Novel therapeutic delivery moieties and uses thereof

Abstract

Provided herein are compounds including delivery moieties for the selective delivery of a therapeutic and / or an oligonucleotide to the lungs. Also provided herein are compounds including a single delivery moiety for the selective delivery of a therapeutic and / or oligonucleotide to the lungs. Also disclosed herein are novel αvβx integrin ligands, such as ligands that associate with αvβ5, αvβ6, and / or αvβ8 integrins.

Description

NOVEL THERAPEUTIC DELIVERY MOIETIES AND USES THEREOFREFERENCE TO A SEQUENCE LISTING

[0001] The present application is being filed along with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file titled “30619_WO” created 15- Oct-2024 and is 252 kilobytes in size. The Sequence Listing information in the ST.26 XML format is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] The present invention relates to novel compounds comprising novel delivery moieties for delivery of oligonucleotides to the lungs, which are useful in the treatment of lung diseases. The present invention also relates to novel delivery moieties that can be linked to an oligonucleotide to aid in the delivery of the oligonucleotide to the lungs and / or lung cells.BACKGROUND OF THE INVENTION

[0003] Compounds comprising oligonucleotides can target genes in a sequence-specific manner for treatment of many different types of diseases involving gene dysregulation. For example, RNA interference (RNAi) compounds, such as small interfering RNAs (siRNAs), and antisense oligonucleotides (ASOs), can be used to knock-down gene expression. In contrast, other oligonucleotide containing compounds may activate a gene using an oligonucleotide, such as short activating RNA (saRNA). By delivering oligonucleotides to a desired tissue of the patient, gene expression can be downregulated, upregulated, or corrected.

[0004] Oligonucleotides can be delivered to the desired tissue through the attachment of delivery moieties. For example, an RNAi compound, such as an oligonucleotide, to the liver can be selectively delivered to the liver by attaching N-acetylgalactosamine (GalNAc) to target the asialoglycoprotein receptor on liver cells. An exemplary compound comprising GalNAc is givosiran, an FDA approved siRNA that targets the ALAS1 gene to treat acute hepatic porphyria. Despite the existence of such compoundsfor delivery to the liver, there remains a need for moieties for the delivery of oligonucleotides to other tissues, such as the lungs.

[0005] While some delivery moieties have been proposed for the selective delivery of compounds comprising oligonucleotides to the lungs, such as in U.S. Patent No. 11,597,701, there is still a need for moieties that can deliver compounds comprising oligonucleotides to the lungs. For example, the delivery moieties proposed in U.S. Patent No 11,597,701 required three ligands for adequate selective delivery of compounds comprising oligonucleotides to the lungs, which can increase the cost associated with the synthesis, use, and storage of these delivery moieties. Thus, there is a need for delivery moieties that can selectively deliver compounds comprising oligonucleotides to the lungs with as little as a single ligand to decrease the overall complexity of the molecule. SUMMARY OF THE INVENTION

[0006] Disclosed herein is a compound of the formula:

[0007] wherein: R1is H or C1to C6alkyl;is (C5to C14aryl)-O-, or (C5to C14heteroaryl)-O- with from 1 to 5 heteroatoms selected from N, O, and S; L comprises a linker or is absent; Z comprises an oligonucleotide; and q is a whole number integer from 1 to 3, or a pharmaceutically acceptable salt thereof.

[0008] Also disclosed herein is a compound of the formula:

[0009] wherein: R1is H or C1to C6alkyl;is (C5to C14aryl)-O-, or (C5to C14heteroaryl)-O- with from 1 to 5 heteroatoms selectedfrom N, O, and S; L comprises a linker or is absent, and Z comprises an oligonucleotide, or a pharmaceutically acceptable salt thereof.

[0010] Also disclosed herein is a compound of the formula:

[0011] ,wherein: R1is H or C1to C6alkyl;is (C5to C14aryl)-O-, or (C5to C14heteroaryl)-O- with from 1 to 5 heteroatoms selected from N, O, and S; L comprises a linker or absent; and Z comprises an oligonucleotide, or a pharmaceutically acceptable salt thereof.

