.83% was calculated. The wt / wt% of the crude product was 76.3% by NMR. 1 H NMR: 400 MHz CDCl3δ 4.75 (s, 1 H), 3.81 (s, 3 H), 3.78 (s, 3 H), 3.10 - 3.14 (m, 2 H), 3.04 - 3.09 (m, 2 H), 2.68 (t, 2 H), 1.82-1.75 (m, 2 H), 1.44 (s, 9 H). [ka]

[0249] To compound 14 (9.33 g, 30.16 mmol, weight from NMR of ∼12 g crude product) in MeOH (40 mL) was added aqueous NaOH (1.45 g, 36.2 mmol) in water (1.5 mL). The mixture was heated to 50 °C and compound 15 (2.76 g, 22.62 mmol) was added. After stirring for 30 min, a second portion of compound 15 (736 mg, 6.03 mmol) was added and the reaction mixture was stirred at 50 °C overnight. The reaction mixture was then concentrated to an oil and partitioned between 2 volumes of EtOAc and 1 volume of H2O. The organic phase was separated and washed with 1 volume of water. The aqueous washes were combined and back-extracted with EtOAc (2x, 1 volume). The combined organic phase was dried over sodium sulfate, filtered and concentrated. The crude product was dried onto approximately 20 g of silica and compound 16 was isolated in 69% yield by separation on silica eluting with a gradient of ethyl acetate in hexane containing 1% triethylamine. 1 H NMR: 400 MHz CDCl3δ 9.09 (dd, 1 H), 8.17 (dd, 1 H), 8.12 (d, 1 H), 7.46 (dd, 1 H), 7.41 (d, 1 H), 4.78 (s, 1 H), 3.24 (q, 2 H), 3.10 (t, 2 H), 2.12 (quin, 2 H), 1.43 (s, 9 H). [ka]

[0250] A solution of compound 16 (5.98 g, 20.8 mmol) in EtOH (50 mL) was charged with palladium (10% on carbon, 2.22 g, 2.08 mmol) and hydrogen at 1 atm. The reaction mixture was stirred at room temperature overnight. Upon completion, the reaction mixture was filtered through Celite® and concentrated. Compound 17 was isolated in 79% yield by separation on silica eluted with a gradient of ethyl acetate in hexanes containing 1% triethylamine. 1H NMR: 400 MHz CDCl3δ 7.05 (d, 1 H), 6.34 (d, 1 H), 5.48 (s, 1 H), 4.81 (s, 1 H), 3.36 - 3.43 (m, 2 H), 3.16 (q, 2 H), 2.68 (t, 2 H), 2.59 (t, 2 H), 1.90 (dt, 2 H), 1.83 (quin, 2 H), 1.44 (s, 9 H). [ka]

[0251] Compound 17 (4.81 g, 16.53 mmol) was dissolved in 6 M aqueous HCl (16.4 mL) and heated at 42° C. for 2 h. An additional portion of 6 M HCl (2.8 mL) was then added and the reaction mixture was stirred for an additional 2 h. Sodium chloride was added to the reaction, followed by 2 N aqueous NaOH until the product precipitated as an oil (pH was >12). The mixture was extracted three times with 2-butanol. The combined organic phases were dried over sodium sulfate, filtered and concentrated. Compound 18 was obtained in 85% yield and was subsequently used without further purification. 1 H NMR: 400 MHz CDCl3δ 7.06 (d, 1 H), 6.35 (d, 1 H), 4.83 (s, 1 H), 3.35 - 3.46 (m, 2 H), 2.75-2.67 (m, 4 H), 2.58 (t, 2 H), 1.88 - 1.95 (m, 2 H), 1.84-1.76 (m, 4 H). [ka]

[0252] To a solution of triphosgene (85 mg, 0.28 mmol) in THF (0.9 mL) in a flame-dried flask at -10°C, a solution of compound 18 (236 mg, 0.62 mmol) and TEA (0.134 mL, 0.96 mmol) in THF (0.5 mL) was added dropwise. The reaction mixture was allowed to warm to room temperature. After TLC showed the reaction was complete, additional TEA (0.134 mL) was added, followed by compound 12 (166 mg, 0.87 mmol) as a solid. The heterogeneous mixture was heated at 50°C for 2 hours with vigorous stirring. Upon completion, the reaction mixture was quenched with one volume of water and extracted three times with EtOAc. The combined organic phase was dried with brine, filtered through sodium sulfate, and concentrated. Compound 19 was obtained assuming 100% yield and was subsequently used without further purification. [ka]

[0253] To crude compound 19 (assumed 400 mg, 0.62 mmol) dissolved in THF (37 mL) was added H2SO4 (2M, 0.6 mL) and the mixture was stirred at room temperature overnight. The next morning, an additional portion of H2SO4 (0.65 equiv.) was added. After 4 h, the reaction was complete. The reaction mixture was diluted with ethyl acetate. The organic phase was separated and the aqueous phase was back-extracted once with ethyl acetate. The combined organic phases were filtered through sodium sulfate and concentrated. Compound 20 was isolated in 75% yield by separation on silica eluting with a gradient of MeOH in DCM. [ka]

[0254] A suspension of compound 20 (251 mg, 0.47 mmol) and Pd / C (10 wt%, 100 mg, 0.094 mmol) in ethanol (9 mL) was charged with H2 to 1 atm and stirred at 35° C. overnight. Upon completion, the palladium was removed by filtration through Celite®. Compound 21 was isolated in 20% yield as the TFA salt by reverse phase HPLC using a C18 5u 19×250 mm BEH column (Waters Corp.) eluted with a gradient of acetonitrile in H2O containing 1% TFA. [ka]

[0255] To a solution of compound 21 (61 mg, 0.097 mmol) in DCM (275 μL) was added TEA (8 μL, 0.24 mmol) followed by NHS-PEG4-N3 (41.4 mg, 0.11 mmol) as a solution in DCM (250 μL). The reaction mixture was stirred for 15 min and LC-MS was checked which indicated the reaction was complete. All volatiles were removed and the residue was dissolved in EtOH (0.4 mL) and water (0.4 mL). LiOH (11.2 mg, 0.47 mmol) was added and the reaction mixture was heated at 40° C. for 2 h. Upon completion, the reaction mixture was concentrated under reduced pressure. Compound 22 (structure 1c) was isolated in 42% yield by reverse phase HPLC using a C18 5u 19×250 mm BEH column (Waters Corp.) eluted with a gradient of acetonitrile in H2O containing 1% TFA.

[0256] Synthesis of structure 2c ((S)-3-(4-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-3-fluorophenyl)-3-(2-oxo-3-(3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl)imidazolidin-1-yl)propanoic acid). [ka]

[0257] To a solution of compound 23 (10 g, 43.4 mmol) in toluene (80 mL) was added compound 6 (21.1 g, 0.17 mol), PPTS (0.55 g, 2.2 mmol), followed by acetic acid (1.24 mL, 21.7 mmol). The reactor was equipped with a Dean-Stark trap and then heated to reflux overnight. Upon completion, the reaction mixture was concentrated, dried over 60 grams of silica, and purified by SiO2 using a gradient of ethyl acetate in hexane to give compound 24 in 66% yield. 1 H NMR: 400 MHz CDCl3δ 8.47 (s, 1 H), 7.68 (d, 1 H), 7.31 - 7.56 (m, 6 H), 6.98 - 7.16 (m, 1 H), 5.23 (s, 2 H), 1.26 (s, 9 H). [ka]

[0258] A flame dried flask was charged with THF (190 mL) and DIPEA (9.07 g, 89.7 mmol), cooled to -20 °C, then n-BuLi (2.5 M, 34.2 mL, 85.6 mmol) was added via cannula. The solution was stirred at -20 °C for 10 min, then cooled to -78 °C. Compound 8 (8 mL, 81.5 mmol) was added dropwise with vigorous stirring. After addition, the mixture was stirred at -78 °C for 30 min. ClTi(iPrO)3 ​​(44.6 g, 0.171 mol) was then added via addition funnel as a solution in THF (40 mL) over 10 min. The reaction was stirred at -78 °C for 30 min. Finally, compound 24 (9.06 g, 27.2 mmol) was added dropwise as a suspension in THF (20 mL) and stirred at -78 °C for 1.25 h until the reaction was complete. To the reaction at -78 °C was added saturated aqueous ammonium chloride. The reaction was then removed from cooling and quenched by slowly thawing the aqueous phase (yellow-orange color disappeared). The mixture was partitioned between EtOAc and saturated aqueous ammonium chloride. The organic phase was separated and the aqueous phase was washed twice with EtOAc. The organic phases were combined, washed through brine, then dried over sodium sulfate, then filtered and concentrated. Separation on silica eluting with a gradient of ethyl acetate in hexanes afforded compound 25 as a single diastereomer in 70% yield. 1 H NMR:400 MHz CDCl3δ 7.31 - 7.48 (m, 5 H), 7.09 (dd, 1 H), 6.89 - 7.04 (m, 2 H), 5.13 (s, 2 H), 4.59 - 4.76 (m, 2 H), 4.13 (q, 2 H), 2.81 (dd, 2 H), 1.21 - 1.25 (m, 12 H). [ka]

[0259] Compound 25 (8.07 g, 19.1 mmol) was added with aqueous HCl (6 M, 20.7 mL, 0.124 mol), followed by MeOH (60 mL). THF was added until a homogeneous solution was obtained, and the reaction mixture was stirred at room temperature for 6 h. The reaction mixture was basified to pH 10 with 2N aqueous NaOH, then extracted three times with EtOAc. The combined organic phases were dried with brine, filtered through sodium sulfate, and concentrated. Compound 26 was obtained in 95% yield, which was subsequently used without further purification. 1 H NMR: 400 MHz CDCl3δ 7.28 - 7.46 (m, 6 H), 7.18 (d, 1 H), 6.99 (t, 1 H), 5.11 (s, 2 H), 4.57 (t, 1 H), 4.09 (q, 2 H), 2.97 - 3.09 (m, 1 H), 2.81 - 2.93 (m, 1 H), 1.18 (t, 3 H). [ka]

[0260] To a mixture of compound 26 (5.76 g, 18.2 mmol) and compound 27 (4.09 g, 23.6 mmol) in THF (40 mL) at 0° C., STAB-H (8.85 g, 41.8 mmol) was added portionwise as a solid. After the last addition, the cooling was removed and the mixture was stirred for about 2.5 h until completion. The reaction mixture was quenched by the addition of a saturated aqueous solution of sodium bicarbonate. The mixture was extracted three times with EtOAc, and the combined organic phases were dried with brine, filtered through sodium sulfate, and concentrated. Compound 28 was isolated in 73% yield by separation on silica eluting with a gradient of ethyl acetate in hexanes. 1H NMR: 400 MHz CDCl3δ 7.30 - 7.49 (m, 5 H), 7.11 (dd, 1 H), 6.88 - 7.02 (m, 2 H), 5.13 (s, 2 H), 4.40 (t, 1 H), 4.10 (q, 2 H), 4.00 (dd, 1 H), 3.35 (s, 3H), 3.31 (s, 3H), 2.47 - 2.75 (m, 4H), 1.20 (t, 3H). [ka]

[0261] To a solution of triphosgene (1.2 g, 4.04 mmol) in THF (24 mL) in a flame-dried flask at -10°C, a solution of compound 19 (3.64 g, 8.99 mmol) and TEA (1.94 mmol, 13.9 mmol) in THF (6 mL) was added dropwise. The reaction mixture was allowed to warm to room temperature. After TLC showed the reaction was complete, additional TEA (3.3 mL, 23.6 mmol) was added, followed by compound 28 (2.61 g, 13.7 mmol) as a solid. The heterogeneous mixture was heated at 50°C with vigorous stirring for 2 h. Upon completion, the reaction mixture was quenched with 1 volume of water and extracted three times with EtOAc. The combined organic phase was dried with brine, filtered through sodium sulfate, and concentrated. Compound 29 was obtained assuming 100% yield, and the crude product was subsequently used without further purification. [ka]

[0262] To compound 29 (5.59 g, 8.97 mmol) dissolved in THF (37 mL) was added water (0.8 mL) and H2SO4 (2M, 8.07 ml, 16.2 mmol) and the reaction mixture was stirred at 28° C. overnight. The next morning, the pH of the mixture was adjusted to 9 using sodium bicarbonate and extracted three times with DCM. The combined organic phases were dried with brine, filtered through sodium sulfate and concentrated. Compound 30 was isolated in 82% yield by separation on silica eluted with a gradient of MeOH in DCM containing 1% TEA. [ka]

[0263] Compound 30 (4.13 g, 7.39 mmol) dissolved in EtOH (30 mL) was charged with Degussa® palladium (10 wt%, 3.15 g, 2.96 mmol) and hydrogen to 50 psi. The mixture was stirred at room temperature overnight. The next day the reaction was 64% complete. The reaction mixture was filtered through Celite® and concentrated. The residue was dissolved in EtOH and charged with palladium (10 wt%, 1.57 g, 1.48 mmol) and hydrogen to 50 psi. After stirring for 48 h, the reaction mixture was heated to 30° C. and stirred for an additional 24 h. Upon completion, the suspension was filtered through Celite® and all volatiles were removed under vacuum. The residue was purified on silica eluting with a gradient of MeOH in DCM to give compound 31 in 72% yield. 1 H NMR: 400 MHz DMSO-d6δ 9.88 (s, 1 H), 7.02 - 7.14 (m, 2 H), 6.86 - 6.93 (m, 2 H), 6.50 - 6.76 (m, 1 H), 6.31 (d, 1 H), 5.17 (t, 1 H), 4.00 (q, 2 H), 3.23 - 3.28 (m, 4 H), 2.79 - 3.18 (m, 7 H), 2.61 (t, 2 H), 2.41 (t, 2 H), 1.65 - 1.78 (m, 4 H), 1.09 (t, 3 H). [ka]

[0264] The solution of DEAD was added dropwise to a solution of PPh3 (699 mg, 2.66 mmol) in THF (0.47 mL) at -10°C. The mixture was warmed to room temperature and added to a neat mixture of compound 31 (600 mg, 1.33 mmol) and HO-PEG4-N3 (466 mg, 3.06 mmol) and stirred overnight. The reaction mixture was then concentrated under reduced pressure and the residue was purified on silica eluted with a gradient of MeOH in DCM to give compound 32 in 50% yield. 1 H NMR: 400 MHz DMSO-d6δ 7.10 - 7.19 (m, 2 H), 6.97 - 7.06 (m, 2 H), 6.18 - 6.31 (m, 2 H), 5.20 (t, 1 H), 4.13 - 4.16 (m, 1 H), 3.98 - 4.04 (m, 2 H), 3.71 - 3.80 (m, 2 H), 3.52 - 3.61 (m, 8 H), 3.38 - 3.37 (m, 5 H), 3.10 - 3.25 (m, 5 H), 2.79 - 3.08 (m, 5 H), 2.59 (t, 2 H), 2.31 - 2.42 (m, 2 H), 1.65 - 1.75 (m, 4 H), 1.10 (t, 3 H). [ka]

[0265] To compound 32 (826 mg, 1.23 mmol) was added EtOH (3 mL) and HO (3 mL), followed by LiOH (97 mg, 4.05 mmol). The mixture was stirred at 30° C. overnight. Upon completion, the mixture was neutralized to pH=5 using 6M aqueous HCl and concentrated. The residue was purified by reverse-phase HPLC using a Phenomenex Gemini C18, 50×250 mm, 10 μm column eluted with a gradient of acetonitrile in water containing 0.1% to give compound 33 (structure 2c) in 81% yield. 1 H NMR: 400 MHz D2O δ 7.30 (d, 1 H), 7.01 - 7.19 (m, 3 H), 6.45 (d, 1 H), 5.24 (t, 1 H), 4.14 - 4.32 (m, 2 H), 3.84 - 3.92 (m, 2 H), 3.59 - 3.77 (m, 10 H), 3.14 - 3.45 (m, 8 H), .02 - 3.12 (m, 1 H), 2.97 (d, 2 H), 2.85 (q, 1 H), 2.50 - 2.72 (m, 4 H), 1.68 - 1.94 (m, 4 H).

