Integrin-targeting ligand and its use

Abstract

Described herein are compounds having affinity for integrins, the synthesis of these compounds, and the use of these compounds as ligands to facilitate the delivery of cargo molecules to cells expressing integrins. 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 the delivery of cargo molecules to cells or tissues expressing integrin αvβ3, integrin αvβ5, or both integrin αvβ3 and integrin αvβ5, such as tumor cells. Compositions and methods of use containing the integrin-targeting ligands are also described.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 663,763, filed on April 27, 2018, and U.S. Provisional Patent Application No. 62 / 790,372, filed on January 9, 2019, and the entire disclosures of both are incorporated herein by reference in their entireties.

[0002] Field of the Invention Compounds having an affinity for integrin, methods for synthesizing such compounds, and the use of such compounds as ligands for delivering cargo molecules in vivo are disclosed herein.

Background Art

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

[0004] In fact, overexpression of integrins, including integrin 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, since inhibition of the αvβ3 receptor has been shown to inhibit angiogenesis and, as a result, suppress the formation of new blood vessels thought to be necessary for tumor growth, attempts have been made to develop αvβ3 inhibitors as promising cancer therapies (see, for example, Brooks et al., 79 Cell 1157-1164 (1994); Mas-Moruno et al., Anticancer Agents Med Chem, 10(10):753-768). However, it has been shown that Cilengitide, a main example of an αvβ3 inhibitor, is ineffective in clinical trials aimed at restricting tumor angiogenesis and progression in patients with glioblastoma (see, for example, Ley et al., Integrin-based Therapeutics: Biological Basis, Clinical Use and New Drugs, 15(3) Nat. Rev. Drug Discov. 173-183 (2016)).

[0005] Generally, the delivery of cargo molecules, including therapeutically effective pharmaceutical compounds or active pharmaceutical ingredients, to desired cells and / or tissues in vivo continues to be a common challenge in the development of therapeutically viable pharmaceutical formulations. There continues to be a need for stable and effective targeting compounds that have an affinity for, and / or selectively bind to, specific cells or tissues (and which can be used or utilized as ligands to facilitate the delivery of therapeutic cargo molecules to those specific cells or tissues). Moreover, there is a particular need for compounds that can selectively target integrin αvβ-3, are suitable for conjugation to cargo molecules, and, in vivo, deliver cargo molecules to cells expressing such integrins, such as tumor cells. With respect to oligonucleotides, particularly oligonucleotide-based therapeutic agents (e.g., oligonucleotide-based compounds such as antisense oligonucleotides or RNAi agents), there continues to be a need for ligands 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] Summary Compounds can be used as ligands for selectively targeting cells or tissues that express integrin αvβ3 and / or αvβ5 to which compounds or other molecules are to be bound (referred to herein as "integrin-targeting ligands", "αvβ3 integrin-targeting ligands", "αvβ3 integrin ligands" or simply "integrin-targeting ligands"). Compounds are described herein that have an affinity for specific integrins, including αvβ3 and αvβ5. 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 one or more cargo molecules to facilitate delivery of the (one or more) cargo molecules to cells or tissues that express integrin αvβ3 and / or αvβ5.

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

[0008] Methods for inhibiting the expression of a target gene in cells in vitro or in vivo are further described herein, where the method comprises 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 therapeutic agents, such as RNAi agents, capable of inhibiting the expression of the target gene in the cell. In some embodiments, methods for inhibiting the expression of a target gene in a subject's cells are described herein, where the subject is administered an effective amount of one or more oligonucleotide-based therapeutic agents (such as RNAi agents) conjugated to one or more integrin-targeting ligands disclosed herein.

[0009] In yet another aspect, compositions comprising integrin-targeting ligands disclosed herein are described herein. The compositions described herein can be pharmaceutical compositions or agents comprising one or more integrin-targeting ligands disclosed herein conjugated to one or more therapeutic cargo molecules, such as RNAi agents, or other cargo molecules or therapeutic agents.

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

[0012] In some embodiments, the integrin-targeting ligands disclosed herein have the following formula:

Chemical Formula

[0013] Any of the integrin-targeting ligands disclosed herein can be linked to a cargo molecule, a reactive group, and / or a protected reactive group. For example, the linkage to a reactive group can be used to facilitate the conjugation of an integrin-targeting ligand to a cargo molecule. The integrin-targeting ligands disclosed herein can enhance the targeting of a cargo molecule to cells expressing integrins, including αvβ3 integrin and / or αvβ5 integrin. The cargo molecule can be, but is not limited to, a pharmaceutically active ingredient or compound, a prodrug, or another substance with known therapeutic benefits. In some embodiments, the cargo molecule can be, but is not limited to, a small molecule, an antibody, an antibody fragment, an immunoglobulin, a monoclonal antibody, a label or marker, a lipid, a natural or modified oligonucleotide, 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, polyethylene glycol, a hapten, digoxigenin, biotin, a radioactive atom or molecule, or a fluorophore. In some embodiments, the cargo molecule includes a pharmaceutically active ingredient or a prodrug. In some embodiments, the cargo molecule is an oligonucleotide-based therapeutic agent, such as an antisense compound or an RNAi agent, or includes them. In some embodiments, the cargo molecule is an oligonucleotide-based compound that is a pharmaceutically active ingredient, or includes them. In some embodiments, the cargo molecule is an RNAi agent that is a pharmaceutically active ingredient, or includes them.

[0014] Described is the use of the αvβ3 / 5 integrin-targeting ligands described herein for targeting and delivering a cargo molecule to cells expressing integrin. The cargo molecule 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, a composition comprising one or more of the 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, a composition for delivering an RNAi agent to cells in vivo is described herein, wherein the RNAi agent is linked to one or more integrin-targeting ligands.

[0016] Compositions comprising one or more integrin-targeting ligands are described. In some embodiments, the composition comprises a pharmaceutically acceptable excipient. In some embodiments, a composition comprising one or more integrin-targeting ligands comprises one or more other pharmaceutical substances or pharmaceutically active ingredients or compounds. In some embodiments, agents comprising one or more integrin-targeting ligands are described herein.

[0017] Compositions comprising one or more of the integrin-targeting ligands disclosed herein can be delivered to a variety of cancer cells, including, 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, HCT116), 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 other patient-derived xenografts, in vivo or in vitro.

[0018] In another aspect, the present disclosure provides methods that include the use of one or more integrin-targeting ligands and / or compositions described herein, and, if desired, formulating 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 manufacturing a ligand and composition described herein, e.g., a medicament.

[0019] Compositions comprising one or more integrin-targeting ligands may be administered in vivo to a subject using routes of administration known in the art to be suitable for such administration, from the perspective 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 example, by intravenous or subcutaneous administration for systemic delivery.

[0020] In some embodiments, methods of delivering one or more desired cargo molecules to clear cell renal carcinoma tumor cells in vivo are disclosed herein, where the method comprises administering to the subject one or more integrin-targeting ligands conjugated to one or more cargo molecules.

[0021] In some embodiments, methods for delivering oligonucleotide-based compounds to tumor cells in vivo are disclosed herein, where the methods include administering to the subject one or more integrin-targeting ligands conjugated to one or more oligonucleotide-based compounds. In some embodiments, methods for delivering RNAi agents to tumor cells in vivo are disclosed herein, where the methods include administering to the subject one or more integrin-targeting ligands conjugated to one or more RNAi agents. In some embodiments, methods for inhibiting the expression of target genes in clear cell renal carcinoma tumor cells in vivo are disclosed herein, where the methods include 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.

Best Mode for Carrying Out the Invention

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

[0024] In some embodiments, the integrin-targeting ligands disclosed herein preferentially direct cargo molecules to cells or tissues that express integrins, including integrin αvβ3 and / or integrin αvβ5, and are conjugated to one or more cargo molecules so as to target them. In some embodiments, the cargo molecules comprise or consist of pharmaceutically active compounds. In some embodiments, the cargo molecules comprise or consist of oligonucleotide-based compounds such as RNAi agents. In some embodiments, the integrin-targeting ligands disclosed herein are conjugated to cargo molecules to direct the cargo molecules to tumor cells in vivo. In some embodiments, the integrin-targeting ligands disclosed herein are conjugated to cargo molecules to direct the cargo molecules to clear cell renal carcinoma tumor cells in vivo.

[0025] Formula I In one aspect, the present invention has the following structure:

Chemical formula

Chemical formula

[0026] In some embodiments of Formula I, R 1 is one of the following:

Chemical formula

Chemical formula

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

[0028] Formula II In some embodiments of Formula I, the integrin-targeting ligand disclosed herein has the following formula:

Chemical formula

[0029] Formula III In some embodiments of Formula I, the integrin - targeting ligand disclosed herein has the following formula:

Chemical Formula

[0030] Formula IV In some embodiments of Formula I, the integrin - targeting ligand disclosed herein has the following formula

Chemical Formula

[0031] Formula V In some embodiments of Formula I, the integrin targeting ligand disclosed herein has the following formula:

Chemical Structure

[0032] Formula VI In some embodiments of Formula I, the integrin-targeting ligand disclosed herein has the following formula:

Chemical Structure

[0033] Formula VII In some embodiments of Formula I, the integrin-targeting ligand disclosed herein has the following formula:

Chemical Structure

[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 contains a cargo molecule. In some embodiments, R 1 is the following:

Chem.

Chem.

[0035] Integrin-targeting ligand precursor In some embodiments, the present invention provides an integrin-targeting ligand precursor that can be used to attach an integrin-targeting ligand to a moiety containing a cargo molecule. The following formula:

Chem.

Chem.

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

[0037] Compound of formula I In some embodiments, the integrin - targeting ligands disclosed herein comprise, consist of, or consist essentially of a structure represented by any of the following structures:

Chemical formula

Chemical formula

Chemical formula

[0038] In some embodiments, the integrin-targeting ligands disclosed herein are 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, 5-30, 5-25, 5-20, 5-15, 5-10, 10-30, 10-25, 10-20, 10-15, 15-30, 15-25, 15-20, 20-30, 20-25, or 25-30 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 ligands disclosed herein are optionally conjugated to one or more cargo molecules via a scaffold comprising at least one attachment point for each ligand and at least one attachment point for each cargo molecule. In some embodiments, the integrin-targeting ligand comprises, consists of, or consists essentially of an integrin-targeting ligand conjugated to one cargo molecule. In some embodiments, the integrin-targeting ligand comprises, consists of, or consists essentially 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 Structures 1a, 2a, 2.1a, 2.2a, 2.3a, 2.4a, 2.5a, 2.6a, 2.8a, 2.9a, 2.10a, 2.11a, 28a, 29a, 30a, 31a, 32a, 33a, 34a, 36a, 37a, 38a, 39a, 40a, and 41a disclosed herein.

[0043] In some embodiments, the integrin-targeting ligands disclosed herein have the following structures:

Chemical formula

[0044] In some embodiments, the integrin-targeting ligand of Structure 1a is linked to one or more cargo molecules (e.g., (single or plural) RNAi agents).

[0045] In some embodiments, the integrin-targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, and has the following structure:

Chemical formula

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

Chemical formula

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

[0048] In some embodiments, the integrin-targeting ligands disclosed herein have the following structure:

Chemical formula

[0049] In some embodiments, the integrin-targeting ligand of Structure 2a is linked to one or more cargo molecules (e.g., (one or more) RNAi agents).

[0050] In some embodiments, the integrin-targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and have the following structure:

Chemical formula

[0051] In some embodiments, the integrin-targeting ligand can be synthesized to include an azide-reactive group and have the following structure: [Chemistry] comprises.

[0052] In some embodiments, the integrin-targeting ligands disclosed herein have the following structure: [Chemistry] comprises.

[0053] In some embodiments, the integrin-targeting ligand of Structure 2.1a is linked to one or more cargo molecules (e.g., (singular or plural) RNAi agents).

[0054] In some embodiments, the integrin-targeting ligand can be synthesized to include an azide-reactive group and has the following structure: [Chemistry] comprises.

[0055] In some embodiments, the integrin-targeting ligands disclosed herein have the following structure: [Chemistry] comprises.

[0056] In some embodiments, the integrin-targeting ligand of Structure 2.2a is linked to one or more cargo molecules (e.g., (singular or plural) RNAi agents).

[0057] In some embodiments, the integrin-targeting ligand can be synthesized to include an azide-reactive group and has the following structure: [Chemistry] comprises.

[0058] In some embodiments, the integrin-targeting ligands disclosed herein have the following structure:

Chem.

[0059] In some embodiments, the integrin-targeting ligand of Structure 2.3a is linked to one or more cargo molecules (e.g., (one or more) RNAi agents).

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

Chem.

[0061] In some embodiments, the integrin-targeting ligands disclosed herein have the following structure:

Chem.

[0062] In some embodiments, the integrin-targeting ligand of Structure 2.4a is linked to one or more cargo molecules (e.g., (one or more) RNAi agents).

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

Chem.

[0064] In some embodiments, the integrin-targeting ligands disclosed herein have the following structure:

Chem.

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

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

Chemical formula

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

Chemical formula

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

[0069] In some embodiments, the integrin-targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure:

Chemical formula

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

Chemical formula

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

[0072] In some embodiments, the integrin targeting ligands disclosed herein have the following structure:

Chemical formula

[0073] In some embodiments, the integrin targeting ligand of Structure 2.7a is linked to one or more cargo molecules (e.g., an (single or plural) RNAi agent).

[0074] In some embodiments, the integrin targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, and has the following structure:

Chemical formula

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

Chemical formula

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

[0077] In some embodiments, the integrin-targeting ligands disclosed herein have the following structure:

Chemical formula

[0078] In some embodiments, the integrin-targeting ligand of Structure 2.8a is linked to one or more cargo molecules (e.g., (single or plural) RNAi agents).

[0079] In some embodiments, the integrin-targeting ligand can be synthesized to contain an azide-reactive group and has the following structure:

Chemical formula

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

[0081] In some embodiments, the integrin-targeting ligands disclosed herein have the following structure:

Chemical formula

[0082] In some embodiments, the integrin-targeting ligand of Structure 2.9a is linked to one or more cargo molecules (e.g., (single or plural) RNAi agents).

[0083] In some embodiments, the integrin-targeting ligand can be synthesized to contain an azide-reactive group and has the following structure:

Chemical formula

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

[0085] In some embodiments, the integrin targeting ligands disclosed herein have the following structure:

Chemical formula

[0086] In some embodiments, the integrin targeting ligand of Structure 2.10a is linked to one or more cargo molecules (e.g., an (single or plural) RNAi agent).

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

Chemical formula

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

[0089] In some embodiments, the integrin targeting ligands disclosed herein have the following structure:

Chemical formula

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

[0091] In some embodiments, the integrin-targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule and has the following structure:

Chemical formula

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

[0093]

Chemical formula

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

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

Chemical formula

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

[0097] In some embodiments, the integrin-targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, and has the following structure:

Chemical formula

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

Chemical formula

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

Chemical formula

[0100] In some embodiments, the integrin-targeting ligand of structure 29a is linked to one or more cargo molecules (e.g., one or more RNAi agents).

[0101] In some embodiments, the integrin-targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, and has the following structure:

Chemical formula

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

Chemical formula

[0103] In some embodiments, the integrin-targeting ligands disclosed herein have the following structure: [Chemical Formula] comprises

[0104] In some embodiments, the integrin-targeting ligand of Structure 30a is linked to one or more cargo molecules (e.g., (one or more) RNAi agents).

[0105] In some embodiments, the integrin-targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, and has the following structure: [Chemical Formula] {wherein X includes a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent)}

[0106] In some embodiments, the integrin-targeting ligand can be synthesized to include an azide-reactive group and has the following structure: [Chemical Formula] comprises

[0107] In some embodiments, the integrin-targeting ligands disclosed herein have the following structure: [Chemical Formula] comprises

[0108] In some embodiments, the integrin-targeting ligand of Structure 31a is linked to one or more cargo molecules (e.g., (one or more) RNAi agents).