[0012] Also disclosed herein are uses of the disclosed compounds in therapy, such as in the treatment of lung disease.

[0013] Also disclosed herein is a compound of the formula:wherein R1is H or C1to C6alkyl andis (C5to C14aryl)-OH, or (C5to C14heteroaryl)-OH with from 1 to 5 heteroatoms selected from N, O, and S. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention relates to novel compounds, including novel delivery moieties and novel compounds comprising a novel delivery moiety and an oligonucleotide, for the selective delivery of oligonucleotides to the lungs, which are useful in the treatment of lung diseases. The novel delivery moieties can be linked to an oligonucleotide to aid in the delivery of the oligonucleotide to a desired location, such as the lungs or lung cells.

[0015] It has been surprisingly found that oligonucleotides can be selectively delivered to lung cells or lung tissue by linking the oligonucleotide to a delivery moiety comprising an integrin αvβ ligand. In particular, it has been surprisingly found that oligonucleotides can be selectively delivered to lung cells or lung tissue by linking the oligonucleotide to a delivery moiety comprising an integrin αvβ6ligand, as αvβ6integrins are highly expressed in lung cells.

[0016] Integrins are a family of cell surface receptor proteins that aid in cell adhesion, cell signaling, and the attachment of cells to the extracellular matrix and other cells. Integrins are essential components of various biological processes, including tissue development, immune responses, wound healing, and cancer metathesis. Integrin heterodimers are formed by the combination of 18 different α subunits and 8 different β subunits. Additionally, several integrins, including αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8 are highly expressed in lung cells. In lung cells, several integrins, such as αvβ6, are expressed in lung epithelial cells and are believed to be involved in extracellular matrix remodeling and TGF-β activation, such as during lung injury and repair processes.

[0017] While not wishing to be bound by theory, it is believed that a delivery moiety can be designed to act as a ligand for one or more αvβxintegrins, such as αvβ6, which will allow for transportation of the delivery moiety through the cell membrane of the lung cells. Additionally, while not wishing to be bound by theory, if the delivery moiety is linked to an oligonucleotide, as further described herein, it can allow for selective delivery of the oligonucleotide to the lungs, as it is believed that αvβxintegrins, such as αvβ6, are highly expressed in lung cells.

[0018] As further described herein, it has been unexpectedly found that the disclosed αvβxligands can allow for the selective delivery of an oligonucleotide to the lungs with as little as a single αvβxligand. This can allow for the synthesis of a simpler delivery moiety that can be easier to handle than a delivery moiety comprising a tris ligand.

[0019] D-L-Z

[0020] Formula I.

[0021] Disclosed herein are novel compounds of Formula I, wherein D is a delivery moiety, the delivery moiety comprising a αvβxintegrin, such as αvβ6ligand, L is a linker or absent, and Z is an oligonucleotide. The oligonucleotide can be connected to the linkerthrough conjugation. For example, the oligonucleotide and / or linker can be alkyne- or NHS-ester functionalized, which can aid in later connection of the linker to the oligonucleotide through conjugation reactions. The compound comprising the oligonucleotide and delivery moiety can be a conjugate compound. Also disclosed herein are pharmaceutically acceptable salts of compounds of Formula I.

[0022]

[0023]

[0024] Also disclosed herein are novel compounds of Formula II, wherein D1and D2, which can be independently selected or identical, are delivery moieties comprising a αvβxintegrin, such as αvβ6ligand, L1and L2, which can be independently selected or identical, or absent, and Z is an oligonucleotide. The oligonucleotide can be connected to the linker through conjugation. For example, the oligonucleotide and / or linkers can be alkyne- or NHS-ester- functionalized, which can aid in later connection of the linkers to the oligonucleotide through conjugation reactions. The compound comprising the oligonucleotide and delivery moiety can be a conjugate compound. Also disclosed herein are pharmaceutically acceptable salts of compounds of Formula II.