[0266] Synthesis of Structure 2.1c ((S)-3-(4-((11-azidoundecyl)oxy)-3-fluorophenyl)-3-(2-oxo-3-(3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl)imidazolidin-1-yl)propanoic acid) [ka]

[0267] To a solution of PPh3 in THF was added the solution of DEAD dropwise at room temperature. The mixture was treated with 31 and OH-(CH2) 11The mixture was transferred to a vial containing a mixture of -N3 and the reaction mixture was stirred at room temperature overnight. Volatiles were removed from the reaction mixture and the crude product was dissolved in EtOH. LiOH was added as a solution in H2O and additional water / EtOH was added until the reaction mixture was homogeneous. After stirring at room temperature for 1.5 h, the mixture was acidified to pH 3 with H2SO4, concentrated and purified by reverse phase HPLC (Phenomenex Gemini C18, 50x250mm, 10μm, acetonitrile / water in 0.1% TFA, gradient elution).

[0268] Synthesis of Structure 2.2c ((S)-3-(4-(2-(1-(6-azidohexanoyl)piperidin-4-yl)ethoxy)-3-fluorophenyl)-3-(2-oxo-3-(3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl)imidazolidin-1-yl)propanoic acid) [ka]

[0269] Compound 35, dissolved in DCM at 0° C., was treated with EDAC and acetonitrile was added to aid solubility. After 5 min, TEA and compound 36 were added, cooling was removed, and stirring was continued for 2 h. Upon completion, saturated ammonium chloride was added and the organic phase was separated, filtered through sodium sulfate, and concentrated. The resulting crude product was subsequently used without further purification. [ka]

[0270] To a solution of PPh3 in THF was added dropwise the solution of DEAD at room temperature with vigorous stirring. The mixture was transferred to a vial containing a mixture of compound 31 and compound 37, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was removed from the volatiles and the crude product was dissolved in EtOH. LiOH was added as a solution in H2O, and additional water was added until the reaction mixture was homogenous. After stirring at room temperature for 1.5 hours, the mixture was acidified to pH 3 with H2SO4, concentrated, and purified by reverse phase HPLC (Phenomenex Gemini C18, 50x250mm, 10μm, acetonitrile / water in 0.1% TFA, gradient elution) to give compound 38 (structure 2.2c).

[0271] Synthesis of Structure 2.3c ((S)-3-(4-(2-((1r,4S)-4-(5-azidopentanamido)cyclohexyl)ethoxy)-3-fluorophenyl)-3-(2-oxo-3-(3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl)imidazolidin-1-yl)propanoic acid) [ka]

[0272] To a suspension of compound 35 in DCM at 0° C., a solution of EDAC in DCM was added. After 5 min, the cooling was removed and compound 39 was added, followed by TEA. The heterogeneous mixture was stirred at room temperature overnight. The next day, the reaction was diluted with DCM and the precipitate was dissolved. The mixture was washed twice with 5% KHSO4 and once with brine. The organic phase was filtered through sodium sulfate and concentrated. The crude residue containing compound 40 was used without further purification. [ka]

[0273] To a solution of PPh3 in THF was added dropwise the solution of DEAD at room temperature with vigorous stirring. The mixture was transferred to a vial containing a mixture of compound 31 and compound 40, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was removed from the volatiles and the crude product was dissolved in EtOH. LiOH was added as a solution in H2O, and additional water was added until the reaction mixture was homogenous. After stirring at room temperature for 1.5 hours, the mixture was acidified to pH 3 with H2SO4, concentrated, and purified by reverse phase HPLC (Phenomenex Gemini C18, 50x250mm, 10μm, acetonitrile / water in 0.1% TFA, gradient elution) to give compound 41 (structure 2.3c).

[0274] Synthesis of Structure 2.4c ((S)-3-(4-(4-(5-azidopentanamido)phenethoxy)-3-fluorophenyl)-3-(2-oxo-3-(3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl)imidazolidin-1-yl)propanoic acid) [ka]

[0275] To a mixture of compound 35 and compound 42 in DCM, EEDQ was added and the solution was stirred at room temperature overnight. The reaction mixture was then diluted with DCM and washed three times with 1M HCl and once with brine. The organic phase was dried over sodium sulfate, filtered and concentrated. Compound 43 was then used without further purification. [ka]

[0276] To a solution of PPh3 in THF was added dropwise the solution of DEAD at room temperature with vigorous stirring. The mixture was transferred to a vial containing a mixture of compound 31 and compound 43, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was removed from the volatiles and the crude product was dissolved in EtOH. LiOH was added as a solution in H2O and additional water was added until the reaction mixture was homogenous. After stirring at room temperature for 1.5 hours, the mixture was acidified to pH 3 with H2SO4, concentrated and purified by reverse phase HPLC (Phenomenex Gemini C18, 50x250mm, 10μm, acetonitrile / water in 0.1% TFA, gradient elution) to give compound 44 (structure 2.4c).

[0277] Synthesis of Structure 2.5c ((S)-3-(4-(4-((5-azidopentyl)oxy)phenethoxy)-3-fluorophenyl)-3-(2-oxo-3-(3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl)imidazolidin-1-yl)propanoic acid) [ka]

[0278] To a solution of 45 and 46 in acetone was added potassium carbonate. The mixture was heated to 65° C. overnight in a sealed vial as a suspension under N2 protection with vigorous stirring. The reaction was then filtered, concentrated, and purified on silica eluting with a gradient of ethyl acetate in hexane to give 47. [ka]

[0279] To a solution of compound 47 in DMF, sodium azide was added and the mixture was stirred overnight at 80° C. in a sealed vial under nitrogen protection. Upon completion, one volume of water was added and the product was extracted with ethyl acetate. The separated organic phase was filtered through sodium sulfate and concentrated. The crude product of compound 48 was used without further purification. [ka]

[0280] To a solution of PPh3 in THF, the solution of DEAD was added dropwise at room temperature with vigorous stirring. The mixture was transferred to a vial containing a mixture of compound 31 and compound 48, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was removed from the volatiles and the crude product was dissolved in EtOH. LiOH was added as a solution in H2O, and additional water was added until the reaction mixture was homogenous, and after stirring at room temperature for 1.5 hours, the mixture was acidified to pH 3 with H2SO4, concentrated, and purified by reverse phase HPLC (Phenomenex Gemini C18, 50x250mm, 10μm, acetonitrile / water in 0.1% TFA, gradient elution) to give compound 49 (structure 2.5c).

[0281] Synthesis of Structure 2.6c ((S)-3-(3-(3-(3-(17-azido-3-oxo-6,9,12,15-tetraoxa-2-azaheptadecyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl)-2-oxoimidazolidin-1-yl)-3-(3-fluoro-4-methoxyphenyl)propanoic acid) [ka]

[0282] To a solution of PPh3 in THF was added dropwise the solution of DEAD at 0°C. After the addition was complete, the mixture was transferred to a vial containing a pure mixture of compound 31 and MeOH. The vial was sealed with N2 and stirred at room temperature overnight. Upon completion, all volatiles were removed and the resulting crude product was purified by silica eluting with a gradient of MeOH in DCM to give compound 50. [ka]

[0283] Bromine was added to a solution of compound 50 in AcOH and the mixture was stirred for 0.5 h. Upon completion, the reaction was diluted with 5 volumes of ethyl acetate and 2.5 volumes of water. The aqueous layer was neutralized to pH 7 with a saturated aqueous solution of sodium bicarbonate and the organic phase was separated. The aqueous layer was extracted twice more with ethyl acetate. The combined organic phases were dried over sodium sulfate, filtered and concentrated. The resulting crude product, compound 51, was subsequently used without further purification. [ka]

[0284] A solution of compound 51, Pd(PPh3)4, and Zn(CN)2 in DMAC was degassed with nitrogen for 30 minutes. The mixture was heated in a sealed vial at 128°C overnight. Upon completion, the mixture was diluted with 5 volumes of EtOAc. The organic phase was then separated and washed twice with water and twice with brine, then the organic phase was filtered through sodium sulfate and concentrated. The residue was purified by silica eluting with 100% EtOAc to give compound 52. [ka]

[0285] To a solution of compound 52 in MeOH, ammonia was added, followed by a slurry of Raney nickel that had been pre-rinsed three times with methanol. The Parr® flask was charged with hydrogen to 60 psi and stirred at room temperature for 16 hours. Upon completion, the suspension was filtered and concentrated. The resulting crude residue was redissolved in DMF. DIEA and NHS-PEG4-N3 were added and the mixture was stirred for 1 hour. Upon completion, all volatiles were removed and the crude residue was redissolved in a mixture of MeOH and THF. LiOH in H2O was added and the mixture was stirred at room temperature for 17 hours. Upon reaction completion, the pH was adjusted to 3 with TFA and the mixture was injected directly onto a semi-preparative reverse phase HPLC (Phenomenex Gemini C18, 250x21.2mm, 5μm, 0.1% TFA in water / ACN, gradient elution) to give compound 53 (structure 2.6c).

[0286] Synthesis of Structure 2.7c ((S)-N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(3-fluoro-4-methoxyphenyl)-3-(2-oxo-3-(3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl)imidazolidin-1-yl)propanamide), Structure 2.8c, Structure 2.9c, and Structure 2.10c [ka]

[0287] To compound 31, solutions of THF, PPh3, and DEAD were added dropwise in succession at 0°C. The mixture was stirred at room temperature for 16 hours. The mixture was then cooled to -20°C for 1 hour and filtered to remove triphenylphosphine oxide. The filtrate was concentrated and the O-alkylated intermediate was isolated by purification on silica eluting with a gradient of ethyl acetate in hexanes containing 1% TEA. The isolated intermediate was then suspended in a mixture of THF and HO, treated with LiOH in HO, and stirred at 35°C for 16 hours. Upon completion, the pH was adjusted to 7 with 2M HCl and all volatiles were removed. The crude product was suspended in HO. Sodium chloride was added and compound 54 was extracted five times with ethyl acetate. The organic phases were combined, filtered through sodium sulfate, and concentrated. Compound 54 was subsequently used without further purification. [ka]

[0288] A solution of compound 54 in DMF was treated with HBTU and stirred for 5 min. Subsequently, DIEA and N3-PEG3-NH2 were added and the mixture was stirred at room temperature for 16 h. Upon completion, the pH was adjusted to 3 with TFA and compound 55 was isolated by direct injection onto a semi-preparative reverse phase HPLC (Phenomenex Gemini C18, 250x21.2mm, 5μm, 250,×21.2mm, 5μm, water / ACN, 0.1% TFA with gradient elution) to give compound 55.

[0289] Using similar procedures, N3-PEG 11 -NH2, N3-PEG 23 -NH2, and N3-PEG 35 Compounds 2.8c, 2.9c, and 2.10c were synthesized using -NH2.

[0290] Synthesis of structure 2.11c ((R)-3-(4-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-3-fluorophenyl)-3-(2-oxo-3-(3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl)imidazolidin-1-yl)propanoic acid). [ka]

[0291] In a 3-L 4-neck round bottom flask purged and maintained with an inert atmosphere of nitrogen, THF (1.50 L), DIPEA (150.00 mL, 716.000 mmol, 0.88 equiv), n-BuLi (430.00 mL, 680.000 mmol, 0.84 equiv) were placed. This was followed by the addition of trimethyl phosphite (195.00 mL) at -60°C and stirring at -60°C for 1 hour. To this was added tert-butyl 2-oxopyrrolidine-1-carboxylate (150.00 g, 809.835 mmol, 1.00 equiv) at 60°C. The resulting solution was stirred in a liquid nitrogen bath at -60°C for 1 hour. The reaction was then quenched by the addition of 350 mL of H2SO4 (2N) and diluted with 1.5 L of H2O. The resulting solution was extracted with 2×1 L of ethyl acetate. The resulting mixture was washed with 1×1 L of HO, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 200 g (crude product) of tert-butyl N-[5-(dimethoxyphosphoryl)-4-oxopentyl]carbamate as a yellow oil. [ka]

[0292] In a 3-L round-bottom flask, tert-butyl N-[5-(dimethoxyphosphoryl)-4-oxopentyl]carbamate (200.00 g, 1500.00 mmol, 1.50 equiv), MeOH (1.50 L), 2-aminopyridine-3-carbaldehyde (53.00 g, 1000.00 mmol, 1.00 equiv), NaOH (50.00 g, 1500.00 mmol, 1.50 equiv) were placed. The resulting solution was stirred in an oil bath at 50° C. for 16 h. The pH value of the solution was adjusted to 8 with NaHCO3 (aq.). The resulting mixture was concentrated. The reaction was then quenched by the addition of 1.5 L of water and extracted with 2×1.5 L of ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. This yielded 160 g (crude) of tert-butyl N-[3-(1,8-naphthyridin-2-yl)propyl]carbamate as a yellow oil. [ka]

[0293] In a 5-L round-bottom flask was placed tert-butyl N-[3-(1,8-naphthyridin-2-yl)propyl]carbamate (160.00 g, 556.787 mmol, 1.00 equiv), MeOH (2.00 L), Rh / C (140.00 g, 1.360 mmol), and H2 (40 Psi). The resulting solution was stirred at 25° C. for 16 h. The solids were filtered off. The resulting mixture was concentrated. This yielded 106 g (65.33%) of tert-butyl N-[3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl]carbamate as a yellow solid. [ka]

[0294] In a 1-L round bottom flask was placed tert-butyl N-[3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl]carbamate (106.00 g, 363.767 mmol, 1.00 equiv), EtOAc (500.00 mL), and HCl in EtOAc (4 M, 400.00 mL). The resulting solution was stirred at 25° C. for 3 h. The resulting solution was diluted with 1 L of HO. The pH was adjusted to 11 using NaOH (aq.). The resulting solution was extracted with 2×1 L of ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. This yielded 56 g (80.48%) of 3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propan-1-amine as a yellow solid. [ka]

[0295] In a 2-L round bottom flask was placed 3-fluoro-4-hydroxybenzaldehyde (140.00 g, 999.194 mmol, 1.00 equiv), ACN (1000 mL), (bromomethyl)benzene (205.08 g, 1199.039 mmol, 1.20 equiv), K2CO3 (414.28 g, 2997.581 mmol, 3.00 equiv). The resulting solution was stirred at 25° C. for 16 hours. The solids were filtered off. The resulting mixture was concentrated. This yielded 230 g (99.98%) of 4-(benzyloxy)-3-fluorobenzaldehyde as a white solid. [ka]

[0296] In a 3-L round bottom flask was placed 4-(benzyloxy)-3-fluorobenzaldehyde (230.00 g, 998.966 mmol, 1.00 equiv), DCM (1600 mL), (S)-2-methylpropane-2-sulfinamide (145.29 g, 1198.762 mmol, 1.20 equiv), and Cs2CO3 (650.97 g, 1997.933 mmol, 2.00 equiv). The resulting solution was stirred in an oil bath at 50° C. for 6 hours. The solids were filtered off. The resulting mixture was concentrated. This yielded 260 g (78.06%) of (S)-N-[[4-(benzyloxy)-3-fluorophenyl]methylidene]-2-methylpropane-2-sulfinamide as a white solid. [ka]