[0109] In some embodiments, the integrin-targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, and has the following structure:

Chemical formula

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

Chemical formula

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

Chemical formula

[0112] In some embodiments, the integrin-targeting ligand of structure 32a is linked to one or more cargo molecules (e.g., one or more RNAi agents).

[0113] In some embodiments, the integrin-targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, and has the following structure:

Chemical formula

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

Chemical formula

[0115] In some embodiments, the integrin-targeting ligands disclosed herein have the following structure:

Chemical formula

[0116] In some embodiments, the integrin-targeting ligand of Structure 33a is linked to one or more cargo molecules (e.g., (single or plural) RNAi agents).

[0117] In some embodiments, the integrin-targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, and has the following structure:

Chemical formula

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

Chemical formula

[0119] In some embodiments, the integrin-targeting ligands disclosed herein have the following structure:

Chemical formula

[0120] In some embodiments, the integrin-targeting ligand of Structure 34a is linked to one or more cargo molecules (e.g., (single or plural) RNAi agents).

[0121] In some embodiments, the integrin-targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, and has the following structure:

Chemical formula

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

Chemical formula

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

Chemical formula

[0124] In some embodiments, the integrin-targeting ligand of Structure 36a is linked to one or more cargo molecules (e.g., one or more RNAi agents).

[0125] In some embodiments, the integrin-targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, and has the following structure:

Chemical formula

[0126] In some embodiments, the integrin-targeting ligand can be synthesized to include an azide reactive group, and has the following structure: [Chemical formula] comprises

[0127] In some embodiments, the integrin - targeting ligand disclosed herein has the following structure: [Chemical formula] comprises

[0128] In some embodiments, the integrin - targeting ligand of Structure 37a is linked to one or more cargo molecules (e.g., (single or plural) RNAi agents).

[0129] In some embodiments, the integrin - targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, and has the following structure: [Chemical formula] {wherein 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 can be synthesized to include an azide - reactive group, and has the following structure: [Chemical formula] comprises

[0131] In some embodiments, the integrin - targeting ligand disclosed herein has the following structure: [Chemical formula] comprises

[0132] In some embodiments, the integrin - targeting ligand of Structure 38a is linked to one or more cargo molecules (e.g., (single or plural) RNAi agents).

[0133] In some embodiments, the integrin-targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, and has the following structure:

Chemical formula

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

Chemical formula

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

Chemical formula

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

[0137] In some embodiments, the integrin-targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, and has the following structure:

Chemical formula

[0138] In some embodiments, the integrin-targeting ligand can be synthesized to include an azide-reactive group, and has the following structure: [Chemical formula] includes

[0139] In some embodiments, the integrin-targeting ligands disclosed herein have the following structure: [Chemical formula] includes

[0140] In some embodiments, the integrin-targeting ligand of Structure 40a is linked to one or more cargo molecules (e.g., (single or plural) RNAi agents).

[0141] In some embodiments, the integrin-targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, and has the following structure: [Chemical formula] {wherein X includes a reactive group, a protected reactive group, or a cargo molecule (e.g., an RNAi agent)} includes

[0142] In some embodiments, the integrin-targeting ligand can be synthesized to include an azide reactive group, and has the following structure: [Chemical formula] includes

[0143] In some embodiments, the integrin-targeting ligands disclosed herein have the following structure: [Chemical formula] includes

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

[0145] In some embodiments, the integrin-targeting ligand can be synthesized to include a reactive group, a protected reactive group, or a cargo molecule, and has the following structure:

Chemical formula

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

Chemical formula

[0147] The azide-reactive groups disclosed in any of Structures 1c, 2c, 2.1c, 2.2c, 2.3c, 2.4c, 2.5c, 2.6c, 2.7c, 2.8c, 2.9c, 2.10c, 2.11c, 28c, 29c, 30c, 31c, 32c, 33c, 34c, 36c, 37c, 38c, 39c, 40c, and 41c can be used to attach the integrin-targeting ligand to the 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 cells expressing integrin.

[0148] As used herein, the term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group having from 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 configuration. 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 valency. The amino group 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 from 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 may include multiple spiro- or fused rings. The cycloalkyl group may optionally be mono-substituted, di-substituted, tri-substituted, tetra-substituted, or penta-substituted at any position as permitted by normal valency.

[0150] As used herein, the term "alkenyl", unless otherwise specified, refers to a straight or branched chain non-aromatic hydrocarbon group containing at least one carbon-carbon double bond and having from 2 to 10 carbon atoms. 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 from 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 portion of an alkenyl group may contain a double bond and optionally may be mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by normal valency. The term "cycloalkenyl" means a monocyclic hydrocarbon group having a specific number of carbon atoms and at least one carbon-carbon double bond.

[0151] As used herein, the term "alkynyl", unless otherwise specified, refers to a straight or branched chain hydrocarbon group containing from 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond. Up to 5 carbon-carbon triple bonds may be included. Thus, "C2-C6 alkynyl" means an alkynyl group having from 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 portion of an alkynyl group may optionally be mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by normal valency.

[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., ethynoyl, propynoyl, butynoyl, pentynoyl, or hexynoyl), aroyl (e.g., benzoyl), heteroaroyl (e.g., pyrrololyl, imidazoloyl, quinolinoyl, or pyridinoyl).

[0154] As used herein, "alkoxycarbonyl" refers to any alkoxy group as defined above, attached via 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 via 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 via 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 pyrimidinyl-oxycarbonyl.

[0157] As used herein, "aryl" or "aromatic" means a stable monocyclic or polycyclic carbocyclic ring having up to 6 atoms in each ring, wherein at least one ring is aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, tetrahydronaphthyl, indanyl, and biphenyl. When an aryl substitution is bicyclic and one ring is non-aromatic, the attachment is understood to be through the aromatic ring. The aryl group is optionally mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by normal valency.

[0158] As used herein, the term "heteroaryl" represents a stable monocyclic or polycyclic ring having up to 7 atoms in each ring, where at least one ring is aromatic and contains 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, benzoxazololonyl, quinolinyl, isoquinolinyl, dihydroisoindolonyl, imidazopyridinyl, isoindolonyl, indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinoline. "Heteroaryl" is also understood to include N-oxide derivatives of any nitrogen-containing heteroaryl. When the heteroaryl substitution is bicyclic and one ring is non-aromatic or does not contain a heteroatom, the bond is understood to be through the aromatic ring or through the heteroatom-containing ring. The heteroaryl group is optionally mono-substituted, di-substituted, tri-substituted, tetra-substituted, or penta-substituted at any position as permitted by normal valency.

[0159] As used herein, the terms "heterocyclic", "heterocyclic ring", or "heterocyclyl" mean a 3- to 14-membered aromatic or non-aromatic heterocyclic ring containing 1 to 4 heteroatoms selected from the group consisting of O, N, and S, including polycyclic groups. As used herein, the term "heterocyclic ring" is also considered to be synonymous with the terms "heterocyclic" and "heterocyclyl" and is understood to have the same definition described herein. "Heterocyclyl" includes the heteroaryl described above, as well as its dihydro and tetrahydro analogs.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, indradinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthopyridinyl, oxadiazolyl, oxooxazolidinyl, oxazolyl, oxazoline, oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyridinonyl, pyrimidinyl, pyrimidinonyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, tetrazolyl, 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, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dioxide thiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl, and their N-oxides. The bond of the heterocyclyl substitution can occur via a carbon atom or via a heteroatom. The heterocyclyl group is optionally, at any position allowed by the normal valency, mono-substituted, di-substituted, tri-substituted, tetra-substituted, or penta-substituted.

[0160] As used herein, terms such as "treating" and "treatment" mean a method or step taken to provide a reduction or alleviation in the number, severity, and / or frequency of one or more symptoms of a disease in a subject. As used herein, "treating" and "treatment" may include the 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 a cell", when referring to a cargo molecule, mean functionally delivering the cargo molecule to the cell. The phrase "functionally delivering" means delivering the cargo molecule to the cell in a manner that enables the cargo molecule to have its expected biological activity. When specifically referring to a cargo molecule that is an RNAi agent, for example, the expected biological activity is sequence-specific inhibition of gene expression.

[0162] Unless otherwise specified, as used herein, the following symbols

Chemical formula

[0163] As used herein, the term "isomer" refers to compounds that have the same molecular formula but differ in the nature or order of bonding of their atoms or in the spatial arrangement of their atoms. Isomers that differ in the spatial arrangement of their atoms are called "stereoisomers". Stereoisomers that are not mirror images of each other 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 portion of a molecule to another second molecule or second portion of a molecule. In the art, the terms linking group and spacer are sometimes used interchangeably. Similarly, as used in the art, the term scaffold is sometimes used interchangeably with linking group. In some embodiments, in some embodiments, the linking group can comprise 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 via a non-covalent bond (e.g., a hydrogen bond or an ionic bond). In some examples where the term “linked” refers to an association between two molecules via a non-covalent bond, the association between the two different molecules has a KD of less than 1×10 -4 M (e.g., less than 1×10 -5 M, less than 1×10 -6 M, or less than 1×10 -7 M) in a physiologically acceptable buffer (e.g., phosphate buffered saline). Unless described otherwise, as used herein, the term linked can refer to a connection between a first compound and a second compound, regardless of the presence or absence of any intervening atoms or groups of atoms.

[0166] One of ordinary skill in the art will readily understand and recognize that the compounds and compositions disclosed herein can 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. Accordingly, as used herein, the structures disclosed herein are envisioned such that certain functional groups, such as OH, SH, or NH, can 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 one of ordinary skill in the art.

[0167] As used in the claims of the present application, the phrase "consisting of" excludes any element, step, or component not specified in the claim. As used in the claims of the present application, the phrase "consisting essentially of" limits the scope of the claim to those specific materials or steps and those that do not materially affect the basic and novel (single or plural) features of the invention recited in the claim.

[0168] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0169] Multivalent 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 "divalent" 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 conjugated directly to the cargo molecule. In some embodiments, the integrin ligands disclosed herein are conjugated to cargo molecules 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 linkers or spacers, can be used to facilitate the attachment of one or more cargo molecules to one or more integrin ligands disclosed herein. Useful scaffolds that are compatible with the ligands disclosed herein are well 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 is a cysteine linker or group, DBCO-PEG 1-24 -NHS, propargyl-PEG 1-24 -NHS, and / or multi-site DBCO and / or propargyl moieties can also be mentioned.

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

Chemical formula

[0173] For example, the use of Scaffold 1 facilitates the efficient 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, as well as a terminal alkyne. The 4-nitrophenol ester can be conjugated 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)), by amide formation. The terminal alkyne is conjugated to azide-modified ligands (both peptides and small molecules) by a copper-catalyzed click chemical reaction.

[0174] In some embodiments, the cargo molecule is an RNAi agent. In some embodiments, the scaffold 1 may be attached to the end of the 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 the 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 any other modification that results in a terminal amine) can be readily conjugated to the scaffold 1 as shown by the representation in the following structure:

Chemical formula

Chemical formula

[0175] At that time, the alkyne group of the above structure 380 can be conjugated to the integrin ligand disclosed herein to form a trimeric integrin targeting group.

[0176] In some embodiments, the scaffold may be synthesized using DBCO (dibenzocyclooctyne), and it has the following structure:

Chemical formula

Chemical formula

[0177] In some embodiments, the triazole group has the following general structure:

Chemical formula

Chemical formula

Chemical formula

[0178] In some embodiments, the scaffold may be synthesized as a phosphoramidite compound, an example of which has the following structure:

Chemical formula

[0179] The trialkyne compound of Structure 400 enables the facile ligation of a tris-ligand to the 5'-end of the sense strand of an RNAi agent by a click reaction of an azide-containing targeting ligand and an alkyne.

[0180] In some embodiments, the 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, where the αvβ3 integrin targeting group is a tris-targeting group and includes three ligands.

[0181] In some embodiments, the αvβ3 tris-targeting group disclosed herein includes three ligands of Structure 2a and has the following structure:

Chemical formula

[0182] In some embodiments, the three - site targeting group disclosed herein comprises three ligands of Structure 2a and has the following structure:

Chem.

[0183] In some embodiments, the three - site targeting group disclosed herein comprises three ligands of Structure 2a and has the following structure:

Chem.

[0184] In some embodiments, the three - site targeting group comprising a glutaric acid linker comprises three ligands of Structure 2a and has the following structure:

Chem.

[0185] In some embodiments, the three - site targeting group disclosed herein comprises three ligands of Structure 2a and has the following structure:

Chem.

Chem.

[0186] In some embodiments, the three - site targeting group disclosed herein comprises three ligands of Structure 2a and has the following structure:

Chem.

Chem.

[0187] In some embodiments, the ανβ3-targeting moiety conjugated to the RNAi agent comprises three ligands of Structure 2a and has the following structure:

Chemical Formula

Chemical Formula

Chemical Formula

[0188] Reactive groups and protected reactive groups Reactive groups are well known in the art and provide for the formation of covalent bonds 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 orthopyridyldisulfide 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 covalent bonds 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 addition cyclization reactions.

[0190] In some embodiments, the integrin-targeting ligands disclosed herein are synthesized as tetrafluorophenyl (TFP) esters, which are substituted by reactive amino groups and can conjugate to cargo molecules. In some embodiments, the integrin-targeting ligands disclosed herein are synthesized as azides, and they can be coupled to propargyl or DBCO, for example, by a click chemistry addition cyclization reaction for attaching cargo molecules.

[0191] Protected reactive groups are also commonly used in the art. A protecting group provides for the temporary chemical conversion of a reactive group to a group that does not react under conditions where the unprotected group would react, for example, providing chemoselectivity in subsequent chemical reactions. 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 ligands described herein, will have a desired effect on cells containing integrin receptors. Cargo molecules can be, but are not limited to, pharmaceutical ingredients, pharmaceuticals, prodrugs, substances having a therapeutic effect, small molecules, antibodies, antibody fragments, immunoglobulins, monoclonal antibodies, labels or markers, lipids, natural or modified nucleic acids or polynucleotides, peptides, polymers, polyamines, proteins, aptamers, toxins, vitamins, PEG, haptens, digoxigenin, biotin, radioactive atoms or molecules, or fluorophores. 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 molecules to cells expressing integrin αvβ3 and / or integrin αvβ5.