[0025]

[0026]

[0027] Also disclosed herein are novel compounds of Formula III, wherein D is a delivery moiety, the delivery moiety comprising a αvβxintegrin, such as αvβ6ligand, L is a linker or absent, and Z is an oligonucleotide. The oligonucleotide can be connected to the linker through conjugation. For example, the oligonucleotide and / or linker can be alkyne- or NHS-ester- functionalized, which can aid in later connection of the linker to the oligonucleotide through conjugation reactions. The compound comprising the oligonucleotide and delivery moiety can be a conjugate compound. Also disclosed herein are pharmaceutically acceptable salts of compounds of Formula III.

[0028]

[0029]

[0030] Also disclosed herein are novel compounds of Formula IV, wherein D is a delivery moiety, the delivery moiety comprising a αvβxintegrin, such as αvβ6ligand, L is a linker or absent, and Z is an oligonucleotide. The oligonucleotide can be connected to the linker through conjugation. For example, the oligonucleotide and / or linker can be alkyne- or NHS-ester- functionalized, which can aid in later connection of the linker to the oligonucleotide through conjugation reactions. The compound comprising the oligonucleotide and delivery moiety can be a conjugate compound. Also disclosed herein are pharmaceutically acceptable salts of compounds of Formula IV.

[0031] The compounds of Formula I, II, III, and IV can be referred to as conjugate compounds as they include an oligonucleotide and a αvβxintegrin, such as αvβ6ligand. Accordingly, the compound can include one delivery moiety attached to either the 3’ or 5’ end of the oligonucleotide, optionally through a linker (Formula I). Additionally, the compound can include two delivery moieties, each delivery moiety attached to the 3’ or the 5’ end of the oligonucleotide, optionally through a linker (Formula II). Additionally, the compound can include three delivery moieties and all delivery moieties are attached to the 3’ or the 5’ end of the oligonucleotide, optionally through a linker (Formula III and IV).

[0032] Delivery Moiety

[0033] Disclosed herein are novel αvβxintegrin ligands, which are represented in Formula V and VI. Also disclosed herein are novel compounds comprising a delivery moiety, the delivery moiety comprising one or more novel αvβxintegrin ligands, which are represented by Formula VII and VIII.

[0034] The compounds provided herein include a delivery moiety, D, D1, and / or D2in Formula I to Formula IV, for delivering an oligonucleotide to a target location, such as the lungs or lung cells. The delivery moiety comprises one or more αvβxintegrin ligands.

[0035] Other suitable delivery moieties for the selective delivery of oligonucleotides to the lungs include any delivery moiety that can bond to an αvβxintegrin, such as an αvβ6integrin ligand. Suitable delivery moieties, D, D1, and / or D2in Formula I-IV, are shown below in Formula V and VI unconnected to an oligonucleotide, and in Formula VII and VIII to show a connection between the ligand and the remaining portion of the conjugate.

[0036] The squiggly line in Formula VII and VIII shows where D, D1, and / or D2can be connected to the linker and / or the oligonucleotide in the case of the delivery moiety- oligonucleotide conjugate or where D, D1, and / or D2can be connected to a leaving group or another suitable atom or molecule in the case of an agonist compound.

[0037]

[0040] In Formula V, R1can be H or C1to C6alkyl. R2can be OH, (C5to C7aryl)-OW, or (C5to C7heteroaryl)-OW with from 1 to 3 heteroatoms selected from N, O, and S. Y1and Y2can be independently selected from N or CH. W can be H or C1to C6alkyl.

[0043] In Formula VI, R1can be H or C1to C6alkyl.can be (C5to C14aryl)-OH, or (C5to C14heteroaryl)-OH with from 1 to 5 heteroatoms selected from N, O, and S.

[0046] In Formula VII, R1can be H or C1to C6alkyl. R2can be O-, (C5to C7aryl)-O-, or (C5to C7heteroaryl)-O- with from 1 to 3 heteroatoms selected from N, O, and S. Y1and Y2 can be independently selected from N or CH.