[0297] In a 3-L round bottom flask purged and maintained with an inert atmosphere of nitrogen was placed THF (2.0 L), Zn (1.02 kg, 15595.945 mmol, 20.00 equiv), CuCl (115.80 g, 1169.696 mmol, 1.50 equiv), ethyl 2-bromoacetate (325.57 g, 1949.498 mmol, 2.50 equiv), (S)-N-[[4-(benzyloxy)-3-fluorophenyl]methylidene]-2-methylpropane-2-sulfinamide (260.00 g, 779.797 mmol, 1.00 equiv). The resulting solution was stirred in a water / ice bath at 0° C. for 30 minutes. The resulting solution was allowed to react with stirring for an additional 2 hours while the temperature was maintained at 50° C. in an oil bath. The solids were filtered off. The resulting mixture was concentrated. The reaction was then quenched by the addition of 2 L of water and extracted with 2×2 L of ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. This yielded 150 g (45.63%) of ethyl (3R)-3-[4-(benzyloxy)-3-fluorophenyl]-3-[[(S)-2-methylpropane-2-sulfinyl]amino]propanoate as a yellow oil. [ka]

[0298] In a 1-L round-bottom flask was placed ethyl (3R)-3-[4-(benzyloxy)-3-fluorophenyl]-3-[[(S)-2-methylpropane-2-sulfinyl]amino]propanoate (150.00 g, 355.847 mmol, 1.00 equiv), 1,4-dioxane in HCl (400.00 mL, 4 M). The resulting solution was stirred at 25° C. for 2 h. The resulting mixture was concentrated. The reaction was then quenched by the addition of 1 L of water. The pH was adjusted to 8 with NaHCO3 (aq.). The resulting solution was extracted with 2×1 L of ethyl acetate dried over anhydrous sodium sulfate and concentrated. This yielded 100 g (88.55%) of ethyl (3R)-3-amino-3-[4-(benzyloxy)-3-fluorophenyl]propanoate as a yellow oil. [ka]

[0299] In a 2-L round-bottom flask was placed ethyl (3R)-3-amino-3-[4-(benzyloxy)-3-fluorophenyl]propanoate (100.00 g, 315.100 mmol, 1.00 equiv), THF (1.00 L), 2,2-dimethoxyacetaldehyde (49.21 g, 472.696 mmol, 1.50 equiv), and NaBH(OAc)3 (133.57 g, 630.199 mmol, 2.00 equiv). The resulting solution was stirred at 25° C. for 2 hours. The reaction was then quenched by the addition of 1 L of water. The resulting solution was extracted with 2×1 L of ethyl acetate dried over Na2SO4 and concentrated under reduced pressure. This yielded 80 g (62.62%) of ethyl (3R)-3-[4-(benzyloxy)-3-fluorophenyl]-3-[(2,2-dimethoxyethyl)amino]propanoate as a yellow oil. [ka]

[0300] In a 2-L three-neck round-bottom flask were placed triphosgene (22.25 g, 74.975 mmol, 0.38 equiv), THF (500 mL), ethyl (3R)-3-[4-(benzyloxy)-3-fluorophenyl]-3-[(2,2-dimethoxyethyl)amino]propanoate (80.00 g, 197.304 mmol, 1.00 equiv), TEA (29.95 g, 295.956 mmol, 1.50 equiv), and 3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propan-1-amine (compound 177, 33.97 g, 177.573 mmol, 0.90 equiv). The resulting solution was stirred in an oil bath at 50° C. for 1 h. The reaction was then quenched by the addition of 1 L of water. The pH was adjusted to 8 with NaHCO3 (aq.). The resulting solution was extracted with 2 x 1 L of ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. This yielded 96 g (78.13%) of ethyl (3R)-3-[4-(benzyloxy)-3-fluorophenyl]-3-[(2,2-dimethoxyethyl)([[3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl]carbamoyl])amino]propanoate as a yellow crude oil. [ka]

[0301] In a 1000 mL round bottom flask was placed ethyl (3R)-3-[4-(benzyloxy)-3-fluorophenyl]-3-[(2,2-dimethoxyethyl)([[3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl]carbamoyl])amino]propanoate (96.00 g, 154.158 mmol, 1.00 equiv), THF (500.00 mL), and H2SO4 (180.00 mL, 2 M). The resulting solution was stirred at 25° C. for 16 h. The pH was adjusted to 8 with NaOH (5 M). The resulting solution was extracted with 2×1 L of dichloromethane dried over anhydrous sodium sulfate and concentrated. The residue was applied to a silica gel column with dichloromethane / methanol (50 / 1). The collected fractions were combined and concentrated. This yielded 73 g (84.76%) of ethyl (3R)-3-[4-(benzyloxy)-3-fluorophenyl]-3-[2-oxo-3-[3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl]-2,3-dihydro-1H-imidazol-1-yl]propanoate as a yellow oil. [ka]

[0302] In a 3-L round-bottom flask, ethyl (3R)-3-[4-(benzyloxy)-3-fluorophenyl]-3-[2-oxo-3-[3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl]-2,3-dihydro-1H-imidazol-1-yl]propanoate (73.00 g, 130.671 mmol, 1.00 equiv), EtOH (1.50 L), Pd(OH)2 / C (60.00 g, 427.259 mmol, 3.27 equiv), and H2 (50 atm) were placed. The resulting solution was stirred at 25° C. for 72 h. The solids were filtered off. The residue was applied to a silica gel column with dichloromethane / methanol (9 / 1). The collected fractions were combined and concentrated. This yielded 41.0415 g (66.75%) of ethyl (3R)-3-(3-fluoro-4-hydroxyphenyl)-3-[2-oxo-3-[3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl]imidazolidin-1-yl]propanoate as a yellow oil.

[0303] LCMS-PH-ARP052-0:[MS+1]+=471

[0304] Optical rotation [α] D 20.0 =+37.5°(C=1g / 100ml in MeOH) H-NMR: (300 MHz, DMSO-d6, ppm) δ 9.84 (s, 1H), 7.07 - 7.00 (m, 2H), 6.95 - 6.850 (m, 2H), 6.24 (d, 2H), 5.18 (t, 1H), 4.06 - 3.96 (m, 2H), 3.32 - 2.75 (m, 10H), 2.60 (t, 2H), 2.37 (t, 2H), 1.77 - 1.67 (m, 4H), 1.10 (t, 3H). [ka]

[0305] To a solution of PPh3 in THF was added dropwise the solution of DEAD at -10°C. The mixture was warmed to room temperature and added to a neat mixture of compound 185 and HO-PEG4-N3 and stirred overnight. The reaction mixture was then concentrated under reduced pressure and the residue was purified by silica eluting with a gradient of MeOH in DCM to give compound 186. [ka]

[0306] To compound 186 was added EtOH and HO followed by LiOH. The mixture was stirred at 30° C. overnight. Upon completion, the mixture was neutralized to pH=5 using 6M aqueous HCl and concentrated. The residue was purified by reverse phase HPLC using a Phenomenex Gemini C18, 50×250 mm, 10 μm column eluted with a gradient of acetonitrile containing 0.1% water to give compound 187 (structure 2.11c).

[0307] Synthesis of Structure 28c (Compound 118a), Structure 29c (Compound 118b), Structure 31c (Compound 119a), and Structure 30c (Compound 119b) [ka]

[0308] To a solution of LHMDS (1.0 M in THF, 95 mL, 95 mmol) and THF (60 mL) was added dropwise a solution of compound 103 (2-methyl-[1,8]naphthyridine (12.5 g, 86.7 mmol)) in THF (180 mL) at -78 °C. After stirring for 30 min, a solution of compound 104 (5-bromo-1-pentene (19.4 g, 130 mmol)) in THF (120 mL) was added dropwise to the reaction mixture. The reaction mixture was warmed to 0 °C and stirred for 4 h. The reaction mixture was quenched with saturated aqueous NH4Cl (100 mL) and deionized water (100 mL) and then extracted with ethyl acetate (2x400 mL). The combined organic phase was dried over Na2SO4, filtered, concentrated, and compound 105 was isolated by CombiFlash® eluting with a gradient of 50-100% ethyl acetate in hexanes. Yield of compound 105: 7.93 g (43%). [ka]

[0309] To a solution of compound 105 (2.50 g, 11.8 mmol) in acetone (67.5 mL), water (7.5 mL), and 2,6 lutidine (2.74 mL, 23.6 mmol) was added 4-methylmorpholine N-oxide (2.07 g, 17.7 mmol) and osmium tetroxide (2.5 wt% in t-butanol, 2.40 g, 0.24 mmol) at room temperature. After stirring for 75 minutes, (diacetoxyiodo)benzene (5.69 g, 17.7 mmol) was added to the reaction mixture. The reaction mixture was stirred for 2 hours and then quenched with saturated aqueous sodium thiosulfate solution (100 mL) and extracted with ethyl acetate (2x100 mL). The combined organic phase was dried over Na2SO4, filtered, concentrated, and compound 106 was isolated by CombiFlash® eluting with a gradient of 0-5% methanol in ethyl acetate. Yield of compound 106: 1.12 g (44%). [ka]

[0310] To a suspension of sodium hydride (60% dispersion in mineral oil, 0.185 g, 4.64 mmol) in THF (9 mL) was added a solution of compound 107 (diethyl (n-methoxy-N-methylcarbamoylmethyl)phosphonate) (1.06 g, 4 mmol) in THF (5 mL) at 0° C. After stirring for 30 min, a solution of compound 106 (0.903 g, 4.21 mmol) in THF (9 mL) was added dropwise. The reaction mixture was stirred for 10 min at 0° C., then quenched with saturated aqueous NH4Cl solution (30 mL) and extracted with ethyl acetate (3×30 mL). The combined organic phase was washed twice with half-saturated aqueous NaHCO3 solution. The organic phase was dried over Na2SO4, filtered and concentrated. Yield of compound 108: 1.40 g (assuming 100% yield and used in subsequent steps without further purification). [ka]

[0311] To a solution of compound 108 (1.31 g, 4.38 mmol) in ethyl acetate (20 mL) was added Pd / C (10% loading, 0.466 g, 0.44 mmol). The reactor was pressurized to 50 PSI with H2. After stirring for 3.5 h, the reaction mixture was filtered through Celite® and rinsed with methanol. The filtrate was concentrated and compound 109 was isolated by CombiFlash® eluting with a gradient of 50-100% ethyl acetate in hexane containing 1% triethylamine. Yield of compound 109: 0.833 g (62%). [ka]

[0312] To a solution of compound 109 (0.833 g, 2.73 mmol) in THF (10 mL) was added DIEA (0.590 mL, 3.41 mmol) and di-tert-butyl dicarbonate (0.744 g, 3.41 mmol). The reaction mixture was heated to 50° C. for 5 h. The reaction was not complete based on LC / MS, so an additional portion of DIEA (0.590 mL, 3.41 mmol) and di-tert-butyl dicarbonate (0.744 g, 3.41 mmol) was added. The reaction mixture was heated at 50° C. for an additional 16 h. The reaction mixture was concentrated and compound 110 was isolated by CombiFlash® eluting with a gradient of 50-100% ethyl acetate in hexanes. Yield of compound 110: 0.934 g (84%). [ka]

[0313] To a solution of n-butyllithium (2.5 M in hexanes, 0.70 mL, 1.8 mmol) and THF (1.5 mL) was added a solution of compound 111 (5-bromo-2-(phenylmethoxy)pyridine) (0.465 g, 1.8 mmol) in THF (0.8 mL) dropwise over 3 min at -78 °C. Compound 110 (0.535 g, 1.3 mmol) was then added as a solution in THF (1 mL). After stirring for 30 min, the reaction was warmed to 0 °C, quenched with saturated aqueous NH4Cl (10 mL), and further acidified to pH 7 with 6 M aqueous HCl. The mixture was extracted with ethyl acetate (3 x 10 mL). The combined organic phase was dried over Na2SO4, filtered, and concentrated. To a solution of the crude product in THF (8 mL) was added DIEA (0.94 mL, 5.4 mmol) and di-tert-butyl dicarbonate (1.18 g, 5.4 mmol). The mixture was stirred at 40° C. overnight. The reaction mixture was concentrated and compound 112 was isolated by CombiFlash® eluting with a gradient of 0-40% ethyl acetate in hexanes. Yield of compound 112: 471 mg (50%). [ka]

[0314] To a suspension of sodium hydride (60% dispersion in mineral oil, 0.106 g, 2.65 mmol) in dimethoxyethane (2 mL) was added compound 113 (triethylphosphonoacetate) (0.593 g, 2.65 mmol) as a solution in dimethoxyethane (1 mL) at 0° C. After stirring for 20 min, the reaction mixture was warmed to room temperature and a solution of compound 112 (0.467 g, 0.88 mmol) in dimethoxyethane (2 mL) was added. The reaction mixture was heated at 70° C. for 4 h. The reaction was quenched with saturated aqueous NH4Cl (10 mL) and the product was extracted with ethyl acetate (3×15 mL). The organic phase was dried over Na2SO4, filtered, concentrated and compound 114 was isolated as a 1:1 mixture of cis:trans isomers by CombiFlash® eluting with a gradient of 0-30% ethyl acetate in hexanes. Yield of compound 114: 392 mg (74%). [ka]

[0315] To a solution of compound 114 (390 mg, 0.65 mmol) in ethanol (6 mL) was added Pd / C (10% loading, 69 mg, 0.07 mmol). The reactor was pressurized to 50 PSI with H2. After stirring for 4 h, the reaction mixture was filtered through Celite® and rinsed with methanol. The filtrate was concentrated and compound 115 was isolated as a racemic mixture by CombiFlash® eluting with a gradient of 0-10% methanol in DCM. Yield of compound 115: 95 mg (29%). Chiral semi-preparative HPLC (250x21 mm Chiralpak® AD column, 5 μm, 90 / 10 hexane / EtOH, 40 mL / min) afforded 42 mg of the first eluting R-isomer (R T = 12-14m, >99% ee, compound 115a) and 40 mg of the second eluting S-isomer (R T=15-18m, >98% ee, compound 115b) was used to isolate the R- and S-isomers. The identities of the R- and S-isomers were assigned based on the order of elution of structurally similar compounds reported by Coleman et al. 47 J. Med. Chem. 4834 (2004).

[0316] Structure 28c ((R)-3-(6-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)pyridin-3-yl)-9-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)nonanoic acid) and 31c ((R)-3-(1-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-6-oxo-1,6-dihydropyridin-3-yl)-9-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)nonanoic acid). [ka]

[0317] To a solution of compound 115a (41 mg, 0.08 mmol) and N3-PEG4-OTs (61 mg, 0.16 mmol) in DMF (0.5 mL) was added cesium carbonate (53 mg, 0.16 mmol). The reaction mixture was stirred at 40° C. for 1 h. The reaction mixture was quenched with aqueous NaHCO3 (1 mL) and then extracted with ethyl acetate (3×3 mL). The organic phase was concentrated under reduced pressure. The crude mixture of N- and O-alkylated regioisomers was subsequently used without further purification. [ka]

[0318] To a solution of compounds 116a and 117a (58 mg, 0.08 mmol, 4:6 mixture of 9a:10a) in THF (1.0 mL) and deionized water (1.0 mL) was added lithium hydroxide (6 mg, 0.25 mmol). The reaction mixture was stirred at room temperature for 1 h and at 35° C. for 2 h. An additional portion of lithium hydroxide (4 mg, 0.16 mmol) was added and the reaction temperature was increased to 40° C. After stirring for 3 h, the final portion of lithium hydroxide (4 mg, 0.25 mmol, total 16 mg, 0.66 mmol) was added. The reaction mixture was stirred at 50° C. for 3 h. The reaction mixture was acidified to pH 7 with 6N aqueous HCl and concentrated under reduced pressure. The regioisomers, compounds 118a and 119a, were separated by CombiFlash® eluting with a gradient of 0-5% methanol in DCM containing 0.5% acetic acid. Compound 118a was further purified by reverse phase HPLC (Thermo Scientific™ Aquasil™ C18, 250x21.2mm, 5μm, 20mL / min, water / ACN in 0.1% TFA, gradient elution) to give 13mg of compound 118a (structure 28c). Compound 119a was purified under the same conditions to give 16mg of compound 119a (structure 31c).