[0193] In some embodiments, one or more cargo molecules are pharmaceutical ingredients or pharmaceutical compositions. In some embodiments, one or more cargo molecules are oligonucleotide-based compounds. As used herein, "oligonucleotide-based compounds" are from about 10 to 50 (e.g., 10 to 48, 10 to 46, 10 to 44, 10 to 42, 10 to 40, 10 to 38, 10 to 36, 10 to 34, 10 to 32, 10 to 30, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 14, 10 to 12, 12 to 50, 12 to 48, 12 to 46, 12 to 44, 12 to 42, 12 to 40, 12 to 38, 12 to 36, 12 to 34, 12 to 32, 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 50, 14 to 48, 14 to 46, 14 to 44, 14 to 42, 14 to 40, 14 to 38, 14 to 36, 14 to 34, 14 to 32, 14 to 30, 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20, 14 to 18, 14 to 16, 16 to 50, 16 to 48, 16 to 46, 16 to 44, 16 to 42, 16 to 40, 16 to 38, 16 to 36, 16 to 34, 16 to 32, 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20, 16 to 18, 18 to 50, 18 to 48, 18 to 46, 18 to 44, 18 to 42, 18 to 40, 18 to 38, 18 to 36, 18 to 34, 18 to 32, 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, 18 to 20, 20 to 50, 20 to 48, 20 to 46, 20 to 44, 20 to 42, 20 to 40, 20 to 38, 20 to 36, 20 to 34, 20 to 32, 20 to 30, 20 to 28, 20 to 26, 20 to 24, 20 to 22, 22 to 50, 22 to 48, 22 to 46, 22 to 44, 22 to 42, 22 to 40, 22 to 38, 22 to 36, 22 to 34, 22 to 32, 22 to 30, 22 to 28, 22 to 26, 22 to 24, 24 to 50, 24 to 48, 24 to 46, 24 to 44, 24 to 42, 24 to 40, 24 to 38, 24 to 36, 24 to 34, 24 to 32, 24 to 30, 24 to 28, 24 to 26, 26 to 50, 26 to 48, 26 to 46, 26 to 44, 26 to 42, 26 to 40, 26 to 38, 26 to 36, 26 to 34, 26 to 32, 26 to 30, 26 to 28, 28 to 50,a nucleotide sequence comprising from 28 to 48, 28 to 46, 28 to 44, 28 to 42, 28 to 40, 28 to 38, 28 to 36, 28 to 34, 28 to 32, 28 to 30, 30 to 50, 30 to 48, 30 to 46, 30 to 44, 30 to 42, 30 to 40, 30 to 38, 30 to 36, 30 to 34, 30 to 32, 32 to 50, 32 to 48, 32 to 46, 32 to 44, 32 to 42, 32 to 40, 32 to 38, 32 to 36, 32 to 34, 34 to 50, 34 to 48, 34 to 46, 34 to 44, 34 to 42, 34 to 40, 34 to 38, 34 to 36, 36 to 50, 36 to 48, 36 to 46, 36 to 44, 36 to 42, 36 to 40, 36 to 38, 38 to 50, 38 to 48, 38 to 46, 38 to 44, 38 to 42, 38 to 40, 40 to 50, 40 to 48, 40 to 46, 40 to 44, 40 to 42, 42 to 50, 42 to 48, 42 to 46, 42 to 44, 44 to 50, 44 to 48, 44 to 46, 46 to 50, 46 to 48, or 48 to 50 nucleotides or nucleotide base pairs. In some embodiments, the oligonucleotide-based compound has a nucleobase sequence that is at least partially complementary to the coding sequence in an expressed target nucleic acid or target gene (e.g., a gene transcript or mRNA of the target gene) within a cell. In some embodiments, the oligonucleotide-based compound can inhibit the expression of the underlying gene upon delivery to a cell that is expressing the gene, and is herein referred to as an "expression-inhibiting oligonucleotide-based compound". 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 RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), ribozymes, interfering RNA molecules, and dicer substrates. In some embodiments, the oligonucleotide-based compound is a single-stranded oligonucleotide-based compound, such as an antisense oligonucleotide. In some embodiments, the oligonucleotide-based compound is a double-stranded oligonucleotide-based compound. In some embodiments, the oligonucleotide-based compound is a double-stranded oligonucleotide that is an RNAi agent.

[0195] In some embodiments, one or more cargo molecules are “RNAi agents,” and as defined herein, an RNAi agent is a chemical composition that includes an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule, and such a molecule can sequence specifically inhibit the degradation of the messenger RNA (mRNA) transcript of a target mRNA or its translation. As used herein, an RNAi agent can operate via the RNA interference mechanism (i.e., induce RNA interference through interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells), or by any alternative mechanism or pathway. When the term is used herein, an RNAi agent is considered to operate primarily via the RNA interference mechanism, but the disclosed RNAi agents are not restricted or limited to any particular pathway or mechanism of action. The RNAi agents disclosed herein consist of a sense strand and an antisense strand, and as the RNAi agents include, but are not limited to: short or small interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), and dicer substrates. The antisense strand of the RNAi agents described herein is at least partially complementary to the targeted mRNA. The RNAi agents can include one or more modified nucleotides and / or one or more non-phosphodiester linkages.

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

[0197] Oligonucleotide-based compounds can generally, and RNAi agents in particular, be composed of modified nucleotides and / or one or more non-phosphodiester linkages. As used herein, "modified nucleotide" is a nucleotide other than a 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 the nucleotides are modified nucleotides. As used herein, modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2'-modified nucleotides, 3' to 3' linkages (inverted) nucleotides, non-natural base-containing nucleotides, bridged nucleotides, peptide nucleic acids, 2',3'-seco nucleotide mimics (non-locked nucleic acid base analogs, locked nucleotides, 3'-O-methoxy (2' nucleoside internucleotide linkage) nucleotides), 2'-F-arabinonucleotides, 5'-Me,2'-fluoronucleotides, morpholinonucleotides, 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'-fluoronucleotides, 2'-deoxynucleotides, 2'-methoxyethyl (2'-O-2-methoxylethyl) nucleotides, 2'-aminonucleotides, and 2'-alkyl nucleotides.

[0198] In addition, one or more nucleotides of an oligonucleotide-based compound, such as an RNAi agent, can be linked by non-standard bonds or backbones (i.e., modified internucleotide bonds or modified backbones). Modified internucleotide bonds can be non-phosphate-containing covalent internucleotide bonds. Modified internucleotide bonds or backbones include, but are not limited to, 5'-phosphorothioate groups, chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkylphosphonates (e.g., methylphosphonate or 3'-alkylene phosphonate), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidate, aminoalkylphosphoramidate, or thionophosphoramidate), thionoalkyl-phosphonate, thionoalkylphosphotriester, morpholino linkages, boranophosphates having a normal 3'-5' linkage, 2'-5' linkage analogs of boranophosphates, or boranophosphates having an inverted polarity in which adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2'.

[0199] Not all positions of a given compound need to be uniformly modified. Conversely, two or more modifications can be incorporated into a single oligonucleotide-based compound or even into its individual nucleotides.

[0200] In some embodiments, the cargo molecule is an RNAi agent for inhibiting HIF-2α (EPAS1) gene expression. The cargo molecule can be an RNAi agent described in International Publication Nos. WO2016 / 196239 and WO2014 / 134255, which are hereby incorporated by reference in their entirety.

[0201] The sense strand and the antisense strand of the RNAi agent can be synthesized and / or modified by methods known in the art. For example, the disclosure of an RNAi agent related to the inhibition of HIF-2α expression can be found, for example, in International Publication No. WO2016 / 196239 (which is incorporated herein by reference in its entirety).

[0202] In some embodiments, one or more cargo molecules can comprise or consist of a PEG moiety that can act as a pharmacokinetic (PK) enhancer or modulator. In some embodiments, one or more cargo molecules can comprise a PEG moiety, and such PEG moiety can have from about 20 to 900 ethylene oxide (CH2-CH2-O) units (e.g., 20 to 850, 20 to 800, 20 to 750, 20 to 700, 20 to 650, 20 to 600, 20 to 550, 20 to 500, 20 to 450, 20 to 400, 20 to 350, 20 to 300, 20 to 250, 20 to 200, 20 to 150, 20 to 100, 20 to 75, 20 to 50, 100 to 850, 100 to 800, 100 to 750, 100 to 700, 100 to 650, 100 to 600, 100 to 550, 100 to 500, 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 200 to 850, 200 to 800, 200 to 750, 200 to 700, 200 to 650, 200 to 600, 200 to 550, 200 to 500, 200 to 450, 200 to 400, 200 to 350, 200 to 300, 250 to 900, 250 to 850, 250 to 800, 250 to 750, 250 to 700, 250 to 650, 250 to 600, 250 to 550, 250 to 500, 250 to 450, 250 to 400, 250 to 350, 250 to 300, 300 to 900, 300 to 850, 300 to 800, 300 to 750, 300 to 700, 300 to 650, 300 to 600, 300 to 550, 300 to 500, 300 to 450, 300 to 400, 300 to 350, 350 to 900, 350 to 850, 350 to 800, 350 to 750, 350 to 700, 350 to 650, 350 to 600, 350 to 550, 350 to 500, 350 to 450, 350 to 400, 400 to 900, 400 to 850, 400 to 800, 400 to 750, 400 to 700, 400 to 650, 400 to 600, 400 to 550, 400 to 500, 400 to 450, 450 to 900, 450 to 850, 450 to 800, 450 to 750, 450 to 700, 450 to 650, 450 to 600, 450 to 550, 450 to 500, 500 to 900, 500 to 850, 500 to 800, 500 to 750, 500 to 700,(having from 500 to 650, 500 to 600, 500 to 550, 550 to 900, 550 to 850, 550 to 800, 550 to 750, 550 to 700, 550 to 650, 550 to 600, 600 to 900, 600 to 850, 600 to 800, 600 to 750, 600 to 700, 600 to 650, 650 to 900, 650 to 850, 650 to 800, 650 to 750, 650 to 700, 700 to 900, 700 to 850, 700 to 800, 700 to 750, 750 to 900, 750 to 850, 750 to 800, 800 to 900, 850 to 900, or 850 to 900 ethylene oxide units). In some embodiments, one or more cargo molecules consist of a PEG moiety having about 455 ethylene oxide units (a 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 can be linear or branched. The PEG moiety can be discrete (monodisperse) or non-discrete (polydisperse). PEG moieties for use as PK enhancing cargo molecules are commercially available. In some embodiments, one or more cargo molecules include a PEG moiety that can act as a PK modulator or enhancer, as well as different cargo molecules, such as pharmaceutically active ingredients or compounds.,

[0203] The integrin ligands described include salts or solvates. Solvates of integrin ligands are interpreted to mean the addition of inert solvent molecules to the integrin ligand that form by virtue of their mutual attraction. Solvates are, for example, monohydrates 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 of the integrin ligand having the corresponding protecting groups.

[0205] The ανβ3 integrin ligand also includes, for example, derivatives, i.e., integrin ligands modified with, for example, an alkyl or acyl group, sugar, or oligopeptide, which are cleaved either in vitro or in vivo.

[0206] In some embodiments, the integrin ligands disclosed herein facilitate the delivery of cargo molecules to the cytosol of cells presenting integrin ανβ3 and / or integrin ανβ5 on their surface, either via ligand-mediated endocytosis, pinocytosis, or any other means. In some embodiments, the integrin ligands disclosed herein facilitate the delivery of cargo molecules to the plasma membrane of cells presenting integrin ανβ3 and / or integrin ανβ5.

[0207] Pharmaceutical composition In some embodiments, the present disclosure provides a pharmaceutical composition comprising, consisting of, or essentially consisting 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 pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance other than the active pharmaceutical ingredient (API, therapeutic product) intentionally included in the drug delivery system. An excipient does not exert, or is not intended to exert, a therapeutic effect at the intended dosage. Excipients assist in the processing of the drug delivery system during manufacture, protect, support, or enhance the stability, bioavailability, or patient acceptability of the API, assist in product identification, and / or enhance any other attribute of the overall safety and effectiveness of the delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0209] Excipients include, but are not limited to: absorption enhancers, antiadhesion agents, antifoaming agents, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery promoters, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, flow promoters, humectants, lubricants, oils, polymers, preservatives, saline solutions, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, isotonic agents, vehicles, water repellents, and wetting agents.

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

[0211] The pharmaceutical compositions can also include preservatives, solubilizing agents, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for changing osmotic pressure, buffers, coating agents, or antioxidants. They can also include other agents having known therapeutic effects.

[0212] The pharmaceutical composition can be administered in many ways depending on whether local or systemic treatment is desired and depending on the area to be treated. Administration can be by any method generally known in the art, for example, but not limited to, locally (e.g., by transdermal patch), intralung (e.g., by inhalation or ventilation 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 an implantable device), intracranial, intrasubstantial, intrathecal, and intraventricular administration. In some embodiments, the pharmaceutical compositions described herein are 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, solutions, emulsions or suspensions. Administration can also be rectally, for example, using suppositories; locally or transdermally, for example, using ointments, creams, gels, or solutions; or parenterally, for example, using injectable solutions.

[0213] A pharmaceutical composition suitable for injectable use includes a sterile aqueous solution (if water-soluble), a dispersion, and a sterile powder for the immediate preparation of a sterile injectable solution or dispersion. In the case of 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 protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium including water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Suitable 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 a dispersion, and by the use of surfactants. In many cases, it will be preferable to include in the composition isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride. Prolonged absorption of the injectable composition is brought about by including in the composition agents that delay absorption, such as aluminum monostearate and gelatin.

[0214] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in a suitable solvent, with one or a combination of the ingredients enumerated above, and, as required, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle that includes a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and freeze-drying, whereby a powder of the active ingredient plus any additional desired ingredients is obtained from its previously sterile-filtered solution.

[0215] Formulations suitable for intra-articular administration can be in the form of a sterile aqueous preparation of any of the ligands described herein, which can be in the form of a microcrystalline form, for example an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems can 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, for example, controlled release formulations including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, as described, for example, in U.S. Patent No. 4,522,811.

[0217] The pharmaceutical composition can contain other additional ingredients commonly found in pharmaceutical compositions. Such additional ingredients include, but are not limited to: antipruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.). As used herein, "pharmacologically effective amount", "therapeutically effective amount" or simply "effective amount" refers to the amount of a pharmaceutically active agent to bring about a pharmacological, therapeutic or prophylactic result.

[0218] Pharmaceuticals containing ανβ3 integrin ligands are also an object of the present invention, as are methods for manufacturing such pharmaceuticals, which include bringing one or more compounds containing an ανβ3 integrin ligand, and optionally one or more substances having a known therapeutic effect, into a pharmaceutically 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 be packaged in a pre-filled syringe or vial.

[0220] Linker group, pharmacokinetic (PK) enhancer, pharmacodynamic (PD) modulator, delivery vehicle, and targeting group In some embodiments, the αvβ3 ligand is conjugated to one or more non-nucleotide groups including, but not limited to, a linker group, a pharmacokinetic (PK) enhancer (also referred to as a PK modulator), a pharmacodynamic (PD) modulator, a delivery polymer, or a delivery vehicle. The non-nucleotide groups can facilitate targeting, delivery, or attachment of a cargo molecule. Examples of scaffolds for targeting and linker groups are disclosed herein. The non-nucleotide groups can be covalently linked to either the 3' and / or 5' terminus of either the sense strand and / or the antisense strand. In embodiments where the cargo molecule is an RNAi agent, the RNAi agent comprises non-nucleotide groups linked to the 3' and / or 5' terminus of the sense strand. In some embodiments, the non-nucleotide group is linked to the 5' terminus of the RNAi agent sense strand. The integrin ligands disclosed herein can be directly or indirectly linked to a cargo molecule via a linker / linker 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 pharmacokinetic or biodistribution properties of the RNAi agent or conjugate to which it is attached to improve the cell- or tissue-specific distribution and cellular 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 improves or modulates the pharmacodynamic properties of the RNAi agent or conjugate to which it is attached to improve the cell- or tissue-specific distribution and cellular uptake of the RNAi agent or conjugate.

[0222] The targeting group or targeting moiety enhances the pharmacokinetic or biodistribution properties of the cargo molecule to which they are attached to improve the cell-specific (optionally including organ-specific) distribution and cellular (or organ-specific) uptake of the cargo molecule. In some embodiments, the targeting group may include an αvβ3 ligand described herein. In some embodiments, the targeting group includes a linker. In some embodiments, the targeting group includes a PK enhancer. In some embodiments, the αvβ3 integrin ligand is linked to the cargo molecule using a PEG linker or a linker such as 1, 2, or 3 abasic and / or ribitol (abasic ribose) residues (which can serve as a linker). The targeting group may include one or more targeting ligands. In some embodiments, the targeting group may include 1 to 4 integrin ligands disclosed herein. In some embodiments, the targeting group is a tris-targeting group and includes 3 integrin ligands disclosed herein.

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

[0224] For example, in some embodiments, an RNAi agent having an NH2-C6 group at the 5'-end of the sense strand of the RNAi agent can be synthesized. The terminal amino group can then be reacted, for example, to form a conjugate with a group containing an integrin targeting ligand. In some embodiments, an RNAi agent having one or more alkyne groups at the 5'-end of the sense strand of the RNAi agent is synthesized. The terminal alkyne group(s) can then be reacted, for example, to form a conjugate with a group containing an αvβ3 integrin targeting ligand.

[0225] In some embodiments, the linker is conjugated to the αvβ3 ligand. The linker facilitates covalent attachment of the αvβ3 ligand to the cargo molecule, PK enhancer, delivery polymer, or delivery vehicle. Examples of linkers 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, etc.