[0047]

[0049] In Formula VIII, R1can be H or C1to C6alkyl.can be (C5to C14aryl)-O-, or (C5to C14heteroaryl)-O- with from 1 to 5 heteroatoms selected from N, O, and S.

[0050] Formula IX and X shows examples of suitable portions of compounds that can be represented by

[0051]

[0055] The novel compounds disclosed herein, in total, can have from 1 to 3 delivery moieties that are attached to the oligonucleotide. The delivery moieties can be attached to either or both sides of the compound. For example, the compound can have a single delivery moiety on the 5’ end, the 3’ end, or both ends of the oligonucleotide attached optionally via a linker. Additionally, the conjugate compound can have multiple delivery moieties attached to the same end of the oligonucleotide through one or more optional linkers.

[0056] Oligonucleotides

[0057] One or more oligonucleotides can be selectively delivered to the lungs using the delivery moieties described herein for diagnostic or therapeutic purposes. The one or more oligonucleotides may comprise DNA or RNA nucleotides, nucleosides, or combination thereof, and may comprise one or more, or all, modified nucleotides, nucleosides, or modified bonds.

[0058] The oligonucleotides can target certain DNA or RNA sequences in a cell to regulate gene expression. In some embodiments, the oligonucleotide decreasesexpression of a target mRNA transcript and / or further decreases target protein expression. Suitable conjugates can have a percent knockdown of the target gene of at least about 20%, at least about 40%, or at least about 50%. The decrease in expression can be durable for about one week, three weeks, and / or four weeks.

[0059] One of skill in the art recognizes that one or more mismatches may be present as between the oligonucleotide and the target nucleotide sequence and still function to regulate gene expression. Accordingly, in an embodiment, the oligonucleotide has 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, or 85 percent identity with the target sequence. The oligonucleotides may also have overhangs of 1-10, 1-5, or 1-3, or 3, 2, or 1 residue(s) at either the 5’ or 3’ end. The oligonucleotide may further include an abasic or inverted abasic moiety or a modified phosphate group. Suitable modifications are known in the art.

[0060] In some embodiments, the oligonucleotide includes 2′-modifications of the sugar residue, e.g., ribose or deoxyribose. For example, some 2’-modifications of ribose or deoxyribose can increase RNA or DNA stability and half-life. Such 2’-modifications include 2’-fluoro, 2’-O-methyl (i.e., 2’-methoxy), 2'-O-alkyl, or 2’-O-methoxyethyl (2’- O-MOE). In some embodiments, the oligonucleotide includes a modified internucleotide linkage, e.g., a phosphorothioate (PS) linkage.

[0061] In certain embodiments the one or more oligonucleotides is / are an RNAi agent such as a small interfering RNA (siRNA), small (also called short) activating RNA (saRNA), microRNA (miRNA), short hairpin RNA (shRNA), or antisense oligonucleotide (ASO). In a suitable embodiment the oligonucleotide is an siRNA. In another suitable embodiment the oligonucleotide is an siRNA comprising a sense strand and an antisense strand.

[0062] In some embodiment, the oligonucleotide is an siRNA comprising an antisense strand targeting a gene of interest in the lungs.

[0063] Suitable genes of interest can include, but are not limited to, any genes that modulation of the gene would improve lung cell function.

[0064] In some embodiments, the gene of interest is selected from α-ENaC, AGER (RAGE), AOC3, APLNR, β-ENaC, CCL24, CTGF, CXCR1, CXCR2, CXCR4, ENPP2, ET-A, ET-B, FGFR1, FGFR2, FGFR3, HIF-1α, IL33, ITGAV, ITGB1, ITGB6, LPAR1,LPAR3, MIF, MKL1, MMP7, Muc5AC, Muc5B, PDGFRα, PDGFRβ, ROCK1, ROCK2, S1PR1, S1PR2, SIPR3, SERPINE1, STAT6, TGFβ1, TGFβ2, TGFβ3, TGFβR1, TGFβR2, TLR7, TLR8, TLR9, TSLP, VEGFR1, VEGFR2, VEGFR3, WISP1, SCNN1A, Muc5B, and / or RAGE. A person of ordinary skill in the art can modify the siRNA antisense strand to target a different gene of interest.