[0319] Structure 29c ((S)-3-(6-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)pyridin-3-yl)-9-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)nonanoic acid) and 30c ((S)-3-(1-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-6-oxo-1,6-dihydropyridin-3-yl)-9-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)nonanoic acid). [ka]

[0320] To a solution of compound 115b (40 mg, 0.08 mmol) and N3-PEG4-OTs (58 mg, 0.16 mmol) in DMF (0.5 mL) was added cesium carbonate (51 mg, 0.16 mmol). The reaction mixture was stirred at 40° C. for 30 min. The reaction mixture was quenched with aqueous NaHCO3 (1 mL) and then extracted with ethyl acetate (3×3 mL). The organic phase was concentrated under reduced pressure. The crude mixture of N- and O-alkylated regioisomers was subsequently used without further purification. [ka]

[0321] To a solution of compounds 116b and 117b (56 mg, 0.08 mmol, 4:6 mixture of 9a:10a) in THF (0.75 mL) and deionized water (0.75 mL) was added lithium hydroxide (6 mg, 0.25 mmol). The reaction mixture was stirred at 45° C. for 2.5 h. An additional portion of lithium hydroxide (6 mg, 0.25 mmol) was added and the reaction mixture was stirred for 2.5 h. The reaction temperature was reduced to 35° C. and the mixture was stirred overnight. The reaction mixture was acidified to pH=7 with 6N aqueous HCl and concentrated under reduced pressure. The regioisomers, compounds 118b and 119b, were separated by CombiFlash® eluting with a gradient of 0-5% methanol in DCM containing 0.5% acetic acid. Compound 118b was further purified by reverse phase HPLC (Thermo Scientific™ Aquasil™ C18, 250x21.2mm, 5μm, 20mL / min, water / ACN in 0.1% TFA, gradient elution) to give 14mg of compound 118b (structure 29c). Compound 119b was purified under the same conditions to give 18mg of compound 119b (structure 30c).

[0322] Synthesis of Structure 32c ((R)-3-(4-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-3-fluorophenyl)-3-(N-methyl-5-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)pentanamido)propanoic acid) [ka]

[0323] To compound 120 (2.75 g, 11.94 mmol) in toluene (80 mL) through 3 Å sieves was added compound 121 (5.79 g, 47.78 mmol), followed by PPTS (300 mg, 1.19 mmol), then AcOH (683 μL, 11.94 mmol). The reaction was brought to reflux overnight. Upon completion, the reaction was quenched by the addition of saturated sodium bicarbonate. The organic layer was diluted with two volumes of ethyl acetate, separated, and filtered through sodium sulfate. The product was isolated by silica eluting with a gradient of ethyl acetate in hexanes (0-30%) to give 2.054 g (54%). [ka]

[0324] A 2.5M solution of n-BuLi (7.76 mL, 19.41 mmol) was added dropwise to DIA (2.85 mL, 20.33 mmol) in THF (15 mL) at -78°C. Stirring was continued for 5 min at -78°C, and ethyl acetate (1.81 mL, 18.48 mmol) was added dropwise. Stirring was continued for an additional 10 min at -78°C, and a solution of chlorotitanium triisopropoxide (9.27 mL, 38.381 mmol) in THF (10 mL) was added dropwise. Stirring was continued for an additional 15 min at -78°C, and a solution of compound 122 (2.054 g, 6.16 mmol) in THF (10 mL) was added dropwise. Stirring was continued for 1.5 h at -78°C. Upon completion, the reaction was quenched by the addition of saturated ammonium bicarbonate. The suspension was diluted with 6 volumes of ethyl acetate and the organic layer was separated, dried over sodium sulfate, filtered and concentrated. The product was isolated by silica eluting with a gradient of ethyl acetate in hexanes to give 1.043 g (53%). [ka]

[0325] To compound 123 (1.043 g, 2.47 mmol) stirred in MeOH (3 mL) was added 4 M HCl solution in dioxane (3.09 mL, 12.37 mmol). Upon completion of deprotection, the solution was diluted with water (8 mL) and washed twice with diethyl ether (6 mL). The aqueous layer was then adjusted to pH 11 with sodium hydroxide. The precipitate was extracted with ethyl acetate and the combined organic extracts were dried over sodium sulfate, filtered and concentrated to give 0.616 g (78.5%) of product 124, which was used without further purification. [ka]

[0326] To compound 125 (92.1 mg, 0.275 mmol) in THF (1.5 mL) at 0° C. was added DCC (68.1 mg, 0.331 mmol). After 5 min, PNP (106.1 mg, 0.331 mmol) was added, the ice bath was removed, and stirring was continued for 1 h. Upon completion, the suspension was cooled at −20° C. for 1 h and the precipitate was removed by filtration. The supernatant was concentrated and gave 129 mg (103%) of crude product 126, which was subsequently used without further purification. [ka]

[0327] A mixture containing compound 124 (148.6 mg, 0.468 mmol) and potassium carbonate (129 mg, 0.937 mmol) in DMF (2 mL) was treated with methyl iodide (66.5 mg, 0.468 mmol) and stirred for 3 h at 50° C. Upon completion of alkylation, all volatiles were removed and the product was isolated by silica eluting with a gradient of ethyl acetate in hexanes buffered with 1% TEA, respectively, to give 94.6 mg (61%). [ka]

[0328] To compound 127 (94.5 mg, 0.285 mmol) in DMF (2 mL) was added DIEA (149 μL, 0.856 mmol), followed by compound 126 (129.9 mg, 0.285 mmol) and the mixture was stirred at 80° C. for 1 h. Upon completion, all volatiles were removed and the crude product was dissolved in MeOH (20 mg), treated with 10% palladium on carbon and the flask was charged with 60 PSI of hydrogen. Upon completion, the suspension was filtered. The supernatant was concentrated and the resulting crude product was subsequently used without further purification. [ka]

[0329] A mixture containing compound 128 (159 mg, 0.285 mmol), bromo-PEG2-azide (74.7 mg, 0.314 mmol), and cesium carbonate (204 mg, 0.627 mmol) in DMF (2 mL) was heated to 60° C. for 2 h. Upon completion, all volatiles were removed and the crude product was treated with 4 M HCl in dioxane (0.5 mL, 2 mmol) and heated to 40° C. for 3 h. Upon completion, all volatiles were removed. The crude product was suspended in a mixture of THF (1 mL), MeOH (1.5 mL), and HO (1.5 mL), treated with lithium hydroxide (83.5 mg, 3.48 mmol), and heated to 40° C. for 16 h. Upon completion, the pH was adjusted to 3 with TFA and the product was isolated by separation through a Phenomenex® Gemini® C18 column (21.2×250 mm, 5 micron) eluting with a gradient of acetonitrile in water containing 0.1% TFA to give 33.1 mg (20%).

[0330] Synthesis of Structure 33c ((R)-1-azido-1,3-(3-fluoro-4-methoxyphenyl)-12-(5-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)pentanoyl)-3,6,9-trioxa-12-azapentadecan-15-oic acid) [ka]

[0331] A mixture containing compound 130 (1.5 g, 9.73 mmol), (R) t-butylsulfinamide (2.36 g, 19.46 mmol), and AcOH (0.14 mL) in toluene (45 mL) was refluxed for 16 h in a flask equipped with a Dean-Stark trap. Upon completion, the reaction was quenched by the addition of saturated sodium bicarbonate. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated. The product was isolated by separation through silica eluting with a gradient of ethyl acetate in hexanes to give 1.714 g (68.4%). [ka]

[0332] A 2.5M solution of n-BuLi (8.324 mL, 20.81 mmol) was added dropwise to DIA (3.056 mL, 21.80 mmol) in THF (18 mL) at -78°C. Stirring was continued for 5 min at -78°C, and ethyl acetate (1.94 mL, 19.82 mmol) was added dropwise. Stirring was continued for an additional 10 min at -78°C, and a solution of chlorotitanium triisopropoxide (9.94 mL, 41.62 mmol) in THF (10 mL) was added dropwise. Stirring was continued for an additional 15 min at -78°C, and a solution of compound 131 (1.70 g, 6.61 mmol) in THF (12 mL) was added dropwise. Stirring was continued for 1.5 h at -78°C. Upon completion, the reaction was quenched by the addition of saturated ammonium bicarbonate. The suspension was diluted with 7 volumes of ethyl acetate and the organic layer was separated, dried over sodium sulfate, filtered and concentrated. The product was isolated on silica eluting with a gradient of ethyl acetate in hexanes to give 0.984 g (43%). [ka]

[0333] To compound 132 (0.975 g, 2.82 mmol) in EtOH (6 mL) at 0° C. was added 4M HCl in dioxane (2.12 mL, 8.47 mmol) and stirred for 30 min. Upon completion, the reaction was diluted with water (15 mL) and washed with diethyl ether. The organic layer was separated and the pH of the aqueous layer was adjusted to 12 with sodium hydroxide. The aqueous layer was washed with 5 volumes of ethyl acetate and the organic layer was separated, filtered through sodium sulfate and concentrated. The product was isolated by separation through silica eluting with a gradient of ethyl acetate in hexane containing 1% TEA to give 0.434 g (64%). [ka]

[0334] To a mixture of compound 133 (0.120 g, 0.497 mmol) and PEG (0.151 g, 0.696 mmol) in THF (2 mL) with 3A molecular sieves was added STAB-H (0.253 g, 1.19 mmol) and the suspension was stirred at room temperature for 16 h. Upon completion, the reaction was quenched by the addition of saturated sodium bicarbonate and the crude product was extracted with three portions of ethyl acetate. The separated organic extracts were combined, dried over sodium sulfate, filtered and concentrated. The resulting crude product was subsequently used without further purification. [ka]

[0335] Compound 134 (0.200 g, 0.597 mmol) in DMF (2 mL) was treated with HATU (0.227 g, 0.597 mmol) and stirred for 5 min. To the activated ester was added DIEA (0.259 mL, 1.49 mmol) followed by compound 125 (0.220 g, 0.497 mmol) in DMF (1 mL) and the resulting mixture was stirred for 1 h. All volatiles were removed and the resulting crude product was treated with neat TFA (3.8 mL) and stirred at 40° C. for 3 h. Upon completion of BOC removal, all volatiles were removed and the crude product was suspended in a mixture of THF (4 mL), water (8 mL), and MeOH (8 mL). The resulting mixture was treated with LiOH (71.6 mg, 2.98 mmol) and heated to 40° C. for 16 h. Upon completion, the pH was adjusted to 3 with TFA and the product was isolated by separation via a Phenomenex® Gemini® c18 column (21.2×250 mm, 5 microns) eluting with a gradient of acetonitrile in water containing 0.1% TFA to give 56.2 mg (18%, 3 steps).

[0336] Synthesis of Structure 34c ((S)-1-azido-13-(3-fluoro-4-methoxyphenyl)-12-(5-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)pentyl)-3,6,9-trioxa-12-azapentadecan-15-oic acid) [ka]

[0337] Compound 136 (0.500 g, 1.45 mmol) in a mixture of THF (9.0 mL) and MeOH (0.5 mL) was treated with lithium borohydride (94.5 mg, 4.34 mmol) at 0° C. The cooling was removed and stirring was continued until gas evolution ceased. The reaction mixture was diluted with 5 volumes of EtOAc. The organic layer was washed with ammonium bicarbonate, dried over sodium sulfate, filtered and concentrated. The product was isolated by elution through silica using a gradient of ethyl acetate in hexanes to give 309 mg (67%). [ka]

[0338] To a solution containing compound 137 (0.305 g, 0.952 mmol) in DCM (9 mL) at 0° C. was added Martin's reagent in several portions. A few drops of water were added, the cooling was removed, and the reaction was stirred for 3 h. Upon completion, the mixture was washed with saturated sodium bicarbonate, then saturated sodium thiosulfate. The separated organic portion was dried over sodium sulfate, filtered, and concentrated. The product 138 was isolated via silica eluting with a gradient of MeOH in DCM to give 140 mg (46%). [ka]

[0339] To a mixture containing compound 1 (85.2 mg, 0.353 mmol) and 138 (134.9 mg, 0.424 mmol) in THF (2.5 mL) with 3 Å molecular sieves was added STAB-H (0.150 g, 0.706 mmol) and the resulting suspension was heated to 40° C. for 16 h. Upon completion, the reaction was diluted with 5 volumes of ethyl acetate and treated with saturated sodium bicarbonate. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated. The product was isolated by separation through silica eluting with a gradient of MeOH in DCM containing 1% TEA to give 64 mg (33%). [ka]

[0340] To a mixture containing compound 140 (60 mg, 0.110 mmol), Ald-PEG3-N3 (71.9 mg, 0.331 mmol) and AcOH (3 μL, 0.0276 mmol) in MeOH (1 mL) with 3 Å molecular sieves, sodium cyanoborohydride (28.9 mg, 0.276 mmol) was added and the reaction was stirred at 40° C. for 3 h. Upon completion, the mixture was cooled to 0° C. and water (0.15 mL) was added and the solution was acidified to pH 7 using HCl in dioxane (4 M). Subsequently, all methanol was removed and 4 M HCl in dioxane (0.138 mL, 0.552 mmol) was added and the mixture was stirred at 40° C. for 2 h. Upon completion of BOC removal, all volatiles were removed and the crude product was suspended in a mixture of THF (1 mL), water (2 mL), and MeOH (2 mL) and treated with lithium hydroxide (26.5 mg, 1.104 mmol). Upon completion of ester removal, the pH was adjusted to 3 by addition of TFA and the product was isolated by separation through a Phenomenex® (21.2×250 mm) C18 column eluted with a gradient of acetonitrile in water containing 0.1% TFA to give 16.4 mg (24%, 3 steps).