[0226] A linker or linking group is a connection between two atoms that links one chemical group (such as an RNAi agent) or segment of interest, via one or more covalent bonds, to another chemical group (such as an αvβ3 integrin ligand, PK enhancer, PD modulator, or delivery polymer) or segment of interest. A labile linkage contains a labile bond. The linkage optionally includes a spacer that increases the distance between 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. Since spacer groups are well known in the art, the foregoing list is not meant to limit the scope of the description.

[0227] In some embodiments, the αvβ3 ligand is linked to the cargo molecule without using an additional linker. In some embodiments, an αvβ3 ligand is designed that has a linker that facilitates presentation of the linkage to the cargo molecule. In some embodiments, when two or more RNAi agents are included in a composition, the 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 a composition, the 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] In the formula, the following:

Chemical formula

[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 (incorporated herein by reference in its entirety).

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

[0233] Internal linking targeted ligand In some embodiments, when the integrin-targeting ligand described herein is bound or linked to an RNAi molecule, the integrin-targeting ligand may be bound to internal nucleotides of the sense 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 to 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 to 4 targeting ligands are conjugated to internal nucleotides on the sense strand of the RNAi agent.

[0234] In some embodiments, the substitution of internal targeting ligands can affect the efficacy or potency of the RNAi agent. In some embodiments of the αvβ3 integrin-targeting ligand bound to the RNAi agent, the targeting group is conjugated to the 5' end of the sense strand, and at least 10 nucleotides are positioned between the trimeric 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 positioned between the trimeric 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 internal nucleotides located on the sense strand of the RNAi agent, there is a gap of at least one nucleotide not conjugated to the targeting ligand located between the two internal nucleotides 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 not conjugated to the targeting ligand are disposed between the two internal nucleotides conjugated to the targeting ligand.

[0236] In some embodiments, the targeting ligand begins at the most distal 3' nucleotide that forms a base pair with a nucleotide on the antisense strand and is conjugated to the second, fourth, and sixth nucleotides on the sense strand numbered from 3' to 5'. 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 forms a base pair 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 illustrate, without limitation, and by way of example, specific embodiments disclosed herein.

[0240] Example 1. Synthesis of integrin targeting ligand. Some of the abbreviations used in the experimental details of the synthesis in the following examples are defined as follows: h or hr = (single or plural) hours; min = minutes; mol = (single or plural) moles; mmol = (single or plural) millimoles; M = molar; μM = micromolar; g = (single or plural) grams; μg = (single or plural) micrograms; rt or RT = room temperature; L = (single or plural) liters; mL = (single or plural) milliliters; wt = weight; Et2O = diethyl ether; THF = tetrahydrofuran; DMSO = dimethyl sulfoxide; 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; BOC = t-butoxycarbonyl; CBZ = benzyloxycarbonyl; TBS = t-butyldimethylsilyl; TBSCl or TBDMSCl = t-butyldimethylsilyl chloride; TFA = trifluoroacetic acid; DMAP = 4-dimethylaminopyridine; NaN3 = sodium azide; Na2SO4 = sodium sulfate; NaHCO3 = sodium bicarbonate; NaOH = sodium hydroxide; MgSO4 = magnesium sulfate; K2CO3 = potassium carbonate; KOH = potassium hydroxide; NH4OH = ammonium hydroxide; 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-azanonadecan-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)

Chem.

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

Chem.

[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 hour. 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 minutes. 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 three times from the oily suspension with diethyl ether. The combined organic phases were filtered through sodium sulfate and concentrated. Next, compound 5 was isolated in 26% yield by separation on silica eluting with a gradient of ethyl acetate in hexane.

Chemical formula

[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 temperature overnight. Upon completion, the mixture was filtered and concentrated under reduced pressure. Next, compound 7 was isolated in 76% yield by separation on silica eluting with a gradient of ethyl acetate in hexane.

Chemical formula

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

Chem.

[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 minutes. At completion, the reaction mixture was diluted with water and washed with diethyl ether. Subsequently, the pH was adjusted to 11 using 2 N 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 then used without further purification. [Chemistry]

[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 in a divided form as a solid. After the addition, 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 adjusted to 9. The product was extracted three times with EtOAc, the organic phases were combined, dried over 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. [Chemistry]

[0248] To DIPEA (7.53 mL, 53.75 mmol) in THF (35 mL), n-BuLi (2.5 M, 19.9 mL, 49.8 mmol) was added via an oven-dried airtight syringe over 2 minutes at -10 °C. The mixture was stirred at -10 °C for 10 minutes, 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 minutes. After aging at -60 °C for about 1 hour, compound 13 (7.37 g, 39.82 mmol) was added dropwise as a solution in THF (15 mL) over 5 minutes at -60 °C. The reaction mixture was stirred at about -60 °C for 1 hour and then at -41 °C for 1.5 hours. The reaction was quenched by the addition of 2.6 equivalents of H2SO4 (2.0 M) and extracted three times with ethyl acetate (~50 mL). The organic phases were combined, dried over brine, filtered through sodium sulfate, and concentrated briefly to weigh the crude product and obtain a sample for NMR. At the time of measuring the dry weight, 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% according to NMR. 1 H NMR: 400 MHz CDCl3 δ 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).

Chemical Structure

[0249] To a solution of compound 14 (9.33 g, 30.16 mmol by weight from NMR of ~12 g of crude product) in MeOH (40 mL) was added an aqueous solution of 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 minutes, a second portion of compound 15 (736 mg, 6.03 mmol) was added and the reaction mixture was stirred at 50 °C overnight. Next, the reaction mixture was concentrated to oil and partitioned between 2 volumes of EtOAc and 1 volume of H2O. The organic phase was separated, washed with 1 volume of water. The aqueous washes were combined and back-extracted with EtOAc (2x, 1 volume). The combined organic phases were dried over sodium sulfate, filtered, and concentrated. The crude product was dried over about 20 g of silica and separated by silica eluting with a gradient of ethyl acetate in hexane containing 1% triethylamine to isolate compound 16 in 69% yield. 1 H NMR: 400 MHz CDCl3 δ 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).

Chemical formula

[0250] To a solution of compound 16 (5.98 g, 20.8 mmol) in EtOH (50 mL) was charged hydrogen at 1 atm with palladium (10% on carbon, 2.22 g, 2.08 mmol). 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 eluting with a gradient of ethyl acetate in hexane containing 1% triethylamine. 11H NMR: 400 MHz CDCl3 δ 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).

Chem.

[0251] Compound 17 (4.81 g, 16.53 mmol) was dissolved in an aqueous solution of 6 M HCl (16.4 mL) and heated at 42 °C for 2 h. Next, an additional portion of 6 M HCl (2.8 mL) was added and the reaction mixture was stirred for an additional 2 h. Sodium chloride was added to the reaction and followed by addition of 2 N aqueous NaOH until the product precipitated as an oil (pH was above 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 used without further purification. 1 1H NMR: 400 MHz CDCl3 δ 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).

Chem.

[0252] A solution of triphosgene (85 mg, 0.28 mmol) in THF (0.9 mL) in a flask dried by heating at -10 °C was added dropwise with a solution of compound 18 (236 mg, 0.62 mmol) and TEA (0.134 mL, 0.96 mmol) in THF (0.5 mL). The reaction mixture was warmed to room temperature. After the completion of the reaction was indicated by TLC, additional TEA (0.134 mL) was added, followed by the addition of 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 an equal volume of water and extracted three times with EtOAc. The combined organic phases were dried over brine, filtered through sodium sulfate, and concentrated. Compound 19 was obtained assuming a 100% yield and used without further purification.

Chemical formula

[0253] To the crude compound 19 (400 mg, assumed to be 0.62 mmol) dissolved in THF (37 mL) was added H2SO4 (2 M, 0.6 mL), and the mixture was stirred overnight at room temperature. The next morning, an additional portion of H2SO4 (0.65 equivalent) was added. After 4 hours, 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.

Chemical formula

[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 up to 1 atm and stirred at 35 °C overnight. Upon completion, palladium was removed by filtration through Celite®. Compound 21 was isolated as the TFA salt in 20% yield by reverse-phase HPLC using a C18 5u 19×250 mm BEH column (Waters Corp.) eluting with a gradient of acetonitrile in H2O containing 1% TFA.

Chemical formula

[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 minutes and checked by LC-MS, which indicated that 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 hours. 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.) eluting 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).

Chemical formula

[0257] To a solution of compound 23 (10 g, 43.4 mmol) in toluene (80 mL) were added compound 6 (21.1 g, 0.17 mol), PPTS (0.55 g, 2.2 mmol), and then acetic acid (1.24 mL, 21.7 mmol). A Dean-Stark trap was attached to the reactor, and then the mixture was heated to reflux temperature 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 afford compound 24 in 66% yield. 1 1H NMR: 400 MHz CDCl3 δ 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). [Chemical formula]

[0258] To the flame-dried flask, THF (190 mL) and DIPEA (9.07 g, 89.7 mmol) were added, and the mixture was cooled to -20 °C. Next, n-BuLi (2.5 M, 34.2 mL, 85.6 mmol) was added via a cannula. The solution was stirred at -20 °C for 10 minutes and then cooled to -78 °C. Compound 8 (8 mL, 81.5 mmol) was added dropwise with vigorous stirring. After the addition, the mixture was stirred at -78 °C for 30 minutes. Next, ClTi(iPrO)3 (44.6 g, 0.171 mol) as a solution in THF (40 mL) was added via an addition funnel over 10 minutes. The reaction mixture was stirred at -78 °C for 30 minutes. Finally, compound 24 (9.06 g, 27.2 mmol) was added dropwise as a suspension in THF (20 mL), and the mixture was stirred at -78 °C for 1.25 hours until the reaction was complete. Aqueous saturated ammonium chloride solution was added to the reaction mixture at -78 °C. Next, the reaction mixture was removed from the cooling bath, and the aqueous phase was gradually thawed and quenched (until the yellow-orange color disappeared). The mixture was partitioned between EtOAc and aqueous saturated ammonium chloride solution. The organic phase was separated, and the aqueous phase was washed twice with EtOAc. The combined organic phases were dried over brine, then over sodium sulfate, filtered, and concentrated. Compound 25 was obtained as a single diastereomer in 70% yield by silica gel separation eluting with a gradient of ethyl acetate in hexane. 1 1H NMR: 400 MHz CDCl3 δ 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). [Chemical Structure]

[0259] To compound 25 (8.07 g, 19.1 mmol), aqueous HCl solution (6 M, 20.7 mL, 0.124 mol) was added, 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 hours. The reaction mixture was basified to pH 10 with 2N aqueous NaOH solution and 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 and used without further purification. 1 H NMR: 400 MHz CDCl3 δ 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).

Chemical Structure

[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 in a divided manner as a solid. After the last addition, cooling was removed and the mixture was stirred for about 2.5 hours until completion. The reaction mixture was quenched by the addition of saturated aqueous sodium bicarbonate. The mixture was extracted three times with EtOAc, 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 hexane. 11H NMR: 400 MHz CDCl3 δ 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, 3 H), 3.31 (s, 3 H), 2.47 - 2.75 (m, 4 H), 1.20 (t, 3 H).

Chem.

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

Chem.

[0262] To the compound 29 (5.59 g, 8.97 mmol) dissolved in THF (37 mL) were added water (0.8 mL) and H2SO4 (2 M, 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 over brine, filtered through sodium sulfate, and concentrated. Compound 30 was isolated in 82% yield by separation on silica eluting with a gradient of MeOH in DCM containing 1% TEA. [Chemical formula]

[0263] To the compound 30 (4.13 g, 7.39 mmol) dissolved in EtOH (30 mL) were added Degussa® palladium (10 wt%, 3.15 g, 2.96 mmol) and hydrogen was charged up 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 palladium (10 wt%, 1.57 g, 1.48 mmol) and hydrogen were charged up to 50 psi. After stirring for 48 hours, the reaction mixture was heated to 30 °C and stirred for an additional 24 hours. Upon completion, the suspension was filtered through Celite® and all volatiles were removed under vacuum. The residue was purified by silica eluting with a gradient of MeOH in DCM to afford compound 31 in 72% yield. 1 H NMR: 400 MHz DMSO-d6 δ 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). [Chemistry]

[0264] A solution of PPh3 (699 mg, 2.66 mmol) in THF (0.47 mL) at -10 °C was added dropwise with a solution of DEAD. The mixture was warmed to room temperature and then 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. Next, the reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel eluting with a gradient of MeOH in DCM, and compound 32 was obtained in a 50% yield. 1 1H NMR: 400 MHz DMSO-d6 δ 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). [Chemistry]

[0265] To compound 32 (826 mg, 1.23 mmol), EtOH (3 mL) and H2O (3 mL) were added, 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 6 M aqueous HCl and then concentrated. The residue was purified by reverse-phase HPLC using a Phenomenex Gemini C18, 50 x 250 mm, 10 μm column eluting with a gradient of acetonitrile in water containing 0.1%, and compound 33 (structure 2c) was obtained 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)

Chemical Structure

[0267] A solution of DEAD was added dropwise to a solution of PPh3 in THF at room temperature. The mixture was then treated with compound 31 and OH-(CH2) 11Transfer to a vial containing the mixture of -N3 and stir the reaction mixture overnight at room temperature. Remove the volatiles from the reaction mixture and dissolve the crude product in EtOH. Add LiOH as a solution in H2O and add additional water / EtOH until the reaction mixture becomes homogeneous. After stirring for 1.5 h at room temperature, acidify the mixture to pH 3 using H2SO4, concentrate it, and purify by reverse-phase HPLC (Phenomenex Gemini C18, 50x250 mm, 10 μm, 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)

Chemical Structure

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

Chemical Structure

[0270] A solution of DEAD was added dropwise to a solution of PPh3 in THF 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. 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 was added until the reaction mixture became homogeneous. After stirring for 1.5 hours at room temperature, 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 obtain 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)

Chemical Structure

[0272] A solution of EDAC in DCM was added to a suspension of compound 35 in DCM at 0 °C. After 5 minutes, the cooling was removed, 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.

Chemical Structure

[0273] A solution of DEAD was added dropwise to a solution of PPh3 in THF 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. 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 was added until the reaction mixture became homogeneous. After stirring for 1.5 hours at room temperature, the mixture was acidified to pH 3 with H2SO4, concentrated, purified by reverse-phase HPLC (Phenomenex Gemini C18, 50x250mm, 10μm, acetonitrile / water in 0.1% TFA, gradient elution), and compound 41 (structure 2.3c) was obtained.

[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)

Chemical formula

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

Chemical formula

[0276] A solution of DEAD was added dropwise to a solution of PPh3 in THF 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. 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 was added until the reaction mixture became 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) 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) [Chemical formula]

[0278] Potassium carbonate was added to a solution of compound 45 and compound 46 in acetone. The mixture was heated at 65 °C overnight in a sealed vial as a suspension with vigorous stirring under N2 protection. Next, the reaction mixture was filtered, concentrated, and purified by silica eluting with a gradient of ethyl acetate in hexane to give compound 47. [Chemical formula]

[0279] Sodium azide was added to a solution of compound 47 in DMF, and the mixture was stirred at 80 °C overnight in a sealed vial under nitrogen protection. At completion, an equal 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.

Chem.

[0280] A solution of DEAD was added dropwise to a solution of PPh3 in THF 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. 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 was added until the reaction mixture became 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, 50x250 mm, 10 μm, 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-azapentadecyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propyl)-2-oxoimidazolidin-1-yl)-3-(3-fluoro-4-methoxyphenyl)propanoic acid)

Chem.

[0282] A solution of DEAD was added dropwise to a solution of PPh3 in THF 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. At the end, all volatiles were removed, and the resulting crude product was purified by silica gel eluting with a gradient of MeOH in DCM to give compound 50.

Chem.

[0283] Bromine was added to a solution of Compound 50 in AcOH, and the mixture was stirred for 0.5 h. At 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 further extracted twice with ethyl acetate. The combined organic phases were dried over sodium sulfate, filtered, and concentrated. The resulting crude product of Compound 51 was used subsequently without further purification.

Chem.

[0284] A solution of Compound 51, Pd(PPh3)4, and Zn(CN)2 in DMAC was degassed with nitrogen for 30 min. The mixture was heated overnight at 128 °C in a sealed vial. At completion, the mixture was diluted with 5 volumes of EtOAc. Next, the organic phase was separated, washed twice with water, washed 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.

Chem.

[0285] A solution of compound 52 in MeOH was added with ammonia, and then a slurry of Raney nickel rinsed three times beforehand with methanol was added. Hydrogen was charged into a Parr (registered trademark) flask up to 60 psi, and the mixture was 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. At the end of the reaction, the pH was adjusted to 3 with TFA, and the mixture was directly injected into preparative reverse-phase HPLC (Phenomenex Gemini C18, 250x21.2mm, 5μm, water / ACN in 0.1% TFA, gradient elution), and compound 53 (Structure 2.6c) was obtained.

[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

Chemical Structure

[0287] To compound 31, a solution of THF, PPh3, and DEAD was added dropwise continuously at 0 °C. The mixture was stirred at room temperature for 16 h. Next, the mixture was cooled to -20 °C for 1 h and filtered to remove triphenylphosphine oxide. The filtrate was concentrated, and the O-alkylation intermediate was isolated by purification on silica eluting with a gradient of ethyl acetate in hexane containing 1% TEA. Next, the isolated intermediate was suspended in a mixture of THF and H2O, treated with LiOH in H2O, and stirred at 35 °C for 16 h. At completion, the pH was adjusted to 7 with 2 M HCl, and all volatiles were removed. The crude product was suspended in H2O. Sodium chloride was added, and compound 54 was extracted 5 times with ethyl acetate. The organic phases were combined, filtered through sodium sulfate, and concentrated. Compound 54 was used without further purification.

Chem.

[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. At completion, the pH was adjusted to 3 with TFA, and compound 55 was isolated by direct injection onto a semi-preparative reversed-phase HPLC (Phenomenex Gemini C18, 250x21.2 mm, 5 μm, 250, ×21.2 mm, 5 μm, water / ACN, gradient elution with 0.1% TFA) to give compound 55.

[0289] Using a similar procedure, compounds 2.8c, 2.9c, and 2.10c were synthesized using N3-PEG 11 -NH2, N3-PEG 23 -NH2, and N3-PEG 35 -NH2, respectively.

[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)

Chem.

[0291] Purge and maintain in a nitrogen inert atmosphere. Place into a 3-L four-necked round-bottom flask THF (1.50 L), DIPEA (150.00 mL, 716.000 mmol, 0.88 equiv), and n-BuLi (430.00 mL, 680.000 mmol, 0.84 equiv). Subsequently, add trimethyl phosphite (195.00 mL) at -60 °C and stir at -60 °C for 1 hour. To this, add tert-butyl 2-oxopyrrolidine-1-carboxylate (150.00 g, 809.835 mmol, 1.00 equiv) at 60 °C. Stir the resulting solution at -60 °C in a liquid nitrogen bath for 1 hour. Next, quench the reaction by adding 350 mL of H2SO4 (2N) and dilute with 1.5 L of H2O. Extract the resulting solution with 2 × 1 L of ethyl acetate. Wash the resulting mixture with 1 × 1 L of H2O, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. This afforded 200 g (crude product) of tert-butyl N-[5-(dimethoxyphosphoryl)-4-oxopentyl]carbamate as a yellow oil.

Chem.

[0292] Into 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), and NaOH (50.00 g, 1500.00 mmol, 1.50 equiv) were added. The resulting solution was stirred at 50 °C in an oil bath for 16 h. The pH value of the solution was adjusted to 8 with NaHCO3(aq.). The resulting mixture was concentrated. Next, the reaction was quenched by the addition of 1.5 L of water, and the mixture was 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 afforded 160 g (crude product) of tert-butyl N-[3-(1,8-naphthyridin-2-yl)propyl]carbamate as a yellow oil.

Chemical formula

[0293] Into a 5-L round-bottom flask, 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) were added. The resulting solution was stirred at 25 °C for 16 h. The solid was filtered off. The resulting mixture was concentrated. This afforded 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.

Chemical formula

[0294] Into a 1-L round-bottom flask, 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) were added. The resulting solution was stirred at 25 °C for 3 hours. The resulting solution was diluted with 1 L of H2O. 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 afforded 56 g (80.48%) of 3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propan-1-amine as a yellow solid.

Chemical formula

[0295] Into a 2-L round-bottom flask, 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), and K2CO3 (414.28 g, 2997.581 mmol, 3.00 equiv) were added. The resulting solution was stirred at 25 °C for 16 hours. The solid was filtered off. The resulting mixture was concentrated. This afforded 230 g (99.98%) of 4-(benzyloxy)-3-fluorobenzaldehyde as a white solid.

Chemical formula

[0296] Into a 3-L round-bottom flask, 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) were added. The resulting solution was stirred in an oil bath at 50 °C for 6 hours. The solid was filtered off. The resulting mixture was concentrated. This gave 260 g (78.06%) of (S)-N-[[4-(benzyloxy)-3-fluorophenyl]methylene]-2-methylpropane-2-sulfinamide as a white solid.

Chemical formula

[0297] A 3-L round-bottom flask purged and maintained under an inert atmosphere of nitrogen was charged with 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), and (S)-N-[[4-(benzyloxy)-3-fluorophenyl]methylene]-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 further reacted with stirring for 2 hours while maintaining the temperature at 50 °C in an oil bath. The solid was filtered off. The resulting mixture was concentrated. Next, the reaction was 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 gave 150 g (45.63%) of ethyl (3R)-3-[4-(benzyloxy)-3-fluorophenyl]-3-[[(S)-2-methylpropane-2-sulfinyl]amino]propanoate as a yellow oil.

Chemical formula

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

Chemical formula

[0299] Into a 2-L round-bottom flask were 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 h. Then, the reaction was 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 then concentrated under reduced pressure. This afforded 80 g (62.62%) of ethyl (3R)-3-[4-(benzyloxy)-3-fluorophenyl]-3-[(2,2-dimethoxyethyl)amino]propanoate as a yellow oil.

Chemical formula

[0300] Into a 2-L three-necked 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), 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 at 50 °C in an oil bath for 1 hour. Next, the reaction was quenched by the addition of 1 L of water. The pH was adjusted to 8 using NaHCO3(aq.). The resulting solution was extracted with 2 × 1 L of ethyl acetate dried over anhydrous sodium sulfate and then concentrated. This afforded 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. [Chemical formula]

[0301] Into a 1000 mL round-bottom flask, 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) were added. The resulting solution was stirred at 25 °C for 16 hours. The pH was adjusted to 8 using NaOH (5 M). The resulting solution was extracted with 2 × 1 L of dichloromethane dried over anhydrous sodium sulfate and then concentrated. The residue was applied to a silica gel column using dichloromethane / methanol (50 / 1). The collected fractions were combined and concentrated. This afforded 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.

Chemical formula

[0302] Into a 3-L round-bottom flask were placed 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). The resulting solution was stirred at 25 °C for 72 hours. The solid was filtered off. The residue was applied to a silica gel column using dichloromethane / methanol (9 / 1). The collected fractions were combined and concentrated. This afforded 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] Specific rotation [α] D 20.0 = +37.5° (in MeOH with C = 1 g / 100 ml) ¹H-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).

Chemical formula

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

[0306] EtOH and H2O were added to compound 186, followed by LiOH. The mixture was stirred at 30 °C overnight. Upon completion, the mixture was neutralized to pH = 5 using 6 M aqueous HCl and then concentrated. The residue was purified by reverse-phase HPLC using a Phenomenex Gemini C18, 50x250 mm, 10 μm column eluting 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) [Chemical formula]

[0308] A solution of LHMDS (1.0 M in THF, 95 mL, 95 mmol) and THF (60 mL) was added dropwise with 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 minutes, 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 hours. The reaction mixture was quenched with saturated aqueous NH4Cl (100 mL) and deionized water (100 mL), and then extracted with ethyl acetate (2 x 400 mL). The combined organic phases were dried over Na2SO4, filtered, concentrated, and compound 105 was isolated by CombiFlash® eluting with a gradient of 50 - 100% ethyl acetate in hexane. Yield of compound 105: 7.93 g (43%).

Chemical formula

[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, then quenched with saturated aqueous sodium thiosulfate (100 mL), and extracted with ethyl acetate (2 x 100 mL). The combined organic phases were 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%).

Chemical formula

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

Chemical formula

[0311] Pd / C (10% added, 0.466 g, 0.44 mmol) was added to a solution of compound 108 (1.31 g, 4.38 mmol) in ethyl acetate (20 mL). The reactor was pressurized to 50 PSI with H2. After stirring for 3.5 hours, 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%).

Chemical formula

[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. Since the reaction was not complete based on LC / MS, additional portions of DIEA (0.590 mL, 3.41 mmol) and di-tert-butyl dicarbonate (0.744 g, 3.41 mmol) were 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%). [Chemical formula]

[0313] To a solution of n-butyllithium (2.5 M in hexanes, 0.70 mL, 1.8 mmol) and THF (1.5 mL) at -78 °C was added dropwise over 3 min a solution of compound 111 (5-bromo-2-(phenylmethoxy)pyridine) (0.465 g, 1.8 mmol) in THF (0.8 mL). Next, compound 110 (0.535 g, 1.3 mmol) was 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 phases were 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%). [Chemical formula]

[0314] To a suspension of sodium hydride (60% dispersion in mineral oil, 0.106 g, 2.65 mmol) in dimethoxyethane (2 mL) was added a solution of compound 113 (triethylphosphonoacetate) (0.593 g, 2.65 mmol) in dimethoxyethane (1 mL) at 0 °C. After stirring for 20 minutes, 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 hours. The reaction was quenched with saturated aqueous NH4Cl (10 mL), and the product was extracted with ethyl acetate (3 x 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 hexane. Yield of compound 114: 392 mg (74%).

Chemical formula

[0315] To a solution of compound 114 (390 mg, 0.65 mmol) in ethanol (6 mL) was added Pd / C (10% added, 69 mg, 0.07 mmol). The reactor was pressurized to 50 PSI with H2. After stirring for 4 hours, 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 (250 x 21 mm Chiralpak® AD column, 5 μm, 90 / 10 hexane / EtOH, 40 mL / min) was used to obtain 42 mg of the first eluting R-isomer (R T = 12 - 14 m, >99% ee, compound 115a) and 40 mg of the second eluting S-isomer (R T= 15 - 18 m, >98% ee, was used to isolate compound 115b). The identities of the R- and S-isomers were assigned based on the elution order of structurally similar compounds reported by Coleman et al. 47 J. Med. Chem. 4834 (2004).

[0316] Structures 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)

Chem.

[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 x 3 mL). The organic phase was concentrated under reduced pressure. The crude mixture of N- and O-alkylated positional isomers was used without further purification.

Chem.

[0318] A solution of compounds 116a and 117a (58 mg, 0.08 mmol, a 4:6 mixture of 9a:10a) in THF (1.0 mL), as well as deionized water (1.0 mL), was added lithium hydroxide (6 mg, 0.25 mmol). The reaction mixture was stirred at room temperature for 1 hour and then at 35 °C for 2 hours. An additional portion of lithium hydroxide (4 mg, 0.16 mmol) was added and the reaction temperature was raised to 40 °C. After stirring for 3 hours, a 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 hours. The reaction mixture was acidified to pH 7 with 6N HCl aqueous solution and concentrated under reduced pressure. The positional isomers, 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, 250x21.2 mm, 5 μm, 20 mL / min, water / ACN in 0.1% TFA, gradient elution) to give 13 mg of compound 118a (structure 28c). Compound 119a was purified under the same conditions to give 16 mg of compound 119a (structure 31c).

[0319] Structures 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)

Chemical Structure

[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 minutes. The reaction mixture was quenched with aqueous NaHCO3 (1 mL) and then extracted with ethyl acetate (3 x 3 mL). The organic phase was concentrated under reduced pressure. The crude mixture of N- and O-alkylated positional isomers was used without further purification. [Chemical formula]

[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), as well as 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 hours. An additional portion of lithium hydroxide (6 mg, 0.25 mmol) was added and the reaction mixture was stirred for 2.5 hours. The reaction temperature was lowered 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 positional isomers, 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, 250 x 21.2 mm, 5 μm, 20 mL / min, water / ACN in 0.1% TFA, gradient elution) to give 14 mg of compound 118b (structure 29c). Compound 119b was purified under the same conditions to give 18 mg 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)pentanamide)propanoic acid)

Chem.

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

Chem.

[0324] To DIA (2.85 mL, 20.33 mmol) in THF (15 mL) at -78 °C, a 2.5 M solution of n-BuLi (7.76 mL, 19.41 mmol) was added dropwise. Stirring was continued at -78 °C for 5 minutes, and then ethyl acetate (1.81 mL, 18.48 mmol) was added dropwise. Stirring was continued at -78 °C for an additional 10 minutes, and then a solution of chlorotitanium triisopropoxide (9.27 mL, 38.381 mmol) in THF (10 mL) was added dropwise. Stirring was continued at -78 °C for an additional 15 minutes, and then a solution of compound 122 (2.054 g, 6.16 mmol) in THF (10 mL) was added dropwise. Stirring was continued at -78 °C for 1.5 hours. Upon completion, the reaction was quenched by the addition of saturated ammonium bicarbonate. The suspension was diluted with six times its volume 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 hexane to give 1.043 g (53%).

Chem.

[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). Subsequently, the aqueous layer was 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, concentrated, and used without further purification to obtain 0.616 g (78.5%) of product 124.

Chemical formula

[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 minutes, PNP (106.1 mg, 0.331 mmol) was added, the ice bath was removed, and stirring was continued for 1 hour. Upon completion, the suspension was cooled at -20 °C for 1 hour, and the precipitate was removed by filtration. The supernatant was concentrated and used without further purification to obtain 129 mg (103%) of crude product 126.

Chemical formula

[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 at 50 °C for 3 hours. Upon completion of alkylation, all volatiles were removed, and the product was isolated by silica eluting with a gradient of ethyl acetate in hexane buffered with 1% TEA each to obtain 94.6 mg (61%).

Chemical formula

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

[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 at 60 °C for 2 hours. 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 at 40 °C for 3 hours. Upon completion, all volatiles were removed. The crude product was suspended in a mixture of THF (1 mL), MeOH (1.5 mL), and H2O (1.5 mL), treated with lithium hydroxide (83.5 mg, 3.48 mmol), and heated at 40 °C for 16 hours. 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 x 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-azapentadecanoic acid) [Chemistry]

[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 hexane to give 1.714 g (68.4%). [Chemistry]

[0332] To DIA (3.056 mL, 21.80 mmol) in THF (18 mL) at -78 °C, a 2.5 M solution of n-BuLi (8.324 mL, 20.81 mmol) was added dropwise. Stirring was continued at -78 °C for 5 min, and ethyl acetate (1.94 mL, 19.82 mmol) was added dropwise. Stirring was continued at -78 °C for an additional 10 min, and a solution of chlorotitanium triisopropoxide (9.94 mL, 41.62 mmol) in THF (10 mL) was added dropwise. Stirring was continued at -78 °C for an additional 15 min, and a solution of compound 131 (1.70 g, 6.61 mmol) in THF (12 mL) was added dropwise. Stirring was continued at -78 °C for 1.5 h. 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 by silica eluting with a gradient of ethyl acetate in hexane to give 0.984 g (43%). [Chemistry]

[0333] To compound 132 (0.975 g, 2.82 mmol) in EtOH (6 mL) at 0 °C, 4 M HCl (2.12 mL, 8.47 mmol) in dioxane was added and the mixture was stirred for 30 minutes. 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 five volumes of ethyl acetate, 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 afford 0.434 g (64%).

Chem.