[0341] Synthesis of Structure 36c ((S)-3-(4-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-3-fluorophenyl)-9-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)nonanoic acid) [ka]

[0342] To a solution of 6-oxoheptanoic acid (9.74 g, 68 mmol) in DCM (30 mL) and MeOH (75 mL) was added conc. H2SO4 (0.18 mL, 3.4 mmol) at room temperature. The reaction mixture was refluxed overnight. The reaction mixture was then concentrated to an oil, redissolved in DCM (150 mL), and washed with saturated aqueous NaHCO3 (2x40 mL) and brine (40 mL). The organic layer was dried over Na2SO4, filtered, and concentrated. The product was used in the next step without further purification. Yield of compound 141: 10.2 g (95%). 1 H NMR (400 MHz, DMSO-d6): δ3.58 (s, 3H), 2.43 (t, 2H), 2.29 (t, 2H), 1.46 (m, 4H). [ka]

[0343] To a solution of compound 141 (10.2 g, 65 mmol) and 2-amino-3-formylpyridine (7.89 g, 65 mmol) in EtOH (80 mL) was added L-proline (3.72 g, 32 mmol). The reaction mixture was heated at reflux overnight. The reaction mixture was then concentrated, dissolved in EtOAc (50 mL) and washed with water (3x30 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash using silica gel as stationary phase and eluted with a gradient of EtOAc in DCM (10-100%). Yield of compound 142: 6.08 g (39%). C 14 H 16 Calculated mass for N2O2 [M+H] + : 245.13, actual value: 245.21. [ka]

[0344] To a solution of compound 142 (6.08 g, 24.9 mmol) in MeOH (50 mL) was added Pd / C (10% loading, Degussa type, 1.99 g, 1.87 mmol). The reaction flask was charged with nitrogen, evacuated, and backfilled with nitrogen three times. This process was repeated with hydrogen, and finally the reactor was charged with hydrogen (1 atm) and stirred at room temperature overnight. The reaction mixture was filtered through Celite®, the pad was rinsed with MeOH, and the filtrate was concentrated. The product, compound 143, was used in the next step without further purification and was assumed to be 100% yield. C 14 H 20 Calculated mass for N2O2 [M+H] + : 249.16, actual value: 249.08. [ka]

[0345] To a solution of dimethylmethylphosphonate (12.3 g, 100 mmol) in anhydrous THF (120 mL) was added n-BuLi solution (2.5 M in hexane, 40 mL, 100 mmol) via syringe pump at -78 °C over 1 h. A solution of compound 143 (6.175 g, 24.9 mmol) in THF (40 mL) was added to the reaction mixture at -78 °C over 45 min. After stirring at -78 °C for 20 min, the reaction mixture was quenched with saturated aqueous NH4Cl (200 mL), warmed to room temperature, and extracted with EtOAc (400 mL). The organic layer was washed with water (200 mL) and brine (200 mL). The organic phase was separated, dried over Na2SO4, filtered, and concentrated. The product was used in the next step without further purification. Yield of compound 144: 7.86 g (93%). C 16 H 25 Calculated mass for N2O4P [M+H] + : 341.17, actual value: 341.17. [ka]

[0346] A suspension of 3-fluoro-4-(phenylmethoxy)-benzaldehyde (0.38 g, 1.65 mmol), compound 144 (0.67 g, 1.98 mmol), and anhydrous potassium carbonate (0.547 g, 3.96 mmol) in THF (13.5 mL) was heated at reflux overnight. Additional 3-fluoro-4-(phenylmethoxy)benzaldehyde (0.19 g, 0.83 mmol) and potassium carbonate (0.23 g, 1.65 mmol) were added and the reaction mixture was refluxed for another 4 h. The mixture was diluted with EtOAc (100 mL) and washed with water (30 mL) and brine (30 mL). The organic phase was separated, dried over Na2SO4, filtered, and concentrated. The residue was purified by CombiFlash using silica gel as stationary phase and eluted with a gradient of MeOH in DCM (0-10%). Yield of compound 145: 446 mg (61%). 28 H 29 Calculated mass for FN2O2 [M+H] + : 445.23, actual value: 445.41. [ka]

[0347] Preparation of R-BINAL: To a slurry of LAH (0.396 g, 10.4 mmol, 0.98 eq) in anhydrous THF (34 mL) was added EtOH (0.492 g, 10.65 mmol, 1.00 eq) as a solution in THF (3.2 mL) over 10 min while maintaining the internal temperature <35° C. After aging for 30 min, R-BINOL (3.05 g, 10.65 mmol, 1.00 eq) was added as a solution in THF (10 mL) while maintaining the internal temperature <35° C. (approximately 10 min). After stirring at room temperature for 2 h, the reaction mixture was cooled to −78° C. in a dry ice / acetone bath.

[0348] Compound 145 (1.18 g, 2.65 mmol) was dried azeotropically with anhydrous toluene (50 mL) and dissolved in anhydrous THF (12 mL). The solution of compound 145 was added dropwise to the solution of R-BINAL via syringe pump over 45 min at -78 °C. After 1.5 h, the reactor was transferred to a very large Dewar, filled with dry ice / acetone, and covered with aluminum foil. The reaction mixture was stirred overnight at -78 °C. Most of the reduction occurred within the first 1.5 h, with only a small amount of additional conversion. The reaction was quenched by the addition of saturated aqueous NH4Cl (150 mL) and allowed to warm to room temperature. The mixture was acidified to pH = 7 using 6N HCl and then extracted with EtOAc (2x250 mL). The combined organic phase was washed with water (125 mL) and brine (125 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash using silica gel as stationary phase and eluted with a gradient of MeOH in DCM (0-5%). Yield of compound 146: 634 mg (53%). Chiral purity was determined by analytical chiral HPLC, Chiralpak AD-H column 4.6x250 mm, 5 micron, EtOH 0.1% diethylamine isocratic, 1.75 mL / min. The first eluting R isomer was 86 area % pure, corresponding to 72% ee. Compound 6 was further purified by chiral semi-preparative HPLC (Chiralpak AD-H 21.2x250 mm, 5 micron, EtOH 0.1% diethylamine, 20 mL / min). Final yield of compound 146: 445 mg (98% ee). 28 H 31 Calculated mass for FN2O2 [M+H] + : 447.25, actual value: 447.30. [ka]

[0349] To a solution of compound 146 (0.325 g, 0.73 mmol) and malonic acid monomethyl ester (0.103 g, 0.87 mmol) in DCM (3 mL) was added a solution of DMAP (9 mg, 0.073 mmol) in DCM. The mixture was cooled to 0° C. and DCC (0.180 g, 0.87 mmol) was added. The cooling bath was removed and the reaction was stirred at room temperature overnight. The reaction mixture was then diluted with DCM (10 mL) and filtered. The filtrate was concentrated and purified by CombiFlash using silica gel as stationary phase and eluting with a gradient of MeOH in DCM (0-5%). Yield of compound 147: 142 mg (37%). C 32 H 35 Calculated mass for FN2O5 [M+H] + :547.26, actual value:547.58. [ka]

[0350] To a solution of compound 147 (0.232 g, 0.42 mmol) in NMP (0.5 mL) was added N,O-bis(trimethylsilyl)acetamide (0.229 g, 1.12 mmol) at room temperature. The mixture was heated at 60° C. for 30 min. Brine (58 μL) was added in two portions over 5 min. The reaction mixture was then heated at 90° C. for 3 h and then at room temperature overnight. The reaction mixture was diluted with EtOAc (12 mL) and washed with water (3 mL). The aqueous layer was back-extracted with EtOAc (12 mL). The combined organic layers were concentrated. The residue was purified by CombiFlash using silica gel as stationary phase and eluted with a gradient of MeOH in DCM. Yield of compound 148: 140 mg (66%). C 31 H 35 Calculated mass for FN2O3 [M+H] + :503.27, actual value:503.29. [ka]

[0351] To a solution of compound 148 (0.169 g, 0.34 mmol) in EtOH (3 mL) was added a slurry of Pd / C (10% loading, 36 mg, 0.034 mmol) in EtOH (1 mL). The reactor was pressurized and vented with hydrogen three times. The reactor was repressurized to 55 psi for 3 h. The reaction mixture was diluted with MeOH (5 mL) and filtered. The filtrate was concentrated and the product, compound 149, was used in the next step without further purification and assumed to be in 100% yield. 24 H 31 Calculated mass for FN2O3 [M+H] + : 415.24, actual value: 415.07. [ka]

[0352] To a solution of compound 149 (139 mg, 0.34 mmol) and azido-PEG4-tosylate (0.188 mg, 0.50 mmol) in DMF (2.5 mL) was added cesium carbonate (164 mg, 0.50 mmol). The reaction mixture was heated at 40° C. for 1 h and then quenched with saturated aqueous NaHCO3 (3 mL). The mixture was extracted with EtOAc (3×10 mL). The combined organic phase was washed with water (2×5 mL). The organic phase was dried over Na2SO4, filtered, concentrated and used in the next step without further purification. C 32 H 46 Calculated mass for FN5O6 [M+H] + : 616.35, actual value: 616.90. [ka]

[0353] To a solution of compound 150 (0.207 mg, 0.34 mmol) in THF (1.5 mL) and water (1.5 mL) was added lithium hydroxide (0.040 g, 1.68 mmol). The reaction mixture was heated at 40° C. overnight. The next morning, the reaction mixture was acidified to pH=7 with 6N HCl and concentrated under reduced pressure. The residue was dissolved in 35% ACN in H2O, 0.1% TFA and purified by RP-HPLC (Thermo Aquasil C18, 250×21 mm, 5 μm, 20 mL / min, gradient of ACN in H2O containing 0.1% TFA). Yield of compound 151 (SM 36): 125 mg (52% over 3 steps). C 31 H 44 Calculated mass for FN5O6 [M+H] + : 602.34, actual value: 602.85.

[0354] Synthesis of structure 37c ((S)-3-(4-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-3-fluorophenyl)-3-(5-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)pentanamido)propanoic acid) [ka]

[0355] Compound 169 (90 mg, 0.268 mmol) in DMF (1.5 mL) was treated with HATU (112 mg, 0.295 mmol) and stirred for 5 min. A mixture containing compound 170 (94 mg, 0.295 mmol) and DIEA (0.154 mL, 0.884 mmol) in DMF (0.5) was subsequently added and stirring was continued for 1 h. Upon completion, all volatiles were removed and compound 171 was isolated by separation through silica eluting with a gradient of MeOH in DCM to give 123 mg (72%). [ka]

[0356] A suspension containing 10% palladium on carbon (21 mg, 0.0194 mmol) and compound 171 (123 mg, 0.194 mmol) in MeOH (2 mL) was charged with 60 PSI of hydrogen and stirred for 1 h. Upon completion, the suspension was filtered through Celite® and concentrated to give 88 mg (83%) of crude product which was subsequently used without further purification. [ka]

[0357] A suspension containing compound 172 (87 mg, 0.160 mmol), Br-PEG3-N3 (50 mg, 0.176 mmol), and cesium carbonate (115 mg, 0.352 mmol) in DMF (1 mL) was heated to 60° C. and stirred for 2 h. Upon completion, all volatiles were removed and compound 173 was isolated by separation through silica eluting with a gradient of MeOH in DCM to give 91 mg (76%). [ka]

[0358] Compound 173 (50 mg, 0.067 mmol) in dioxane (0.5 mL) was treated with a solution of 4 M HCl in dioxane (0.671 mmol, 0.168 mL) and stirred at 40° C. for 3 h. Upon completion, all volatiles were removed. The crude product was dissolved in a mixture of HO (0.4 mL), THF (0.2 mL), and MeOH (0.4 mL), treated with LiOH (8 mg, 0.356 mmol), and stirred at 40° C. for 16 h. Upon completion, the pH was adjusted to 3 with TFA, and the product was isolated by separation via a Phenomenx Gemini C18 column (21.2×250 mm, 5 micron) eluting with a gradient of acetonitrile in water containing 0.1% TFA to give 25 mg (60%, 2 steps).

[0359] Synthesis of structure 38c ((S)-3-(2-(3-((2-(2-(2-azidoethoxy)ethoxy)ethyl)amino)-3-oxopropyl)pyrimidin-5-yl)-9-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)nonanoic acid) and structure 39c ((S)-3-(2-(1-azido-12-oxo-3,6,9-trioxa-13-azahexadecan-16-yl)pyrimidin-5-yl)-9-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)nonanoic acid) [ka]

[0360] To a solution of 5-bromo-2-iodopyrimidine (8.00 g, 28.1 mmol) in anhydrous THF (95 mL) was added a solution of i-PrMgBr in THF (0.75 M, 56 mL, 42.0 mmol) at -78 °C while maintaining the internal temperature < -70 °C (about 15 min). The resulting solution was then stirred for 15 min, after which a solution of CuCN·2LiCl in THF (1 M, 31 mL, 31.0 mmol) was added, followed by allyl bromide (5.10 g, 42 mmol) as a solution in THF (10 mL). The reaction mixture was allowed to warm to room temperature and stirred for 1 h. The reaction mixture was quenched with MeOH (40 mL) and concentrated. The residue was purified by CombiFlash using silica gel as stationary phase and eluted with a gradient of EtOAc in hexanes (0-20%). Yield of compound 152: 4.13 g (74%). Mass calculated for C7H7BrN2 [M+H] + : 198.99, actual value: 199.05. [ka]

[0361] To a solution of compound 152 (7.70 g, 38.7 mmol) in THF (115 mL) was added a solution of 9-BBN in THF (0.5 M, 131 mL, 65.8 mmol) at 0 °C over 30 min. The reaction mixture was allowed to warm to room temperature and stirred overnight. To the reaction mixture was added a slurry of NaHCO3 (48.7 g, 580 mmol) in water (100 mL), followed by a slurry of NaBO3 monohydrate (46.3 g, 464 mmol) in water (100 mL) at 0 °C. The cooling bath was removed and the mixture was stirred vigorously for 1 h. The reaction mixture was transferred to a separatory funnel and the layers were separated. The aqueous layer was extracted with EtOAc (200 mL). The organic phases were combined and washed with brine (100 mL). The brine layer was back extracted with EtOAc (100 mL). The combined organic phases were dried over Na2SO4, filtered and concentrated to give ∼15 g of a crude yellow oil. The crude product was purified by CombiFlash using silica gel as stationary phase and eluted with a gradient of EtOAc in hexanes (50-100%). Yield of compound 153: 3.44 g (41%). Mass calculated for C7H9BrN2O [M+H] + : 217.00, actual value: 216.97. [ka]

[0362] To a solution of compound 153 (3.44 g, 15.8 mmol) in DCM (40 mL) was added a solution of imidazole (1.73 g, 25.4 mmol) and TBDPSCl (5.23 g, 19.0 mmol) in DCM (12 mL) at 0° C. The reaction was warmed to room temperature and stirred overnight. The reaction mixture was diluted with DCM (75 mL) and washed with water (50 mL) and brine (50 mL). The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash using silica gel as stationary phase and eluted with a gradient of EtOAc in hexanes (0-8%). Yield of compound 154: 5.56 g (77%). C 23 H 27 Calculated mass for BrN2OSi [M+H] +: 455.12, actual value: 455.44. [ka]

[0363] To a solution of compound 154 (6.07 g, 13.3 mmol) in THF (150 mL) at -75°C was added a solution of nBuLi in THF (2.5 M, 5.6 mL, 14.0 mmol) dropwise while maintaining the internal temperature <-70°C (approximately 10 min). After 3 min, a solution of ethyl formate (1.04 g, 1.13 mL, 14.0 mmol) in THF (5 mL) was added dropwise while maintaining the internal temperature <-70°C. The mixture was stirred at -78°C for 20 min, then it was further diluted with THF (5 mL) and quenched with HCl in dioxane (4 M, 3.67 mL, 14.7 mmol) while maintaining the internal temperature <-65°C. The cooling bath was removed and the reaction was allowed to warm to ambient temperature and concentrated. The residue was purified by CombiFlash using silica gel as stationary phase and eluted with a gradient of EtOAc in hexanes (0-20%). Yield of compound 155: 1.79 g (33%). 1 H NMR(400 MHz, CDCl3):δ10.09 (s, 1H), 9.06 (s, 2H), 7.64 (m, 4H), 7.38 (m, 6H), 3.77 (t, 2H), 3.20 (t, 2H), 2.17 (q, 2H), 1.03 (s, 9H). [ka]

[0364] To a solution of compound 144 (1.68 g, 4.15 mmol) and compound 155 (1.70 g, 4.98 mmol) in THF (25 mL) was added K2CO3 (0.861 g, 6.23 mmol). The reaction mixture was heated to 40 °C for 2.5 h and then to 50 °C for 12 h. The reaction mixture was diluted with EtOAc (100 mL) and washed with water (50 mL) and brine (50 mL). The organic phase was dried over Na2SO4, filtered, and concentrated. The residue was purified by CombiFlash using silica gel as stationary phase and eluted with a gradient of EtOAc in hexanes (0-100%) containing 1% triethylamine. Yield of compound 156: 2.04 g (79%). C 38 H 46 Calculated mass for N4O2Si [M+H] + : 619.35, actual value: 619.69. [ka]

[0365] Preparation of R-BINAL: LAH (1.169 g, 30.8 mmol) was slurried in anhydrous THF (90 mL). int EtOH was added as a solution in THF (6M, 5.2 mL, 31.4 mmol) while maintaining the temperature at <40° C. The mixture was aged at 35° C. for 40 min and then cooled to 30° C. int A solution of R-(BINOL) (9.00 g, 31.4 mmol) in THF (45 mL) was added while maintaining <40° C. The mixture was aged at 50° C. for 1 h, cooled to ambient temperature, then heated to 50° C. and TMEDA (14.1 mL, 11.0 g, 94.3 mmol) was added. The mixture was aged at 50° C. for 1 h, cooled to ambient temperature, then used with compound 156.