[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) using 3A molecular sieves, STAB-H (0.253 g, 1.19 mmol) was added and the suspension was stirred at room temperature for 16 hours. Upon completion, the reaction was quenched by the addition of saturated sodium bicarbonate and the crude product was extracted with ethyl acetate in three portions. The separated organic extracts were combined, dried over sodium sulfate, filtered and concentrated. The crude product obtained was used without further purification subsequently.

Chem.

[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 minutes. To the activated ester, DIEA (0.259 mL, 1.49 mmol) was added, followed by the addition of compound 125 (0.220 g, 0.497 mmol) in DMF (1 mL), and the resulting mixture was stirred for 1 hour. All volatiles were removed, and the resulting crude product was treated with neat TFA (3.8 mL) and stirred at 40 °C for 3 hours. 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 at 40 °C for 16 hours. Upon completion, the pH was adjusted to 3 with TFA, and the product was isolated by preparative separation via a Phenomenex® Gemini® c18 column (21.2 x 250 mm, 5 micron) 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-azapentadecanoic acid)

Chemical Structure

[0337] Compound 136 (0.500 g, 1.45 mmol) in a mixture of THF (9.0 mL) and MeOH (0.5 mL) at 0 °C was treated with lithium borohydride (94.5 mg, 4.34 mmol). 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 hexane to give 309 mg (67%).

Chemical formula

[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 portioned into several portions. A few drops of water were added, the cooling was removed, and the reaction was stirred for 3 h. At completion, the mixture was washed with saturated sodium bicarbonate and then with saturated sodium thiosulfate. The separated organic portion was dried over sodium sulfate, filtered, and concentrated. Product 138 was separated by elution through silica using a gradient of MeOH in DCM to give 140 mg (46%).

Chemical formula

[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) using 3 Å molecular sieves was added STAB-H (0.150 g, 0.706 mmol), and the resulting suspension was heated at 40 °C for 16 h. At 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 by elution through silica using a gradient of MeOH in DCM containing 1% TEA to give 64 mg (33%).

Chemical formula

[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) using 3 Å molecular sieve, sodium cyanoborohydride (28.9 mg, 0.276 mmol) was added, and the reaction mixture 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 (4 M) in dioxane. Subsequently, all the methanol was removed, 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 via a Phenomenex® (21.2 x 250 mm) C18 column eluting 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)

Chemical Structure

[0342] 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. Next, the reaction mixture was concentrated to oil, redissolved in DCM (150 mL), and washed with saturated aqueous NaHCO3 (2 x 40 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 1H NMR (400 MHz, DMSO-d6): δ 3.58 (s, 3H), 2.43 (t, 2H), 2.29 (t, 2H), 1.46 (m, 4H).

Chemical Structure

[0343] L-Proline (3.72 g, 32 mmol) was added 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). The reaction mixture was heated at reflux temperature overnight. Next, the reaction mixture was concentrated, dissolved in EtOAc (50 mL), and washed with water (3 x 30 mL). The organic phase was dried over Na2SO4, filtered, and concentrated. The residue was purified by CombiFlash using silica gel as the 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 [M + H] for N2O2 + : 245.13, found: 245.21.

Chemical Structure

[0344] To a solution of compound 142 (6.08 g, 24.9 mmol) in MeOH (50 mL) was added Pd / C (10% added, Degussa type, 1.99 g, 1.87 mmol). Nitrogen was introduced into the reaction flask, evacuated, and backfilled three times with nitrogen. This process was repeated with hydrogen, and finally hydrogen (1 atm) was introduced into the reactor and stirred overnight at room temperature. 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 in 100% yield. C 14 H 20 Calculated mass [M + H] for N2O2 + : 249.16, found: 249.08.

Chemical Structure

[0345] To a solution of dimethyl methylphosphonate (12.3 g, 100 mmol) in anhydrous THF (120 mL) was added n-BuLi solution (2.5 M in hexanes, 40 mL, 100 mmol) via a syringe pump over 1 hour at -78 °C. A solution of compound 143 (6.175 g, 24.9 mmol) in THF (40 mL) was added to the reaction mixture over 45 minutes at -78 °C. After stirring at -78 °C for 20 minutes, the reaction mixture was quenched with saturated aqueous NH4Cl solution (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 [M + H] for N2O4P + : 341.17, found: 341.17.

Chemical Structure

[0346] A suspension of 3-fluoro-4-(phenylmethoxy)-benzaldehyde (0.38 g, 1.65 mmol), compound 144 (0.67 g, 1.98 mmol), and potassium carbonate anhydrous (0.547 g, 3.96 mmol) in THF (13.5 mL) was heated at reflux temperature 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 an additional 4 hours. 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 the stationary phase and eluted with a gradient of MeOH in DCM (0 - 10%). Yield of compound 145: 446 mg (61%). C 28 H 29 Calculated mass [M + H] for FN2O2 + : 445.23, found: 445.41.

Chemical formula

[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 minutes while maintaining an internal temperature < 35 °C. After aging for 30 minutes, R-BINOL (3.05 g, 10.65 mmol, 1.00 eq) was added as a solution in THF (10 mL) while maintaining an internal temperature < 35 °C (for about 10 minutes). After stirring at room temperature for 2 hours, 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 by azeotroping with anhydrous toluene (50 mL) and then dissolved in anhydrous THF (12 mL). The solution of Compound 145 was added dropwise via a syringe pump to the solution of R-BINAL at -78 °C over 45 minutes. After 1.5 hours, the reactor was transferred to a very large Dewar, filled with dry ice / acetone, and covered with aluminum foil. The reaction mixture was stirred at -78 °C overnight. Most of the decrease occurred within the first 1.5 hours, with very little additional conversion. The reaction was quenched by the addition of saturated aqueous NH4Cl (150 mL) and warmed to room temperature. The mixture was acidified to pH = 7 using 6N HCl and then extracted with EtOAc (2 x 250 mL). The combined organic phases were 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 the stationary phase and eluted with a gradient of MeOH in DCM (0 - 5%). Yield of Compound 146: 634 mg (53%). The chiral purity was measured by analytical chiral HPLC, Chiralpak AD-H column 4.6 x 250 mm, 5 micron, EtOH 0.1% diethylamine isocratic, 1.75 mL / min. The first eluting R isomer was 86 area % pure and corresponded to 72% ee. Compound 6 was further purified by chiral preparative HPLC (Chiralpak AD-H 21.2×250 mm, 5 micron, EtOH 0.1% diethylamine, 20 mL / min). Final yield of Compound 146: 445 mg (98% ee). C 28 H 31 Calculated mass [M+H] for FN2O2 + : 447.25, found: 447.30.

Chemical Structure

[0349] To a solution of compound 146 (0.325 g, 0.73 mmol) and monomethyl malonate (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. Next, the reaction mixture was diluted with DCM (10 mL) and filtered. The filtrate was concentrated and purified by CombiFlash eluting with a gradient of MeOH in DCM (0 - 5%) using silica gel as the stationary phase. Yield of compound 147: 142 mg (37%). C 32 H 35 Calculated mass [M + H] for FN2O5 + : 547.26, found: 547.58.

Chem.

[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. Next, the reaction mixture was 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 the stationary phase and eluting with a gradient of MeOH in DCM. Yield of compound 148: 140 mg (66%). C 31 H 35 Calculated mass [M + H] for FN2O3 + : 503.27, found: 503.29.

Chem.

[0351] A solution of compound 148 (0.169 g, 0.34 mmol) in EtOH (3 mL) was added to a slurry of Pd / C (10% added, 36 mg, 0.034 mmol) in EtOH (1 mL). The reactor was pressurized and purged with hydrogen three times. The reactor was repressurized to 55 psi for 3 hours. 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, assuming a 100% yield. C 24 H 31 Calculated mass [M + H] for FN2O3 + : 415.24, found: 415.07.

Chem.

[0352] Cesium carbonate (164 mg, 0.50 mmol) was added 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). The reaction mixture was heated at 40 °C for 1 hour and then quenched with saturated aqueous NaHCO3 (3 mL). The mixture was extracted with EtOAc (3 x 10 mL). The combined organic phases were washed with water (2 x 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 [M + H] for FN5O6 + : 616.35, found: 616.90.

Chem.

[0353] A solution of compound 150 (0.207 mg, 0.34 mmol) in THF (1.5 mL) and water (1.5 mL) was added to 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 H2O, 35% ACN in 0.1% TFA and purified by RP-HPLC (Thermo Aquasil C18, 250x21 mm, 5 μm, gradient of ACN in H2O containing 0.1% TFA at 20 mL / min). Yield of compound 151 (SM 36): 125 mg (52% over 3 steps). C 31 H 44 Calculated mass [M+H] for FN5O6 + : 602.34, found: 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)pentaamide)propanoic acid)

Chemical formula

[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 minutes. 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 hour. At 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%).

Chemical formula

[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 hydrogen at 60 PSI and stirred for 1 hour. At completion, the suspension was filtered through Celite® and concentrated to afford 88 mg (83%) of the crude product which was used without further purification.

Chem.

[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 hours. At completion, all volatiles were removed and compound 173 was isolated by separation through silica eluting with a gradient of MeOH in DCM to afford 91 mg (76%).

Chem.

[0358] Compound 173 (50 mg, 0.067 mmol) in dioxane (0.5 mL) was treated with a solution of 4 M HCl (0.671 mmol, 0.168 mL) in dioxane and stirred at 40 °C for 3 hours. At completion, all volatiles were removed. The crude product was dissolved in a mixture of H2O (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 hours. At completion, the pH was adjusted to 3 with TFA and the product was isolated by separation through a Phenomenx Gemini C18 column (21.2 x 250 mm, 5 micron) eluting with a gradient of acetonitrile in water containing 0.1% TFA to afford 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-azahexadec-16-yl)pyrimidin-5-yl)-9-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)nonanoic acid) [Chemical formula]

[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, and simultaneously, the internal temperature was maintained <-70 °C (for about 15 minutes). Next, the resulting solution was stirred for 15 minutes, then a solution of CuCN·2LiCl in THF (1 M, 31 mL, 31.0 mmol) was added, and then allyl bromide (5.10 g, 42 mmol) was added as a solution in THF (10 mL). The reaction mixture was warmed to room temperature and stirred for 1 hour. The reaction mixture was quenched with MeOH (40 mL) and concentrated. The residue was purified by CombiFlash using silica gel as the stationary phase and eluted with a gradient of EtOAc in hexane (0 - 20%). Yield of Compound 152: 4.13 g (74%). Calculated mass [M+H] for C7H7BrN2 + : 198.99, found: 199.05. [Chemical formula]

[0361] A solution of compound 152 (7.70 g, 38.7 mmol) in THF (115 mL) was added dropwise with a solution of 9-BBN in THF (0.5 M, 131 mL, 65.8 mmol) at 0 °C over 30 minutes. The reaction mixture was warmed 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 hour. The reaction mixture was transferred to a separatory funnel and the layers were separated. The aqueous layer was extracted with EtOAc (200 mL). The combined organic phases were 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 an unpurified, yellow oil. The crude product was purified by CombiFlash using silica gel as the stationary phase and eluted with a gradient of EtOAc in hexane (50 - 100%). Yield of compound 153: 3.44 g (41%). Calculated mass [M + H] for C₇H₉BrN₂O + : 217.00, found: 216.97. [Chemical formula]

[0362] A solution of compound 153 (3.44 g, 15.8 mmol) in DCM (40 mL) was added with imidazole (1.73 g, 25.4 mmol) and a solution of 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 the stationary phase and eluted with a gradient of EtOAc in hexane (0 - 8%). Yield of compound 154: 5.56 g (77%). C 23 H 27 Calculated mass [M + H] for BrN₂OSi +: 455.12, Measured value: 455.44.

Chem.

[0363] To a solution of compound 154 (6.07 g, 13.3 mmol) in THF (150 mL) at -75 °C, a solution of nBuLi in THF (2.5 M, 5.6 mL, 14.0 mmol) was added dropwise while maintaining the internal temperature <-70 °C (for about 10 minutes). After 3 minutes, 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 minutes, and then, while maintaining the internal temperature <-65 °C, it was further diluted with THF (5 mL) and quenched with HCl in dioxane (4 M, 3.67 mL, 14.7 mmol). The cooling bath was removed, the reaction was warmed to ambient temperature, and concentrated. The residue was purified by CombiFlash using silica gel as the stationary phase and eluted with a gradient of EtOAc in hexane (0 - 20%). Yield of compound 155: 1.79 g (33%). 1 H NMR (400 MHz, CDCl3): δ 10.09 (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).

Chem.

[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 at 40 °C for 2.5 h and then at 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 the stationary phase and eluted with a gradient of EtOAc in hexane (0 - 100%) containing 1% triethylamine. Yield of Compound 156: 2.04 g (79%). C 38 H 46 Calculated mass [M + H] for N4O2Si + : 619.35, found: 619.69.

Chemical Structure

[0365] Preparation of R-BINAL: LAH (1.169 g, 30.8 mmol) was slurried in anhydrous THF (90 mL). To the slurry was added EtOH as a solution in THF (6 M, 5.2 mL, 31.4 mmol) while maintaining <40 °C. The mixture was aged at 35 °C for 40 min and then cooled to 30 °C. T int While maintaining <40 °C, a solution of R-(BINOL) (9.00 g, 31.4 mmol) in THF (45 mL) was added. 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, and then used with Compound 156. int

[0366] ​A solution of compound 16 (1.16 g, 1.88 mmol) in THF (12 mL) was added to a solution of R-BINAL in THF (~0.2 M, 110 mL, 22.0 mmol) at -78 °C over 5 minutes. After 30 minutes, the reaction mixture was quenched with saturated aqueous NH4Cl, warmed to room temperature, and the product was extracted with EtOAc (3 x 125 mL). The organic layer was dried over Na2SO4, filtered, and concentrated. The residue was purified by CombiFlash using silica gel as the stationary phase and eluted with a gradient of MeOH in EtOAc (0 - 5%) containing 1% triethylamine. Yield of compound 157: 0.96 g (82%). The chiral purity was measured by analytical chiral HPLC (Chiralpak AD-H column 4.6 x 250 mm, 5 micron, 25% EtOH, 75% hexane, 0.1% diethylamine isocratic, 2 mL / min). The second eluting R isomer was ~95 area % pure, corresponding to ~90% ee. C 38 H 48 Calculated mass [M+H] for N4O2Si + : 621.36, found: 621.71.

Chemical Structure

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

Chemical Structure

[0368] A slurry of Pd / C (addition level: 10 wt%, 0.138 g, 0.13 mmol) in EtOH (4 mL) was added to a solution of compound 158 (0.893 g, 1.30 mmol) in EtOH (10 mL). H2 at 50 psi was introduced into the reaction mixture, and the mixture was 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 [M+H] for N4O3Si + : 693.42, found: 693.82.

Chemical formula

[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 addition to a solution of DMAP (20 mg / mL in THF, 155 μL, 0.0031 g, 0.026 mmol). The mixture was heated at 60 °C for 6 h. The reaction mixture was concentrated, and the residue was purified by CombiFlash using silica gel as the stationary phase and eluting with a gradient of EtOAc in hexane (0 - 50%). Yield of compound 160: 0.721 g (71%). C 47 H 64 Calculated mass [M+H] for N4O5Si + : 793.47, found: 794.28.

Chemical formula

[0370] A solution of compound 160 (0.621 g, 0.783 mmol) in THF (6 mL) was added to 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 hours. The reaction mixture was diluted with EtOAc (30 mL) and washed with saturated aqueous NH4Cl (2 x 10 mL). The organic layer was concentrated. The residue was purified by CombiFlash using silica gel as the stationary phase and eluting with a gradient of EtOAc in hexane (50 - 100%). Yield of compound 21: 0.362 g (83%). The chiral purity was measured by analytical chiral HPLC, Chiralpak AD-H column 4.6 x 250 mm, 5 micron, 20% EtOH, 80% hexane, 0.1% diethylamine, isocratic, 1.5 mL / min. The second eluting R isomer was 93% pure and corresponded to 86% ee. Compound 161 was further purified by chiral preparative HPLC (Chiralpak AD-H 21.2 x 250 mm, 5 micron, 20% EtOH, 80% hexane, 0.1% diethylamine, 60 mL / min). Final yield of compound 161: 308 mg (99% ee). C 31 H 46 Calculated mass [M + H] for C + N4O5: 555.36, found: 555.72.

Chem.

[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 the addition of water (0.30 mL). After 2 hours, the reaction mixture was concentrated. The residue was purified by RP-HPLC (Phenomenex Gemini C18 21.2 × 250 mm, 5 micron, 0.1% TFA in water / ACN, gradient of 30 - 80% ACN). Yield of compound 162: 0.030 g (97%). C 31 H 44 Calculated mass [M + H] for C+ : Calculated value: 569.34, measured value: 569.68. [Chem.]

[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, TBTU (32 mg, 0.099 mmol) was added, and then DIEA (35 μL, 26 mg, 0.203 mmol) was added. The reaction mixture was warmed to room temperature and stirred for 30 minutes. The reaction mixture was concentrated, and the product, compound 163, was used in the next step without purification. C 37 H 56 Calculated mass [M+H] for N8O7 + : 725.44, measured value: 725.77. [Chem.]

[0373] To a solution of compound 163 (42 mg, 0.058 mmol) in THF (0.30 mL), a 1 M solution of LiOH (0.174 mL, 0.174 mmol) was added. The reaction mixture was heated at 40 °C for 1 hour. An additional portion of LiOH was added (0.174 mL, 0.174 mmol). After 3 hours, the reaction was quenched and an additional portion of LiOH was added (0.174 mL, 0.174 mmol). The reaction was stirred for an additional 2 hours (9 equivalents of LiOH, total 5 hours). The reaction mixture was neutralized to pH = 5 using 3 N HCl and concentrated. The residue was dissolved in TFA:water [95:5] and stirred at room temperature for 2 hours. The reaction mixture was concentrated, and the residue was purified by RP-HPLC (Phenomenex Gemini C18 21.2x250 mm, 5 micron, water / ACN containing 0.1% TFA, 20-50% ACN gradient). Yield of compound 164 (structure 38c): 23 mg (66%). C 30 H 44 Calculated mass [M+H] for N8O5 +: 597.35, measured value: 597.85.

Chem.

[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 addition of 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 hours. The reaction mixture was concentrated and purified by RP-HPLC (Phenomenex Gemini C18 21.2 x 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 [M + H] for N7O4 + : 580.36, measured value: 580.66.

Chem.

[0375] To a solution of compound 165 (18 mg, 0.031 mmol) in EtOH (100 μL) was added a slurry of Pd / C (10% added, 3.3 mg, 0.003 mmol) in EtOH (170 μL). H2 was charged into the reactor, then evacuated three times, and then H2 (1 atm) was charged. After 30 minutes, 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 [M + H] for N5O4 + : 554.37, measured value: 554.73.

Chem.

[0376] To a solution of compound 166 (17 mg, 0.031 mmol) and azido-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. C 40 H 62 Calculated mass [M+H] for N8O8 + : 783.48, found: 783.84.

Chem.

[0377] To a solution of compound 167 (24 mg, 0.031 mmol) in THF (180 μL) was added 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 3 N HCl and then 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 21.2x250 mm, 5 micron, 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 [M+H] for N8O6 + : 655.40, found: 656.01.

[0378] Example 2. Synthesis of a tris-integrin targeting ligand, an RNAi agent, and conjugation of the integrin targeting ligand to a cargo molecule (RNAi agent) One or more integrin-targeting ligands can be conjugated to one or more RNAi agents useful for inhibiting the expression of one or more targeted genes in cells expressing integrin. The integrin-targeting ligands disclosed herein facilitate the delivery of the RNAi agent to target cells and / or tissues. Example 1 above describes the synthesis of specific integrin-targeting ligands disclosed herein. General procedures for the synthesis of specific integrin-targeting ligand-RNAi agent conjugates, exemplified in the non-limiting examples described herein, are described below.

[0379] A. Synthesis of RNAi agent. RNAi agents can be synthesized using methods widely known in the art. In the synthesis of the RNAi agents exemplified in the examples described herein, the sense and antisense strands of the RNAi agents were synthesized based on the phosphoramidite technology on a solid phase used for oligonucleotide synthesis. Depending on the scale, MerMade96E® (Bioautomation), a MerMade12® (Bioautomation), or OP Pilot 100 (GE Healthcare) was used. The 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 RNAs 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-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).Inverse 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, and 5'-(4,4'-dimethoxy-trityl)-2',3'-seco-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. Commercially available TFA aminolink phosphoramidite was also purchased (ThermoFisher).

[0380] In some embodiments, the integrin-targeting ligands disclosed herein are conjugated to an RNAi agent by linking the components to a scaffold containing a trialkyne group, or to a modified nucleotide containing a propargyl group as shown in Table B above. In some embodiments, the trialkyne group is added by using a trialkyne-containing phosphoramidite, and it can be added at the 5' end of the sense strand of the RNAi agent. When used with the RNAi agents shown in the specific examples herein, the trialkyne-containing phosphoramidite is dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other amidites are dissolved in anhydrous acetonitrile (50 mM), and molecular sieves (3 Å) are further 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 10 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, MA, USA) in anhydrous acetonitrile was used.

[0381] Alternatively, when the integrin-targeting ligand is conjugated to the RNAi agent via a trialkyne scaffold instead of using the phosphoramidite method, the trialkyne-containing compound can be introduced after synthesis (see, e.g., item E below). When used with the RNAi agents shown in the specific examples described herein, when a trialkyne group was conjugated to the 5'-end of the sense strand after synthesis, the 5'-end 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 further 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, MA, USA) in anhydrous acetonitrile was used.

[0382] In the examples described herein; the following shows the modified nucleotide sequences for duplex synthesis: Duplex 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) Duplex 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] Regarding the modified nucleotide sequences listed above, a, c, g, and u represent 2’-O-methyladenosine, cytidine, guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent 2’-fluoroadenosine, cytidine, guanosine, or uridine, respectively; aAlk, cAlk, gAlk, and uAlk represent 2’-O-propynyladenosine, cytidine, guanosine, or uridine, respectively; (invAb) represents an inverted abasic residue (inverted abasic deoxyribonucleotide); s represents a phosphorothioate linkage; (NH2-C6) represents the following: [Chemical formula] and (C6-S-Mal-L) represents the following: [Chemical formula] {wherein, as shown in the following examples, L is a PEG chain or ethyl} represents. Regarding the embodiments of the present specification, when looking at each strand 5’→3’, an inverted abasic was inserted so that the 3’ position of deoxyribose was linked at the 3’ end of the aforementioned monomers of each strand.

[0384] B. Cleavage and deprotection of the support-bound oligomer. After completion of 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) at 30 °C for 1.5 h. The solution was evaporated and the solid residue was reconstituted in water (see below).

[0385] C. Purification. The 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 and contained 20% acetonitrile. Buffer B was Buffer A with 1.5 M sodium chloride added. A UV trace was recorded at 260 nm. The appropriate fractions were pooled and then passed through 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 combined and mixed with an equimolar amount of RNA solutions (sense and antisense) in 1×PBS (phosphate buffered saline, 1×, Corning, Cellgro) to form the RNAi agent. Some of the RNAi agents were lyophilized and stored at -15 to -25 °C. The duplex concentration was determined by measuring the solution absorbance using a UV-Vis spectrometer in 1×PBS. Next, the duplex concentration was determined by multiplying the solution absorbance at 260 nm by the conversion factor and dilution factor. 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 to the trialkyne scaffold. Either before or after annealing, the 5' or 3' amine-functionalized sense strand of the RNAi agent can be conjugated to a trialkyne scaffold. Examples of trialkyne backbone structures that can be used in forming the constructs disclosed herein include the following: [Chemical formula]

[0388] The following describes the conjugation of the trialkyne scaffold to the annealed duplex: The amine-functionalized duplex was dissolved in 90% DMSO / 10% H2O at ~50 - 70 mg / mL. 40 eq of triethylamine was added, followed by 3 eq of trialkyne-PNP. Upon completion, the conjugate was immersed twice in a solvent system of 1× phosphate buffered saline / acetonitrile (1:14 ratio) and then dried.

[0389] F. Conjugation of integrin-targeting ligands. Either before or after annealing, conjugate a 5’ or 3’ triazole-functionalized sense strand to an integrin-targeting ligand. The following example describes the conjugation of an αvβ3 / 5 integrin-targeting ligand to an annealed double strand: Prepare stock solutions of 0.5 M Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 0.5 M copper(II) sulfate pentahydrate (Cu(II)SO4·5H2O), and 2 M sodium ascorbate in deionized water. Make a 75 mg / mL solution of the integrin-targeting ligand in DMSO. Add 25 μL of 1 M Hepes pH 8.5 buffer to a 1.5 mL centrifuge tube containing the trialkyne-functionalized double strand (3 mg, 75 μL, 40 mg / mL in deionized water, ~15,000 g / mol). After vortexing, add 35 μL of DMSO and vortex the solution. Add the integrin-targeting ligand to the reaction (6 eq / double strand, 2 eq / alkyne, ~15 μL) and vortex the solution. Check the pH using pH paper and confirm that it is ~8. In a 1.5 mL conical centrifuge tube, mix 50 μL of 0.5 M THPTA with 10 μL of 0.5 M Cu(II)SO4·5H2O, vortex, and incubate at room temperature for 5 minutes. After 5 minutes, add the THPTA / Cu solution (7.2 μL, 6 eq, 5:1 THPTA:Cu) to the reaction vial and vortex. Immediately thereafter, add 2 M ascorbate (5 μL, 50 eq / double strand, 16.7 / alkyne) to the reaction vial and vortex. When the reaction is complete (typically complete in 0.5 - 1 hour), purify the reaction immediately by non-denaturing anion exchange chromatography.

[0390] G. Functionalization of the cysteine ring thiol groups. In some embodiments, a cysteine linker was used to facilitate the conjugation of an integrin targeting ligand to the RNAi agent. Either before or after annealing, the 5' or 3' trialkyne-Cys(Stbu)-PEG2 functionalized sense strand was functionalized with a maleimide-containing moiety or, as shown in the following structure, reduced and left with a free thiol: [Chemical formula]

[0391] The following example describes the modification of a trialkyne-Cys(Stbu)-PEG2-double strand with N-ethylmaleimide: The trialkyne-Cys(Stbu)-PEG2-double strand (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. A 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 disulfide reduction by RP-HPLC denaturation, the conjugate was precipitated 3 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 minutes. After completion of the reaction, the conjugate was precipitated 3 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 As shown in Table 1 below, IC50 binding data were obtained for the integrin targeting ligands of Structures 1c, 2c, and 3c, as well as for the RGD mimetic peptide:

[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 showing a particular preference with respect to binding to αvβ3 integrin (IC50 = 0.3 nM and 0.8 nM, respectively). Further, each of Structures 1, 2, and 3 showed a slightly increased binding activity to αvβ3 integrin as compared to an RGD mimetic peptide (see, for example, the mimetic RGD ligand structure disclosed in U.S. Patent No. 9,487,556). Moreover, while the RGD mimetic ligand has been shown to have binding activity, the integrin-targeting ligands of the present disclosure have high stability with respect to both serum stability in vivo and chemical stability ex vivo as compared to such peptide-based RGD mimetic ligands.

[0395] Example 4. Renal Tumor-Bearing Mouse Model (Orthotopic Xenograft) Generation of Clear Cell Renal Cell Carcinoma (ccRCC) A498 Cells Expressing SEAP A pCR3.1 expression vector expressing a reporter gene (SEAP) that secretes alkaline phosphatase under the CMV promoter was prepared by directional cloning of the SEAP coding sequence PCR-amplified from the pSEAP2 basic vector manufactured by Clontech. Convenient restriction sites were added onto 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. The selected A498-SEAP clones were evaluated for SEAP expression and integration stability.

[0396] Transplantation of Clear Cell Renal Cell Carcinoma (ccRCC) A498 Cells Expressing SEAP Female athymic nude mice were anesthetized with ~3% isoflourane and placed in the right lateral position. A small, 0.5 - 1 cm longitudinal abdominal incision was made on the left flank. Using a moistened cotton swab, the left kidney was lifted from the peritoneum and gently fixed. Immediately before 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. Next, the filled syringe was attached to a syringe pump (Harvard Apparatus, model PHD2000), and preparations were made to remove air. The tip of the 27-gauge needle catheter attached to the syringe was inserted just beneath the renal capsule near the caudal pole, and then the tip of the needle was carefully advanced 3 - 4 mm cephalad 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 renal parenchyma using the syringe pump. The needle was left in the kidney for 15 - 20 seconds to ensure complete injection. Next, the needle was removed from the kidney, and a cotton swab was placed over the injection site for 30 seconds to prevent cell leakage or bleeding. Next, the kidney was gently placed back in its original abdominal location, and the abdominal wall was closed. Serum was collected every 7 - 14 days after transplantation, and tumor growth was observed using a commercially available SEAP assay kit. For most of the studies, tumor-bearing mice were used 5 - 6 weeks after transplantation when tumor measurements were typically about 4 - 8 mm at that time.

[0397] Measurement of HIF2 mRNA expression For the studies reported in the examples herein, on specific days after injection, the mice were euthanized, and total RNA was isolated from the 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) only.

[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-made 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). The ΔΔC T method was used to calculate relative gene expression.

[0399] Example 5. In Vivo Administration of an Integrin-Targeted Ligand as an RNAi Agent Targeting HIF-2α (EPAS1) in Mice Bearing Renal Carcinoma The RNAi agent containing a sense strand and an antisense strand was synthesized by the phosphoramidite technique on a solid phase according to the general methods known in the art and commonly used in oligonucleotide synthesis as described in Example 2 of this specification. The RNAi agent contained an antisense strand having 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 and encodes half of a transcription factor involved in gene induction regulated by oxygen, which is induced when oxygen levels are reduced (a state known as hypoxia) and is known to be often overexpressed in clear cell renal carcinoma cells. The Hif2α RNAi agent was designed to reduce or inhibit the translation of the messenger RNA (mRNA) transcript of Hif2α in a sequence-specific manner, and as a result, inhibit the expression of the EPAS1 gene. The Hif2α RNAi agent consisted of modified nucleotides and two or more non-phosphodiester bonds.

[0400] On the first day of the test, according to the following dosing groups, it was administered by tail vein injection to kidney cancer-bearing mice (see Example 4):

[0401] [Table 4]

[0402] An RNAi agent of Example 5 was synthesized, which has a nucleotide sequence directed to target the human Hif2α gene and contains 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-mimicking 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 "monovalent" 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 terminal end above the 5'-end of the sense strand. Each integrin-targeting ligand having an azide-reactive group (which is then conjugated to the DBCO component of the linker) was synthesized (see, for example, Example 1).

[0403] The following structure:

Chemical formula

Chemical formula

Chemical formula

[0404] For Groups 6 and 7, four integrin-targeting ligands were the following:

Chemical formula

[0405] As mentioned in Table 2 above, in some groups, a 40 kDa or 20 kDa PEG moiety was attached to serve as a PK enhancer to extend the circulation time of the drug - product conjugate. The 40 kDa or 20 kDa PEG moiety was conjugated using the reagent of the following formula:

[0406] In each group, three tumor - bearing mice were administered (n = 3). The mice were sacrificed on day 8 after injection, and total RNA was isolated from the renal tumors according to the procedure described in Example 4. Next, relative human HIF2α mRNA expression was quantified by probe - based quantitative PCR (RT - qPCR) normalized to human Cyclophilin A (PPIA) expression and expressed as a fraction of the vehicle 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 a reduction in mRNA expression in mice as compared to the control. The inclusion of the 40 kDa PEG moiety as an enhancer of PK improved the inhibition of the expression of the targeted gene in most cases. Moreover, a comparison of groups 3, 4, and 5 showed that the integrin-targeting ligands of structure 1a described herein are comparable to RGD mimetic peptide-based ligands known to have affinity for αvβ3, and further, that the ligands of structure 2a showed an improvement of about 10% in knockdown over the RGD mimetic ligands. For example, group 3 (RGD mimetic) had a knockdown of about 60% (0.400); group 4 (structure 1a) had a knockdown of about 61% (0.390); and group 5 (structure 2a) had a knockdown of about 69% (0.308).