[0366] To a solution of R-BINAL (~0.2M, 110 mL, 22.0 mmol) in THF was added a solution of compound 16 (1.16 g, 1.88 mmol) in THF (12 mL) at -78 °C over 5 min. After 30 min, the reaction mixture was quenched with saturated aqueous NH4Cl, warmed to room temperature, and the product was extracted with EtOAc (3x125 mL). The organic layer was dried over Na2SO4, filtered, and concentrated. The residue was purified by CombiFlash using silica gel as stationary phase and eluted with a gradient (0-5%) of MeOH in EtOAc containing 1% triethylamine. Yield of compound 157: 0.96 g (82%). Chiral purity was determined by analytical chiral HPLC (Chiralpak AD-H column 4.6x250mm, 5 micron, 25% EtOH, 75% hexane, 0.1% diethylamine isocratic, 2mL / min). The second eluting R isomer was ~95% area pure, corresponding to ~90% ee. 38 H 48 Calculated mass for N4O2Si [M+H] + : 621.36, actual value: 621.71. [ka]

[0367] To a solution of compound 157 (0.925 g, 1.49 mmol) in triethyl orthoacetate (9.25 mL) was added a solution of propionic acid in trimethyl orthoacetate (0.15 M, 0.55 mL, 0.08 mmol). The reaction mixture was heated in a sealed vial at 140° C. for 1.5 h. The reaction mixture was concentrated and the residue was purified by CombiFlash using silica gel as stationary phase and eluting with a gradient (0-50%) of EtOAc in hexane containing 1% triethylamine. Yield of compound 158: 0.898 g (87%). 42 H 54 Calculated mass for N4O3Si [M+H] + : 691.41, actual value: 691.93. [ka]

[0368] To a solution of compound 158 (0.893 g, 1.30 mmol) in EtOH (10 mL) was added a slurry of Pd / C (extent of loading: 10 wt%, 0.138 g, 0.13 mmol) in EtOH (4 mL). The reaction mixture was charged with 50 psi of H2 and stirred for 4.5 h. The reaction mixture was filtered, concentrated, and used in the next step without further purification. Yield of compound 159: 0.885 g (99%). C 42 H 56 Calculated mass for N4O3Si [M+H] + : 693.42, actual value: 693.82. [ka]

[0369] A solution of Boc anhydride (0.836 g, 3.83 mmol) in THF (2.5 mL) was added to compound 159 (0.885 g, 1.28 mmol), followed by a solution of DMAP (20 mg / mL in THF, 155 μL, 0.0031 g, 0.026 mmol). The mixture was heated to 60° C. for 6 h. The reaction mixture was concentrated and the residue was purified by CombiFlash using silica gel as stationary phase and eluting with a gradient of EtOAc in hexanes (0-50%). Yield of compound 160: 0.721 g (71%). C 47 H 64 Calculated mass for N4O5Si [M+H] + : 793.47, actual value: 794.28. [ka]

[0370] To a solution of compound 160 (0.621 g, 0.783 mmol) in THF (6 mL) was added a solution of TBAF in THF (1 M, 1.2 mL, 1.2 mmol) at 0° C. The reaction mixture was warmed to room temperature and stirred for 2 h. The reaction mixture was diluted with EtOAc (30 mL) and washed with saturated aqueous NH4Cl (2×10 mL). The organic layer was concentrated. The residue was purified by CombiFlash using silica gel as stationary phase and eluting with a gradient of EtOAc in hexanes (50-100%). Yield of compound 21: 0.362 g (83%). Chiral purity was determined by analytical chiral HPLC, Chiralpak AD-H column 4.6×250 mm, 5 micron, 20% EtOH, 80% hexanes, 0.1% diethylamine, isocratic, 1.5 mL / min. The second eluting R isomer was 93% pure, corresponding to 86% ee. Compound 161 was further purified by chiral semi-preparative HPLC (Chiralpak AD-H 21.2x250mm, 5 micron, 20% EtOH, 80% Hexane, 0.1% diethylamine, 60mL / min). Final yield of compound 161: 308mg (99% ee). 31 H 46 Calculated mass for N4O5 [M+H] + : 555.36, actual value: 555.72. [ka]

[0371] To a solution of compound 161 (0.030 g, 0.054 mmol) in ACN (0.30 mL) was added BAIB (0.042 g, 0.130 mmol) and TEMPO (2.5 mg, 0.016 mmol) at room temperature, followed by water (0.30 mL). After 2 h, the reaction mixture was concentrated. The residue was purified by RP-HPLC (Phenomenex Gemini C18 21.2×250 mm, 5 micron, 0.1% TFA water / ACN, gradient 30-80% ACN). Yield of compound 162: 0.030 g (97%). C 31 H 44 Calculated mass for N4O6 [M+H]+ :569.34, actual value:569.68. [ka]

[0372] To a solution of compound 162 (33 mg, 0.058 mmol) and amino-PEG2-azide (15 mg, 0.087 mmol) in DMF (0.5 mL) at 0° C. was added TBTU (32 mg, 0.099 mmol) followed by DIEA (35 μL, 26 mg, 0.203 mmol). The reaction mixture was warmed to room temperature and stirred for 30 min. The reaction mixture was concentrated and the product, compound 163, was used in the next step without purification. 37 H 56 Calculated mass for N8O7 [M+H] + : 725.44, actual value: 725.77. [ka]

[0373] To a solution of compound 163 (42 mg, 0.058 mmol) in THF (0.30 mL) was added a 1 M solution of LiOH (0.174 mL, 0.174 mmol). The reaction mixture was heated at 40° C. for 1 h. An additional portion of LiOH was added (0.174 mL, 0.174 mmol). After 3 h, the reaction was stopped and an additional portion of LiOH was added (0.174 mL, 0.174 mmol). The reaction was stirred for an additional 2 h (9 equiv. LiOH, 5 h total). The reaction mixture was neutralized to pH=5 using 3N HCl and concentrated. The residue was dissolved in TFA:water [95:5] and stirred at room temperature for 2 h. The reaction mixture was concentrated and the residue was purified by RP-HPLC (Phenomenex Gemini C18 21.2x250mm, 5 micron, water / ACN containing 0.1% TFA, 20-50% ACN gradient). Yield of compound 164 (structure 38c): 23 mg (66%). 30 H 44 Calculated mass for N8O5 [M+H] +:597.35, actual value:597.85. [ka]

[0374] To a solution of compound 161 (30 mg, 0.054 mmol) in THF (150 μL) was added diphenylphosphoryl azide (35 μL, 45 mg, 0.162 mmol) at 0° C., followed by DBU (12 μL, 12 mg, 0.081 mmol). The reaction mixture was warmed to room temperature and stirred overnight. The next morning, the reaction mixture was heated at 60° C. for 7 h. The reaction mixture was concentrated and purified by RP-HPLC (Phenomenex Gemini C18 21.2×250 mm, 5 micron, 0.1% TFA water / ACN, 32-60% ACN gradient). Yield of compound 165: 14 mg (44%). C 31 H 45 Calculated mass for N7O4 [M+H] + :580.36, actual value:580.66. [ka]

[0375] To a solution of compound 165 (18 mg, 0.031 mmol) in EtOH (100 μL) was added a slurry of Pd / C (10% loading, 3.3 mg, 0.003 mmol) in EtOH (170 μL). The reactor was charged with H2, then evacuated three times, then charged with H2 (1 atm). After 30 min, the reaction mixture was filtered, concentrated, and used in the next step without further purification. Yield of compound 166: 17 mg (99%). C 31 H 47 Calculated mass for N5O4 [M+H] + :554.37, actual value:554.73. [ka]

[0376] To a solution of compound 166 (17 mg, 0.031 mmol) and azide-PEG3-NHS ester (14 mg, 0.040 mmol) in DMF (170 μL) was added DIEA (16 μL, 12 mg, 0.092 mmol) at room temperature. The reaction mixture was stirred at room temperature for 1 h, concentrated, and then used in the next step without purification. 40 H 62 Calculated mass for N8O8 [M+H] + : 783.48, actual value: 783.84. [ka]

[0377] To a solution of compound 167 (24 mg, 0.031 mmol) in THF (180 μL) was added a 1 M solution of LiOH (153 μL, 0.153 mmol). The reaction mixture was heated at 40° C. After 1 h, an additional portion of LiOH (153 μL, 0.153 mmol, 5 eq) was added. The reaction mixture was stirred at 40° C. for 3 h and then at room temperature overnight. The reaction mixture was neutralized to pH=5 using 3N HCl and concentrated. The residue was dissolved in TFA:water [95:5] and it was stirred at room temperature for 3 h. The reaction mixture was concentrated and the residue was purified by RP-HPLC (Phenomenex Gemini C18 21.2×250 mm, 5 microns, water / ACN containing 0.1% TFA, 15-45% ACN gradient). Yield of compound 168 (structure 39c): 9.8 mg (49%). C 33 H 50 Calculated mass for N8O6 [M+H] + : 655.40, actual value: 656.01.

[0378] Example 2. Tridentate integrin targeting ligands, synthesis of RNAi agents, and conjugation of integrin targeting ligands to cargo molecules (RNAi agents) Integrin targeting ligand can be conjugated to one or more RNAi agents that are useful for inhibiting the expression of one or more targeted genes in cells that express integrin.The integrin targeting ligand disclosed herein facilitates the delivery of RNAi agents to target cells and / or tissues.The synthesis of specific integrin targeting ligands disclosed herein is described in Example 1 above.The general methodology for the synthesis of specific integrin targeting ligand-RNAi agent conjugates exemplified in the non-limiting examples described herein is described below.

[0379] A. Synthesis of RNAi agents. RNAi agents can be synthesized using methods generally known in the art. In the synthesis of RNAi agents illustrated in the examples described herein, the sense and antisense strands of the RNAi agents were synthesized based on the phosphoramidite technology on solid phase used for oligonucleotide synthesis. Depending on the scale, a MerMade96E® (Bioautomation), a MerMade12® (Bioautomation), or an OP Pilot 100 (GE Healthcare) was used. Synthesis was carried out 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 phosphoramidites were used: (5'-O-dimethoxytrityl-N6-(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, 5'-O-dimethoxy-trityl-N4-(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino) phosphoramidite, (5'-O-dimethoxytrityl-N2-(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino) phosphoramidite, The following protective groups were used: 5'-dimethoxytrityl-2'-O-methyl-inosine-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 had the same protecting groups as the 2'-O-methylamidite. 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-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-guanosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-isobutyryl-2',3'-seco-guanosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-(4,4'-dimethoxytrityl)-2',3'-seco-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. TFA aminolink phosphoramidites were also purchased commercially (ThermoFisher).

[0380] In some examples, the integrin targeting ligands disclosed herein are conjugated to RNAi agents by linking the moiety to a scaffold containing a trialkyne group, or to modified nucleotides containing a propargyl group as shown in Table B above. In some examples, the trialkyne group is added by using a trialkyne-containing phosphoramidite, which can be added at the 5' end of the sense strand of the RNAi agent. When used with the RNAi agents shown in certain examples herein, the trialkyne-containing phosphoramidite was dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), whereas all other amidites were dissolved in anhydrous acetonitrile (50 mM) with the addition of molecular sieves (3 Å). 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 min (RNA), 90 s (2'OMe), and 60 s (2'F). To introduce phosphorothioate linkages, a 100 mM solution of 3-phenyl-1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was used.

[0381] Alternatively, when an integrin targeting ligand is conjugated to an RNAi agent via a trialkyne scaffold instead of using phosphoramidite methods, the trialkyne-containing compound can be introduced post-synthetically (see, for example, item E below). When used with the RNAi agents shown in the specific examples described herein, when a trialkyne group was 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. TFA aminolink 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. The coupling times were 12 minutes (RNA), 90 seconds (2'OMe), and 60 seconds (2'F). To introduce phosphorothioate linkages, a 100 mM solution of 3-phenyl-1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Inc., Leominster, Mass., USA) in anhydrous acetonitrile was used.

[0382] In the examples described herein; the following shows modified nucleotide sequences for double stranded synthesis: Double-stranded AD04545: Modified antisense strand sequence (5'→3): usUfsusCfaUfgAfaAfuCfgUfuAfcGfuUfsg (SEQ ID NO: 1) Modified sense strand sequence (5'→3): (NH2-C6)scsaacguaaCfGfAfuuucaugaasa(invAb) (SEQ ID NO: 2) Double-stranded AD04546: Modified antisense strand sequence (5'→3): usUfsusCfaUfgAfaAfuCfgUfuAfcGfuUfsg (SEQ ID NO: 3) Modified sense strand sequence (5'→3): (NH2-C6)scsaacguaaCfGfAfuuucaugaasa(invAb)(C6-S-Mal-X) (SEQ ID NO: 4) Double-stranded AD05971: Modified antisense strand sequence (5'→3): usUfsusCfaUfgAfaAfuCfgUfuAfcGfuUfsg (SEQ ID NO:5) Modified sense strand sequence (5'→3): (NH2-C6)scsaacguaaCfGfAfuuuAlkcaAlkugAlkaaAlksa(invAb) (C6-S-Mal-C-18-diacid moiety) (SEQ ID NO: 6)

[0383] With respect to the modified nucleotide sequences listed above, a, c, g, and u represent 2'-O-methyl adenosine, cytidine, guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, cytidine, guanosine, or uridine, respectively; aAlk, cAlk, gAlk, and uAlk represent 2'-O-propargyl adenosine, cytidine, guanosine, or uridine, respectively; (invAb) represents an inverted abasic residue (inverted abasic deoxyribonucleotide); s represents a phosphorothioate linkage; and (NH2-C6) represents the following: [ka] and (C6-S-Mal-L) represents the following: [ka] where L is a PEG chain or ethyl, as shown in the examples below. For the embodiments herein, when looking at each strand 5'→3', an inverted abasic has been inserted such that the 3' position of the deoxyribose is linked at the 3' end of the preceding monomer of each strand.

[0384] B. Cleavage and deprotection of the support-bound oligomer. After completing the solid-phase synthesis, the dried solid support was treated with a 1:1 volume solution of 40 wt % methylamine in water 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).

[0385] C. Purification. Crude oligomers were 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. Buffer B was the same as buffer A plus 1.5 M sodium chloride. UV traces were recorded at 260 nm. Appropriate fractions were pooled and then run on a size-exclusion HPLC using a GE Healthcare XK 26 / 40 column packed with Sephadex G-25 fine, with 100 mM ammonium bicarbonate (pH 6.7) and 20% acetonitrile, or filtered water, as the running buffer.

[0386] D. Annealing. Complementary strands were mixed together in equimolar amounts of RNA solution (sense and antisense) in 1x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form the RNAi agent. Some RNAi agents were lyophilized and stored at -15 to -25 °C. Duplex concentrations were determined by measuring the solution absorbance in 1x PBS using a UV-Vis spectrometer. The solution absorbance at 260 nm was then multiplied by the conversion factor and dilution factor to determine the duplex concentration. All conversion factors used were 0.037 mg / (mL cm) or, for some experiments, the conversion factor was calculated from the experimentally determined extinction coefficient.

[0387] E. Conjugation of Trialkine Scaffolds. Either before or after annealing, the 5' or 3' amine-functionalized sense strand of the RNAi agent can be conjugated to the trialkyne scaffold. Illustrative trialkyne backbone structures that can be used in forming the constructs disclosed herein include the following: [ka]

[0388] The following describes the conjugation of a trialkyne scaffold to the annealed duplex: Amine-functionalized duplexes were dissolved in 90% DMSO / 10% HO at ~50-70mg / mL. 40eq of triethylamine was added, followed by 3eq of trialkyne-PNP. Upon completion, the conjugate was immersed twice in a solvent system of 1x phosphate buffered saline / acetonitrile (1:14 ratio) and allowed to dry.

[0389] F. Conjugation of integrin-targeting ligands. Conjugate the 5' or 3' tridentate alkyne functionalized sense strand to the integrin targeting ligand either before or after annealing. The following example describes the conjugation of an αvβ3 / 5 integrin targeting ligand to an annealed duplex: Stock solutions of 0.5 M Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 0.5 M Cu(II) sulfate pentahydrate (Cu(II)SO4·5H2O), and a 2 M solution of sodium ascorbate were prepared in deionized water. A 75 mg / mL solution of the integrin targeting ligand in DMSO was made. Add 25 μL of 1 M Hepes pH 8.5 buffer to a 1.5 mL centrifuge tube containing the tri-alkene functionalized duplex (3 mg, 75 μL, 40 mg / mL in deionized water, ~15,000 g / mol). After vortexing, 35 μL of DMSO was added and the solution was vortexed. Integrin targeting ligand was added to the reaction (6 eq / duplex, 2 eq / alkyne, ~15 μL) and the solution was vortexed. pH was checked using pH paper to ensure pH was ~8. In a 1.5 mL split 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 eq, 5:1 THPTA:Cu) was added to the reaction vial and vortexed. Immediately after, 2 M ascorbate (5 μL, 50 eq per duplex, 16.7 per alkyne) was added to the reaction vial and vortexed. Upon reaction completion (typically in 0.5-1 h), the reaction was immediately purified by non-denaturing anion exchange chromatography.

[0390] G. Functionalization of Cysteine ​​Ringer-like Thiol Groups. In some embodiments, a cysteine ​​linker was used to facilitate conjugation of the integrin targeting ligand to the 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 and left as a free thiol, as shown in the following structure: [ka]

[0391] The following example describes the modification of a trialkyne-Cys(Stbu)-PEG2-duplex with N-ethylmaleimide: Trialkyne-Cys(Stbu)-PEG2-duplex (35 mg) was dissolved in 500 μL of deionized H2O. HEPES buffer (1 M, pH 8.5, 82 μL) was added to the reaction and the solution was vortexed. 1 M dithiothreitol (DTT, 100 eq, 236 μL) solution was added and the solution was placed on a vortex shaker for 3 hours. After confirmation of reduction of disulfides by denaturation on RP-HPLC, the conjugate was precipitated three times in 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, N-ethylmaleimide (3 mg, 10 eq) was added to the solution and placed on a vortex mixer for ∼15 min. After reaction completion, the conjugate was precipitated three times in a solvent system of 1× phosphate buffered saline / acetonitrile (1:14 ratio), desalted, and dried.

[0392] Example 3. Binding activity of integrin-targeting ligands IC50 binding data was obtained for integrin targeting ligands of structures 1c, 2c, and 3c, as well as the RGD mimetic peptide, as shown in Table 1 below:

[0393] [Table 3]

[0394] As shown in Table 1 above, each of structures 1, 2, and 3 showed strong binding to αvβ3 integrin and αvβ5 integrin, for example, structures 2 and 3 showed a particular preference for binding to αvβ3 integrin (IC50=0.3nM and 0.8nM, respectively). Furthermore, each of structures 1, 2, and 3 showed slightly increased binding activity to αvβ3 integrin compared to RGD mimetic peptides (see, for example, mimetic RGD ligand structures disclosed in U.S. Patent No. 9,487,556). Moreover, while RGD mimetic ligands have been shown to have binding activity, the integrin-targeting ligands of the present disclosure have high stability, both in vivo serum stability and ex vivo chemical stability, compared to such peptide-based RGD mimetic ligands.

[0395] Example 4. Renal tumor-bearing mouse model (orthotopic xenograft) Generation of SEAP-expressing clear cell renal cell carcinoma (ccRCC) A498 cells The pCR3.1 expression vector expressing the alkaline phosphatase secreted reporter gene (SEAP) under the CMV promoter was prepared by directional cloning of the PCR-amplified SEAP coding sequence from the pSEAP2 basic vector from Clontech. Convenient restriction sites were added on the primers used to amplify the SEAP coding sequence for cloning into the pCR3.1 vector (Invitrogen). The resulting construct, pCR3-SEAP, was used to generate an A498 ccRCC cell line expressing SEAP. Briefly, the pCR3-SEAP plasmid was transfected into A498 ccRCC cells by electroporation according to the manufacturer's recommendations. Stable transfectants were selected by G418 resistance. Selected A498-SEAP clones were evaluated for SEAP expression and integration stability.

[0396] Implantation of clear cell renal cell carcinoma (ccRCC) A498 cells expressing SEAP Female athymic nude mice were anesthetized with 3% isoflurane and placed in the right lateral position. A small, 0.5-1 cm longitudinal abdominal incision was made in the left flank. Using a moistened cotton swab, the left kidney was elevated from the peritoneum and gently secured. Just prior to injection, a 1.0 ml syringe was filled with the cell / Matrigel mixture and a 27-gauge needle catheter was attached to the syringe tip. The filled syringe was then attached to a syringe pump (Harvard Apparatus, model PHD2000) and prepared for air removal. The tip of the 27-gauge needle catheter attached to the syringe was inserted just below the renal capsule near the caudal pole, and the tip of the needle was then carefully advanced 3-4 mm rostrally along the capsule. A 10 μl aliquot of a 2:1 (volume:volume) cell / Matrigel® mixture containing approximately 300,000 cells was slowly injected into the kidney parenchyma using a syringe pump. The needle was left in the kidney for 15-20 seconds to ensure the injection was complete. The needle was then removed from the kidney and a cotton swab was placed over the injection site for 30 seconds to prevent cell leakage or bleeding. The kidney was then gently placed back into the abdomen and the abdominal wall was closed. Serum was collected every 7-14 days after implantation and tumor growth was monitored using a commercially available SEAP assay kit. For most studies, tumor-bearing mice were used 5-6 weeks after implantation, at which time tumor measurements were usually around 4-8 mm.

[0397] Measurement of HIF2 mRNA expression For the studies reported in the Examples herein, mice were euthanized on specific days after injection and total RNA was isolated from renal tumors using Trizol reagent according to the manufacturer's recommendations. Relative HiF2α mRNA levels were measured by RT-qPCR as described below and compared to mice treated with delivery buffer (isotonic glucose) alone.

[0398] In preparation for quantitative PCR, total RNA was isolated from tissue samples homogenized with TriReagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's protocol. Approximately 500 ng of RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Life Technologies). For human (tumor) Hif2α (EPAS1) expression, pre-manufactured TaqMan gene expression assays for human Hif2α (catalog #4331182) and CycA (PPIA) (catalog #: 4326316E) were used in triplicate biplex reactions using TaqMan Gene Expression Master Mix (Life Technologies) or VeriQuest Probe Master Mix (Affymetrix). Quantitative PCR was performed by using a 7500 Fast or StepOnePlus real-time PCR system (Life Technologies). ΔΔC T Relative gene expression was calculated using the method.

[0399] Example 5. In vivo administration of integrin-targeting ligands as RNAi agents targeting HIF-2α (EPAS1) in mice bearing renal cancer The RNAi agents, including sense and antisense strands, were synthesized by phosphoramidite technology on solid phase according to general techniques known in the art and commonly used for oligonucleotide synthesis, as described in Example 2 herein. The RNAi agents included an antisense strand with a nucleobase sequence at least partially complementary to the HIF-2α (Hif2α or EPAS1) gene. EPAS1 is a member of the HIF (hypoxia-inducible factor) gene family, encoding half of a transcription factor involved in oxygen-regulated gene induction, which is known to be induced when oxygen levels are reduced (a condition known as hypoxia) and is frequently overexpressed in clear cell renal carcinoma cells. The Hif2α RNAi agents were designed to reduce or inhibit the translation of messenger RNA (mRNA) transcripts of Hif2α in a sequence-specific manner, thereby inhibiting the expression of the EPAS1 gene. The Hif2α RNAi agents consisted of modified nucleotides and two or more non-phosphodiester bonds.

[0400] On study day 1, kidney cancer-bearing mice (see Example 4) were dosed by tail vein injection according to the following dosing groups:

[0401] [Table 4]

[0402] The RNAi agent of Example 5 was synthesized with a nucleotide sequence directed to target the human Hif2α gene and with a functionalized amine reactive group (NH2-C6) at the 5' end of the sense strand to facilitate conjugation to an integrin targeting ligand (or, in group 3, to an RGD mimetic peptide-based ligand). The modified sequence of the RNAi agent was shown in Example 2 above. For groups 4 and 5, a single integrin targeting ligand (referred to herein as a "monodentate" ligand) was conjugated to the RNAi agent via a DBCO-PEG5-NHS ester linker (BroadPharm). The linker was conjugated to the primary amine at the end above the 5' end of the sense strand. Each integrin targeting ligand was synthesized with an azide reactive group (which was then conjugated to the DBCO component of the linker) (see, for example, Example 1).

[0403] The following structure: [ka] {wherein: [ka] The RNAi agents were synthesized having PK modulators referred to as "20 kDA PEG moieties" or "40 kDA PEG moieties" with the C6-SS-C6 group shown in Table A (which is then combined with the following compounds: [ka] The PK modulator was attached to the 3' end of the sense strand by reducing the PEG-100 (where PEG represents a 20 kDa or 40 kDa PEG chain) which undergoes a Michael addition.

[0404] For groups 6 and 7, the four integrin targeting ligands were as follows: [ka] The DBCO-functionalized PAMAM-G1 is conjugated via a tetradentate scaffold containing a cystamine core, which has the general structure represented by:

[0405] As noted above in Table 2, in some groups, 40 kDa or 20 kDa PEG moieties were attached to serve as PK enhancers to extend the circulation time of the drug-product conjugate. The 40 kDa or 20 kDa PEG moieties were attached using reagents of the following formula:

[0406] Three tumor-bearing mice were administered in each group (n=3). Mice were sacrificed on the eighth day of the study after injection, and total RNA was isolated from the renal tumors according to the procedure described in Example 4. Relative human HIF2α mRNA expression was then quantified by probe-based quantitative PCR (RT-qPCR) normalized to human Cyclophilin A (PPIA) expression, and expressed as a fraction of the solvent control group (isotonic glucose) (geometric mean, + / - 95% confidence interval), as described in Example 4.

[0407] [Table 5]

[0408] As shown in Table 3 above, each of the Hif2α RNAi agents showed reduced mRNA expression in mice compared to the control. Inclusion of a 40kDa PEG moiety as a PK enhancer improved the inhibition of targeted gene expression in most cases. Moreover, comparison of groups 3, 4, and 5 showed that the integrin-targeting ligands of structure 1a described herein were comparable to RGD mimetic peptide-based ligands known to have affinity for αvβ3, and furthermore, the ligands of structure 2a showed about 10% improvement in knockdown over the RGD mimetic ligands. For example, group 3 (RGD mimetic) had about 60% knockdown (0.400); group 4 (structure 1a) had about 61% knockdown (0.390); and group 5 (structure 2a) had about 69% knockdown (0.308).

[0409] Importantly, the data also demonstrated ligand dependency, as inclusion of an integrin targeting ligand as disclosed herein demonstrated improvement when compared to the same construct without the ligand. For example, Group 6 (tridentate integrin targeting ligand structure 2a) demonstrated approximately 72% knockdown (0.289) when compared to Group 2 (no integrin targeting ligand), which demonstrated only approximately 44% knockdown (0.563).

[0410] Additionally, Group 6 showed a small improvement over Group 5, indicating a slight preference for the multidentate ligand over the monodentate ligand; however, both forms were active and delivered the RNAi agent to the kidney (as indicated by inhibition of gene expression by the RNAi agent).

[0411] Example 6. In vivo administration of an integrin-targeting ligand conjugated to an RNAi agent targeting HIF-2α in mice bearing renal cancer The RNAi agents, including the sense and antisense strands, were synthesized by phosphoramidite technology on solid phase according to the general methods known in the art and commonly used for oligonucleotide synthesis, as described in Example 2 herein. The RNAi agents had the respective modified nucleotide sequences described in Example 2 herein and were designed to target Hif2α (EPAS1).

[0412] On study day 1, kidney cancer-bearing mice (see Example 4) were dosed by tail vein injection according to the following dosing groups:

[0413] [Table 6]

[0414] RNAi agents were synthesized having nucleotide sequences directed to target the human Hif2α gene and containing a functionalized amine reactive group (NH2-C6) at the 5' end of the sense strand to facilitate conjugation to an integrin-targeting ligand (or to an RGD-mimetic peptide for groups 2, 3, and 4). For groups 5 and 6, a single integrin-targeting ligand (a "monodentate" ligand) was synthesized using the following DBCO-PEG5-NHS ester: [ka] The RNAi agent was conjugated via

[0415] Three tumor-bearing mice were administered in each group (n=3). Mice were sacrificed on the eighth day of the study after injection, and total RNA was isolated from the renal tumors according to the procedure described in Example 4. Relative human HIF2α mRNA expression was then quantified by probe-based quantitative PCR (RT-qPCR) normalized to human Cyclophilin A (PPIA) expression, and expressed as a fraction of the solvent control group (isotonic glucose) (geometric mean, + / - 95% confidence interval), as described in Example 4.

[0416] [Table 7]

[0417] As shown in Table 5 above, each of the Hif2α RNAi agents showed reduced mRNA expression in mice compared to the control. Moreover, groups 5 and 6 (containing the integrin targeting ligand of structure 2a disclosed herein) showed improved knockdown of Hif2α mRNA compared to the RGD mimetic peptide-based ligands of groups 2 and 3 (e.g., group 6 (about 73% knockdown (0.271) at 15 mg / kg RNAi agent) compared to group 3 (about 67% knockdown (0.330) at 15 mg / kg RNAi agent)).

[0418] Example 7. In vivo administration of an integrin-targeting ligand conjugated to an RNAi agent targeting HIF-2α in mice bearing renal cancer The RNAi agents, including the sense and antisense strands, were synthesized by phosphoramidite technology on solid phase according to the general methods known in the art and commonly used for oligonucleotide synthesis, as described in Example 2 herein. The RNAi agents had the respective modified nucleotide sequences described in Example 2 herein and were designed to target Hif2α (EPAS1).

[0419] On study day 1, kidney cancer-bearing mice (see Example 4) were dosed by tail vein injection according to the following dosing groups:

[0420] [Table 8]

[0421] RNAi agents were synthesized that have nucleotide sequences directed to target the human Hif2α gene and contain a functionalized amine-reactive group (NH2-C6) at the 5' end of the sense strand to facilitate conjugation to integrin-targeting ligands. For groups 2-7, the following compounds were synthesized: [ka] was used to functionalize the conjugate with a tridentate scaffold.

[0422] For group 8, an alkyne-PEG4-NHS ester was used to link a monodentate integrin targeting ligand to the 5' amine on the sense strand. Groups 4-7 used integrin targeting ligands with various PEG lengths, as described herein.