[0409] Importantly, the data also showed ligand-dependence, as the inclusion of the integrin-targeting ligands disclosed herein showed improvement when compared to the same construct without the ligand. For example, group 6 (trimeric integrin-targeting ligand structure 2a) showed a knockdown of about 72% (0.289) when compared to group 2 (without integrin-targeting ligand), which showed only about 44% knockdown (0.563).

[0410] Furthermore, group 6 showed a small improvement over group 5, indicating a slight preference for the multimeric ligand over the monomeric ligand; however, both forms were active and delivered the RNAi agent to the kidney (as shown by the inhibition of gene expression by the RNAi agent).

[0411] Example 6. In Vivo Administration of Integrin-Targeting Ligands Conjugated to an RNAi Agent Targeting HIF-2α in Mice Bearing Renal Carcinoma An RNAi agent containing a sense strand and an antisense strand was synthesized by the phosphoramidite technique on a solid phase according to general methods known in the art and commonly used in oligonucleotide synthesis, as described in Example 2 of this specification. The RNAi agent has each of the modified nucleotide sequences described in Example 2 of this specification and is designed to target Hif2α (EPAS1).

[0412] On the first day of the test, according to the following dosing groups, it was administered by tail vein injection to kidney cancer-bearing mice (see Example 4):

[0413]

Table 6

[0414] An RNAi agent was synthesized that has a nucleotide sequence directed to target the human Hif2α gene and contains 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 in Groups 2, 3, and 4). For Groups 5 and 6, a single integrin-targeting ligand ("monovalent" ligand) was conjugated to the RNAi agent via the following DBCO-PEG5-NHS ester:

Chemical formula

[0415] In each group, it was administered to 3 cancer-bearing mice (n = 3). The mice were sacrificed on the 8th day of the test after injection, and total RNA was isolated from the kidney tumors according to the procedure described in Example 4. Next, relative human HIF2α mRNA expression was 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).

[0416]

Table 7

[0417] As shown in Table 5 above, each of the Hif2α RNAi agents showed a reduction in mRNA expression in mice compared to the control. Moreover, groups 5 and 6 (containing the integrin-targeting ligand of Structure 2a disclosed herein) showed an improvement in the knockdown of Hif2α mRNA compared to the RGD-mimicking peptide-based ligands of groups 2 and 3 (e.g., comparison of group 6 (approximately 73% knockdown (0.271) with a 15 mg / kg RNAi agent) to group 3 (approximately 67% knockdown (0.330) with a 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 Carcinoma The RNAi agent containing the sense strand and the antisense strand was synthesized by the phosphoramidite technique on a solid phase according to general methods known in the art and commonly used in oligonucleotide synthesis, as described in Example 2 herein. The RNAi agent had each of the modified nucleotide sequences described in Example 2 herein and was designed to target Hif2α (EPAS1).

[0419] On the first day of the test, according to the following dosing groups, mice bearing renal carcinoma (see Example 4) were administered by tail vein injection:

[0420]

Table 8

[0421] An RNAi agent was synthesized that has a nucleotide sequence directed to target the human Hif2α gene and contains a functionalized amine-reactive group (NH2-C6) at the 5' end of the sense strand to facilitate conjugation to an integrin-targeting ligand. For groups 2-7, the following compounds:

Chemical formula

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

[0423] In each group, it was administered to 3 tumor-bearing mice (n = 3). The mice were sacrificed on day 8 of the test after injection, and total RNA was isolated from the renal tumors according to the procedure described in Example 4. Next, relative human HIF2α mRNA expression was quantified by probe-based quantitative PCR (RT-qPCR) normalized to human Cyclophilin A (PPIA) expression and expressed as a fraction of the vehicle 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 a reduction in mRNA expression in mice compared to the control. For example, group 2 (containing a dose of an RNAi agent conjugated to a trivalent integrin-targeting ligand of structure 2a (containing a PEG4 group) at 7.5 mg / kg) showed an approximately 64% knockdown (0.361) of Hif2α. Furthermore, PEG 36All constructs with increased PEG chain length up to (e.g., groups 6 and 7) showed knockdown, but no benefit was seen when compared to the PEG4 chain present in construct 2a.

[0426] Example 8. In Vivo Dose Response Study of Injected Integrin-Targeting Ligand Conjugated to an RNAi Agent Targeting HIF-2α in Mice Bearing Renal Cancer The RNAi agent containing sense and antisense strands was synthesized by phosphoramidite technology on solid phase according to general methods known in the art and commonly used in oligonucleotide synthesis as described in Example 2 herein. The RNAi agent had the respective modified nucleotide sequences described in Example 2 herein and was designed to target Hif2α (EPAS1).

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

[0428] [Table 10]

[0429] An RNAi agent was synthesized that had a nucleotide sequence 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 an integrin-targeting ligand. Each group had the following structure: [Chemical Structure] and had a tridentate integrin ligand of structure 2a as shown.

[0430] In each group, it was administered to 3 tumor-bearing mice (n = 3). The mice were sacrificed on day 8 of the test after injection, and total RNA was isolated from the kidney tumors according to the procedure described in Example 4. Next, relative human HIF2α mRNA expression was quantified by probe-based quantitative PCR (RT-qPCR) normalized to human Cyclophilin A (PPIA) expression and expressed as a fraction of the vehicle control group (isotonic glucose) (geometric mean, + / - 95% confidence interval) as described in Example 4.

[0431]

Table 11

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

[0433] Example 9. Persistence of knockdown of an RNAi agent targeting HIF-2α conjugated to an integrin-targeting ligand in mice bearing renal cancer The RNAi agents containing sense and antisense strands were synthesized by phosphoramidite technology on a solid phase according to general procedures known in the art and commonly used in 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 day 1 of the test, it was administered by tail vein injection to mice bearing renal cancer (see Example 4) 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, it was administered to 2 mice in group 1 and 3 mice in group 1A. For the RNAi agent-integrin targeting ligand-containing groups (i.e., groups 2, 3, 4, and 5), it was administered to 4 tumor-bearing mice in each group (n = 4). Total RNA was isolated from renal tumors according to the procedure described in Example 4. Next, relative human HIF2α mRNA expression was 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 a reduction in mRNA expression compared to the control at the 22-day time point (approximately 70% knockdown (0.299) at the 22-day time point).

[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 agent containing a sense strand and an antisense strand was synthesized by phosphoramidite technology on a solid phase according to general methods known in the art and commonly used in oligonucleotide synthesis as described in Example 2 of this specification. The RNAi agent had the respective modified nucleotide sequences described in Example 2 of this specification and was designed to target Hif2α (EPAS1).

[0441] On the first day of the test, according to the following administration groups, it was administered by tail vein injection to kidney cancer-bearing mice (see Example 4):

[0442]

Table 14

[0443] An RNAi agent was synthesized that had a nucleotide sequence 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 an integrin-targeting ligand.

[0444] In each group, it was administered to 3 cancer-bearing mice (n = 3). The mice were sacrificed on the 8th day of the test after injection, and total RNA was isolated from the kidney tumors according to the procedure described in Example 4. Next, relative human HIF2α mRNA expression was 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).

[0445]

Table 15

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

[0447] Example 11. In Vivo Administration of an Integrin-Targeting Ligand Conjugated to an RNAi Agent Targeting HIF-2α in Mice Bearing αvβ3 KO A498 Renal Carcinoma Clear cell renal cell carcinoma (ccRCC) A498 tumor cells express both αvβ3 integrin and αvβ5 integrin, and according to flow cytometry analysis, αvβ3 expression is approximately 4-fold higher than αvβ5 expression. To evaluate the contribution of αvβ5 in this model, αvβ3 knockout (KO) A498 cells were synthesized by gene editing technology. Knockout of integrin αvβ3 was confirmed by genomic sequencing and immunohistochemical staining of αvβ3, and it showed that the staining was negative in αvβ3 KO A498 cells. Renal cancer-bearing mice with A498 WT (having both αvβ3 and αvβ5) cells and αvβ3 KO A498 cells were prepared as previously described in Example 4.

[0448] On day 1 of the test, it was administered to the renal cancer-bearing mice by tail vein injection. In each group described in Table 13 below, it was administered to 3 cancer-bearing mice (n = 3). The mice were sacrificed on day 8 of the test after injection, and total RNA was isolated from the renal tumors as described in Example 4. Next, relative human HIF2α mRNA expression was quantified by probe-based quantitative PCR (RT-qPCR) normalized to human Cyclophilin A (PPIA) expression and expressed as a fraction of the vehicle 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 (approx. 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 substantially 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 agent containing the sense strand and the antisense strand was synthesized by the phosphoramidite technique on a solid phase according to general methods known in the art and commonly used in oligonucleotide synthesis, as described in Example 2 herein. The RNAi agent had the respective modified nucleotide sequences described in Example 2 herein and was designed to target Hif2α (EPAS1).

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

[0453]

Table 17

[0454] An RNAi agent was synthesized that had a nucleotide sequence 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 an integrin-targeting ligand.

[0455] The following structure:

Chemical Structure

Chem.

Chem.

[0456] In each group, it was administered to 3 tumor-bearing mice (n = 3). The mice were sacrificed on the 8th day after the injection, and total RNA was isolated from the kidney tumors according to the procedure described in Example 4. Next, the relative human HIF2α mRNA expression was 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).

[0457]

Table 18

[0458] As shown in Table 16 above, each of the Hif2α RNAi agent-integrin targeting ligand conjugates showed a reduction in 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 An RNAi agent containing a sense strand and an antisense strand was synthesized by the phosphoramidite technique on a solid phase according to general methods known in the art and commonly used in oligonucleotide synthesis, as described in Example 2 herein. The RNAi agent has each of the modified nucleotide sequences described in Example 2 herein and is designed to target Hif2α (EPAS1).

[0460] On the first day of the test, according to the following dosing groups, it was administered by tail vein injection to kidney cancer-bearing mice (see Example 4):

[0461]

Table 19-1

Table 19-2

[0462] In each group, it was administered to 3 cancer-bearing mice (n = 3). The mice were sacrificed on the 8th day of the test after injection, and total RNA was isolated from the kidney tumors according to the procedure described in Example 4. Next, relative human HIF2α mRNA expression was 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).

[0463]

Table 20

[0464] As shown in Table 18 above, each of the Hif2α RNAi agent-integrin targeting ligand conjugates having a structure containing the integrin targeting ligands of structures 2a and 32a showing the maximum inhibitory activity showed a reduction in mRNA expression in mice compared to the control.

[0465] Example 14. In Vivo Administration of an Integrin-Targeting Ligand Conjugated to an RNAi Agent Targeting HIF-2α in Mice Bearing Renal Cancer An RNAi agent containing a sense strand and an antisense strand was synthesized by phosphoramidite technology on a solid phase according to general procedures known in the art and commonly used in oligonucleotide synthesis, as described in Example 2 herein. The RNAi agent had the respective modified nucleotide sequences described in Example 2 herein and was designed to target Hif2α (EPAS1).

[0466] On the first day of the test, according to the following dosing groups, renal cancer-bearing mice (see Example 4) were administered by tail vein injection:

[0467] [Table 21]

[0468] In each group, three cancer-bearing mice were administered (n = 3). The mice were sacrificed on the eighth day of the test after injection, and total RNA was isolated from the renal tumors according to the procedure described in Example 4. Next, relative human HIF2α mRNA expression was 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).

[0469] [Table 22]

[0470] As shown in Table 20 above, each of the Hif2α RNAi agent-integrin targeting ligand conjugates showed a reduction in 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 agent containing the sense strand and the antisense strand was synthesized by the phosphoramidite technique on a solid phase according to general methods known in the art and commonly used in oligonucleotide synthesis, as described in Example 2 of this specification. The RNAi agent had each of the modified nucleotide sequences described in Example 2 of this specification and was designed to target Hif2α (EPAS1).

[0472] On the first day of the test, according to the following administration groups, mice bearing renal cancer (see Example 4) were administered by tail vein injection:

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

[0474] In each group, it was administered to 3 cancer-bearing mice (n = 3). The mice were sacrificed on the 8th day of the test after injection, and total RNA was isolated from the renal tumors according to the procedure described in Example 4. Next, the relative human HIF2α mRNA expression was 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 in Table 20 above, each of the Hif2α RNAi agent-integrin targeting ligand conjugates showed a reduction in mRNA expression in mice compared to the control.

[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 agent containing the sense strand and the antisense strand was synthesized by the phosphoramidite technique on a solid phase according to general methods known in the art and commonly used in oligonucleotide synthesis, as described in Example 2 herein. The RNAi agent had each of the modified nucleotide sequences described in Example 2 herein and was designed to target Hif2α (EPAS1).

[0478] On the first day of the test, according to the following dosing groups, mice bearing renal cancer (see Example 4) were administered by tail vein injection:

[0479]

Table 25-1

Table 25-2

Table 25-3

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

[0481]

Table 26-1

Table 26-2

[0482] As shown in Table 24 above, each of the Hif2α RNAi agent-integrin targeting ligand conjugates showed a reduction in mRNA expression in mice compared to the control. 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 as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. The following: 【Chemical 1】 A compound or a pharmaceutically acceptable salt thereof, comprising an integrin-targeting ligand and an RNAi agent having the structure of: {wherein the following: 【Chemical 2】 represents the point of connection of the integrin target ligand to the remainder of the compound} A compound or a pharmaceutically acceptable salt thereof.

2. The compound according to Claim 1, wherein the compound comprises one integrin target ligand of Structure 2a.

3. The compound according to Claim 1, wherein the compound comprises two integrin target ligands of Structure 2a.

4. The compound according to Claim 1, wherein the compound comprises three integrin target ligands of Structure 2a.

5. The compound according to Claim 1, wherein the compound comprises four integrin target ligands of Structure 2a.

6. The following formula: 【Chemical Formula 3】 A compound or a pharmaceutically acceptable salt thereof, comprising the structure of and an RNAi agent, {wherein the following: 【Chemical Formula 4】 represents the point of connection of the structure to the remainder of the compound} A compound or a pharmaceutically acceptable salt thereof.

7. A composition comprising the compound according to any one of Claims 1 to 6 and a pharmaceutically acceptable excipient.

8. A composition comprising the compound according to any one of Claims 1 to 6, wherein the RNAi agent is capable of inhibiting the expression of a target gene in cells expressing integrin.

9. The composition according to Claim 7 or 8, wherein the integrin is integrin αvβ3, αvβ5, or both αvβ3 and αvβ5.

10. A composition for delivering an RNAi agent to cells expressing integrin αvβ3, αvβ5, or both αvβ3 and αvβ5 in a subject, comprising the compound according to any one of Claims 1 to 6.

11. A composition for delivering an RNAi agent to cells or tissues of a subject in vivo, comprising the compound according to any one of Claims 1 to 6.

12. A composition for inhibiting the expression of a target gene of cells expressing integrin αvβ3, integrin αvβ5, or both integrin αvβ3 and αvβ5 in vivo, comprising an effective amount of the compound according to any one of Claims 1 to 6.

13. The composition according to claim 12, wherein the target gene is EPAS1 (HIF2α).

14. The composition according to claim 12 or claim 13, wherein the cell is a clear cell renal carcinoma tumor cell.

15. For the delivery of an RNAi agent to cells comprising αvβ3, αvβ5, or both αvβ3 and αvβ5, in vitro or ex vivo Use of a compound according to any one of claims 1 to 6, or a composition according to any one of claims 7 to 9.

16. The use according to claim 15, wherein the cell is a kidney cell.

17. Use of a composition according to any one of claims 7 to 9 for the manufacture of a medicament for the treatment of a disease or disorder, wherein the disease or disorder can be treated or ameliorated by delivery of an RNAi agent to cells comprising αvβ3, αvβ5, or both αvβ3 and αvβ5.

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