[0423] Three tumor-bearing mice were administered in each group (n=3). Mice were sacrificed on the eighth day of the study after injection, and total RNA was isolated from the renal tumors according to the procedure described in Example 4. Relative human HIF2α mRNA expression was then quantified by probe-based quantitative PCR (RT-qPCR) normalized to human Cyclophilin A (PPIA) expression, and expressed as a fraction of the solvent control group (isotonic glucose) (geometric mean, + / - 95% confidence interval), as described in Example 4.

[0424] [Table 9]

[0425] As shown in Table 7 above, each of the Hif2α RNAi agents showed reduced mRNA expression in mice compared to the control. For example, Group 2 (containing a dose of 7.5 mg / kg of RNAi agent conjugated to a tridentate integrin targeting ligand of structure 2a (containing a PEG4 group)) showed approximately 64% knockdown (0.361) of Hif2α. Furthermore, PEG 36While all of the constructs with increased PEG group length up to 100 (eg, groups 6 and 7) showed knockdown, no benefit was seen when compared to the PEG4 group present in structure 2a.

[0426] Example 8. Dose-response study of in vivo administration of integrin-targeting ligands conjugated to RNAi agents targeting HIF-2α in mice bearing renal cancer. The RNAi agents, including the sense and antisense strands, were synthesized by phosphoramidite technology on solid phase according to the general methods known in the art and commonly used for oligonucleotide synthesis, as described in Example 2 herein. The RNAi agents had the respective modified nucleotide sequences described in Example 2 herein and were designed to target Hif2α (EPAS1).

[0427] On study day 1, kidney cancer-bearing mice (see Example 4) were dosed by tail vein injection according to the following dosing groups:

[0428] [Table 10]

[0429] RNAi agents were synthesized that had nucleotide sequences directed to target the human Hif2α gene and contained a functionalized amine-reactive group (NH2-C6) at the 5' end of the sense strand to facilitate conjugation to integrin-targeting ligands. Each group had the following structure: [ka] The compound has a tridentate integrin ligand of structure 2a, represented by the formula:

[0430] Three tumor-bearing mice were administered in each group (n=3). Mice were sacrificed on the eighth day of the study after injection, and total RNA was isolated from the renal tumors according to the procedure described in Example 4. Relative human HIF2α mRNA expression was then quantified by probe-based quantitative PCR (RT-qPCR) normalized to human Cyclophilin A (PPIA) expression, and expressed as a fraction of the solvent control group (isotonic glucose) (geometric mean, + / - 95% confidence interval), as described in Example 4.

[0431] [Table 11]

[0432] As shown in Table 9 above, Hif2α RNAi agents conjugated to integrin targeting ligands of structure 2a disclosed herein showed reduced mRNA expression in mice compared to controls across all dosage levels.

[0433] Example 9. Sustained knockdown of RNAi agents targeting HIF-2α conjugated to integrin-targeting ligands in mice bearing renal cancer The RNAi agents, including the sense and antisense strands, were synthesized by phosphoramidite technology on solid phase according to the general methods known in the art and commonly used for oligonucleotide synthesis, as described in Example 2 herein. The RNAi agents had the respective modified nucleotide sequences described in Example 2 herein and were designed to target Hif2α (EPAS1).

[0434] On study day 1, kidney cancer-bearing mice (see Example 4) were dosed by tail vein injection according to the following dosing groups:

[0435] [Table 12]

[0436] Mice in groups 1 and 2 were euthanized on day 5 after injection; mice in group 3 were euthanized on day 8 after injection; mice in group 4 were euthanized on day 15 after injection; and mice in groups 1A and 5 were euthanized on day 22 after injection.

[0437] For the vehicle control group, two mice were administered in group 1 and three mice were administered in group 1A. For the RNAi agent-integrin targeting ligand-containing groups (i.e., groups 2, 3, 4, and 5), four tumor-bearing mice were administered in each group (n=4). Total RNA was isolated from renal tumors according to the procedure described in Example 4. Relative human HIF2α mRNA expression was then quantified by probe-based quantitative PCR (RT-qPCR) normalized to human Cyclophilin A (PPIA) expression and expressed as a fraction of the solvent control group (isotonic glucose) (geometric mean, + / - 95% confidence interval), as described in Example 4.

[0438] [Table 13]

[0439] As shown in Table 11 above, the Hif2α RNAi agent continued to show reduced mRNA expression at day 22 compared to the control (approximately 70% knockdown (0.299) at day 22).

[0440] Example 10. In vivo administration of an integrin-targeting ligand conjugated to an RNAi agent targeting HIF-2α in mice bearing renal cancer The RNAi agents, including the sense and antisense strands, were synthesized by phosphoramidite technology on solid phase according to the general methods known in the art and commonly used for oligonucleotide synthesis, as described in Example 2 herein. The RNAi agents had the respective modified nucleotide sequences described in Example 2 herein and were designed to target Hif2α (EPAS1).

[0441] On study day 1, kidney cancer-bearing mice (see Example 4) were dosed by tail vein injection according to the following dosing groups:

[0442] [Table 14]

[0443] An RNAi agent was synthesized having a nucleotide sequence directed to target the human Hif2α gene and containing a functionalized amine-reactive group (NH2-C6) at the 5' end of the sense strand to facilitate conjugation to integrin-targeting ligands.

[0444] Three tumor-bearing mice were administered in each group (n=3). Mice were sacrificed on the eighth day of the study after injection, and total RNA was isolated from the renal tumors according to the procedure described in Example 4. Relative human HIF2α mRNA expression was then quantified by probe-based quantitative PCR (RT-qPCR) normalized to human Cyclophilin A (PPIA) expression, and expressed as a fraction of the solvent control group (isotonic glucose) (geometric mean, + / - 95% confidence interval), as described in Example 4.

[0445] [Table 15]

[0446] As shown in Table 13 above, each of the Hif2α RNAi agent-integrin targeting ligand conjugates showed reduced mRNA expression in mice compared to controls. For example, Group 2 (containing a 7.5 mg / kg dose of RNAi agent conjugated to a tridentate integrin targeting ligand of structure 2a) showed approximately 65% ​​knockdown (0.351) of Hif2α mRNA.

[0447] Example 11. In vivo administration of integrin-targeting ligands conjugated to RNAi agents targeting HIF-2α in mice bearing αvβ3 KO A498 renal carcinoma Clear cell renal cell carcinoma (ccRCC) A498 tumor cells express both αvβ3 and αvβ5 integrins, and αvβ3 expression is approximately 4 times higher than αvβ5 by flow cytometry analysis. To evaluate the contribution of αvβ5 in this model, αvβ3 knockout (KO) A498 cells were synthesized by gene editing technology. The knockout of integrin αvβ3 was confirmed by genome sequencing and immunohistochemical staining of αvβ3, which showed that staining was negative in αvβ3 KO A498 cells. Renal cancer-bearing mice with A498 WT (both αvβ3 and αvβ5) cells and αvβ3 KO A498 cells were prepared as described above in Example 4.

[0448] On study day 1, kidney cancer-bearing mice were administered by tail vein injection. Three tumor-bearing mice were administered (n=3) in each group as described in Table 13 below. Mice were sacrificed on study day 8 after injection, and total RNA was isolated from kidney tumors as described in Example 4. Relative human HIF2α mRNA expression was then quantified by probe-based quantitative PCR (RT-qPCR) normalized to human Cyclophilin A (PPIA) expression, and expressed as a fraction of the solvent control group (isotonic glucose) as described in Example 4 (geometric mean, + / -95% confidence interval).

[0449] [Table 16]

[0450] As shown in Table 14 above, the Hif2α RNAi agent-integrin ligand conjugate showed a reduction in Hif2α mRNA expression in A498 WT (wild type) tumors compared to the control (approximately 71% (0.295) knockdown). In contrast, as expected, the reduction in Hif2α mRNA expression was less effective in A498 αvβ3 KO tumors; however, the reduction was still substantial, a 38% (0.621) knockdown. This indicates that both integrin αvβ3 and integrin αvβ5 contribute to the delivery of the RNAi agent.

[0451] Example 12. In vivo administration of an integrin-targeting ligand conjugated to an RNAi agent targeting HIF-2α in mice bearing renal cancer The RNAi agents, including the sense and antisense strands, were synthesized by phosphoramidite technology on solid phase according to the general methods known in the art and commonly used for oligonucleotide synthesis, as described in Example 2 herein. The RNAi agents had the respective modified nucleotide sequences described in Example 2 herein and were designed to target Hif2α (EPAS1).

[0452] On study day 1, kidney cancer-bearing mice (see Example 4) were dosed by tail vein injection according to the following dosing groups:

[0453] [Table 17]

[0454] An RNAi agent was synthesized having a nucleotide sequence directed to target the human Hif2α gene and containing a functionalized amine-reactive group (NH2-C6) at the 5' end of the sense strand to facilitate conjugation to integrin-targeting ligands.

[0455] The following structure: [ka] {wherein: [ka] The RNAi agents were synthesized having PD modulators also referred to as "Mal-C18-diacid moieties" with the C6-SS-C6 group shown in Table A (which then reacts with the following compound: [ka] The PD modulator was conjugated to the 3' end of the sense strand by reducing (which undergoes a Michael addition with

[0456] Three tumor-bearing mice were administered in each group (n=3). Mice were sacrificed on the eighth day of the study after injection, and total RNA was isolated from the renal tumors according to the procedure described in Example 4. Relative human HIF2α mRNA expression was then quantified by probe-based quantitative PCR (RT-qPCR) normalized to human Cyclophilin A (PPIA) expression, and expressed as a fraction of the solvent control group (isotonic glucose) (geometric mean, + / - 95% confidence interval), as described in Example 4.

[0457] [Table 18]

[0458] As shown above in Table 16, each of the Hif2α RNAi agent-integrin targeting ligand conjugates demonstrated reduced mRNA expression compared to the control.

[0459] Example 13. In vivo administration of an integrin-targeting ligand conjugated to an RNAi agent targeting HIF-2α in mice bearing renal cancer The RNAi agents, including the sense and antisense strands, were synthesized by phosphoramidite technology on solid phase according to the general methods known in the art and commonly used for oligonucleotide synthesis, as described in Example 2 herein. The RNAi agents had the respective modified nucleotide sequences described in Example 2 herein and were designed to target Hif2α (EPAS1).

[0460] On study day 1, kidney cancer-bearing mice (see Example 4) were dosed by tail vein injection according to the following dosing groups:

[0461] [Table 19-1] [Table 19-2]

[0462] Three tumor-bearing mice were administered in each group (n=3). Mice were sacrificed on the eighth day of the study after injection, and total RNA was isolated from the renal tumors according to the procedure described in Example 4. Relative human HIF2α mRNA expression was then quantified by probe-based quantitative PCR (RT-qPCR) normalized to human Cyclophilin A (PPIA) expression, and expressed as a fraction of the solvent control group (isotonic glucose) (geometric mean, + / - 95% confidence interval), as described in Example 4.

[0463] [Table 20]

[0464] As shown in Table 18 above, each of the Hif2α RNAi agent-integrin targeting ligand conjugates having structures including the integrin targeting ligands of Structure 2a and Structure 32a, which showed the greatest inhibitory activity, showed reduced mRNA expression in mice compared to controls.

[0465] Example 14. In vivo administration of an integrin-targeting ligand conjugated to an RNAi agent targeting HIF-2α in mice bearing renal cancer The RNAi agents, including the sense and antisense strands, were synthesized by phosphoramidite technology on solid phase according to the general methods known in the art and commonly used for oligonucleotide synthesis, as described in Example 2 herein. The RNAi agents had the respective modified nucleotide sequences described in Example 2 herein and were designed to target Hif2α (EPAS1).

[0466] On study day 1, kidney cancer-bearing mice (see Example 4) were dosed by tail vein injection according to the following dosing groups:

[0467] [Table 21]

[0468] Three tumor-bearing mice were administered in each group (n=3). Mice were sacrificed on the eighth day of the study after injection, and total RNA was isolated from the renal tumors according to the procedure described in Example 4. Relative human HIF2α mRNA expression was then quantified by probe-based quantitative PCR (RT-qPCR) normalized to human Cyclophilin A (PPIA) expression, and expressed as a fraction of the solvent control group (isotonic glucose) (geometric mean, + / - 95% confidence interval), as described in Example 4.

[0469] [Table 22]

[0470] As shown above in Table 20, each of the Hif2α RNAi agent-integrin targeting ligand conjugates demonstrated reduced mRNA expression compared to the control.

[0471] Example 15. In vivo administration of an integrin-targeting ligand conjugated to an RNAi agent targeting HIF-2α in mice bearing renal cancer The RNAi agents, including the sense and antisense strands, were synthesized by phosphoramidite technology on solid phase according to the general methods known in the art and commonly used for oligonucleotide synthesis, as described in Example 2 herein. The RNAi agents had the respective modified nucleotide sequences described in Example 2 herein and were designed to target Hif2α (EPAS1).

[0472] On study day 1, kidney cancer-bearing mice (see Example 4) were dosed by tail vein injection according to the following dosing groups:

[0473] [Table 23-1] [Table 23-2] [Table 23-3] [Table 23-4]

[0474] Three tumor-bearing mice were administered in each group (n=3). Mice were sacrificed on the eighth day of the study after injection, and total RNA was isolated from the renal tumors according to the procedure described in Example 4. Relative human HIF2α mRNA expression was then quantified by probe-based quantitative PCR (RT-qPCR) normalized to human Cyclophilin A (PPIA) expression, and expressed as a fraction of the solvent control group (isotonic glucose) (geometric mean, + / - 95% confidence interval), as described in Example 4.

[0475] [Table 24-1] [Table 24-2]

[0476] As shown above in Table 20, each of the Hif2α RNAi agent-integrin targeting ligand conjugates demonstrated reduced mRNA expression in mice compared to controls.

[0477] Example 16. In vivo administration of an integrin-targeting ligand conjugated to an RNAi agent targeting HIF-2α in mice bearing renal cancer The RNAi agents, including the sense and antisense strands, were synthesized by phosphoramidite technology on solid phase according to the general methods known in the art and commonly used for oligonucleotide synthesis, as described in Example 2 herein. The RNAi agents had the respective modified nucleotide sequences described in Example 2 herein and were designed to target Hif2α (EPAS1).

[0478] On study day 1, kidney cancer-bearing mice (see Example 4) were dosed by tail vein injection according to the following dosing groups:

[0479] [Table 25-1] [Table 25-2] [Table 25-3]

[0480] Four tumor-bearing mice were administered in each group (n=4), except for group 4, which had only three mice because one mouse was determined to have received an incomplete injection. Mice were sacrificed on study day 8 after injection, and total RNA was isolated from renal tumors according to the procedure described in Example 4. Relative human HIF2α mRNA expression was then quantified by probe-based quantitative PCR (RT-qPCR) normalized to human Cyclophilin A (PPIA) expression, and expressed as a fraction of the solvent control group (isotonic glucose) (geometric mean, + / - 95% confidence interval), as described in Example 4.

[0481] [Table 26-1] [Table 26-2]

[0482] As shown above in Table 24, each of the Hif2α RNAi agent-integrin targeting ligand conjugates demonstrated reduced mRNA expression in mice compared to controls. Other embodiments

[0483] Although the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

[Claim 1] The following: 【Chemical 1】 or a pharmaceutically acceptable salt thereof, comprising an integrin targeting ligand and an RNAi agent of the structure: wherein: 【Chemistry 2】 indicates the point of connection of the integrin-targeting ligand to the RNAi agent. A compound or a pharmaceutically acceptable salt thereof.

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