[0065] In some embodiments, the sense strand and the antisense strand are each between 15-40 nucleotides, e.g., 18-25 nucleotides, in length. The sense strand and the antisense strand form a duplex, optionally with one or more 5’ or 3’ nucleotide overhangs.

[0066] In some embodiments, one or more nucleotides of the sense strand and / or the antisense strand are independently modified nucleotides, which means the sense strand and the antisense strand can have different modified nucleotides. In some embodiments, one or more nucleotides of the sense strand are modified nucleotides. In some embodiments, each nucleotide of the sense strand is a modified nucleotide. In some embodiments, one or more nucleotides of the antisense strand are modified nucleotides. In some embodiments, each nucleotide of the antisense strand is a modified nucleotide. In some embodiments, the modified nucleotide is a 2'-fluoro modified nucleotide, 2'-O- methyl modified nucleotide, or 2'-O-alkyl modified nucleotide. In some embodiments, each nucleotide of the sense strand and the antisense strand is independently a modified nucleotide, e.g., a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide, or 2'- O-alkyl modified nucleotide.

[0067] In some embodiments, the antisense strand includes a phosphate analog, e.g., 5’- vinylphosphonate (5’-VP), at the 5’ end.

[0068] In some embodiments, the sense strand has an abasic moiety or inverted abasic moiety, at position 9, 10 or 11.

[0069] In some embodiments, the sense strand and the antisense strand have one or more modified internucleotide linkages. In some embodiments, the modified internucleotide linkage is phosphorothioate linkage. In some embodiments, the sense strand has four or five phosphorothioate linkages. In some embodiments, the antisense strand has four or five phosphorothioate linkages. In some embodiments, the sense strand and the antisense strand each has four or five phosphorothioate linkages. In some embodiments, the sensestrand has four phosphorothioate linkages and the antisense strand has four phosphorothioate linkages.

[0070] In some embodiments, Z can also include a branching moiety so that multiple linkers and multiple delivery moieties can be attached to the same location of the oligonucleotide, such as, for example, in FIG.3.

[0071] In some embodiments, the branching moiety can be attached to the oligonucleotide and / or one or more linkers through well-known means of conjugation chemistry, such as amidation reactions.

[0072] Linker

[0073] The present invention can also include a linker, L, L1, and / or L2, to connect the one or more oligonucleotide to the delivery moiety and / or the αvβxintegrin, such as αvβ6integrin, ligand. Suitable linkers include any molecule that can connect the delivery moiety and / or αvβxintegrin, such as αvβ6ligand, to the one or more oligonucleotide. The linker can directly connect the αvβxintegrin, such as αvβ6ligand, to the oligonucleotide or there can be an additional conjugation linker.

[0074] Suitable linkers can include C1to C20linear alkyl, C1to C20branched alkyl, C1to C20alkenyl, cycloalkyl with from 3 to 7 carbon atoms, heterocyclic compounds with from 3 to 7 membered rings, aryl with from 3 to 7 carbon atoms in the aryl ring, heteroaryl with from 3 to 7 atoms in the heteroaryl ring, -[OCH2CH2]n-, wherein n is a whole number integer from 1 to 20, and / or combinations thereof.

[0075] In some embodiments, the linker is a compound of Formula XI. n can be a whole number integer from 1 to 20, from 6 to 20, from 10 to 20, from 12 to 18, or from 12 to 15. A and B are connection points for either the oligonucleotide, the conjugation portion, and / or the delivery moiety. For example, connection point A can connect to an oligonucleotide and a conjugation portion (i.e. the oligonucleotide is conjugated to the linker) and connection point B can connect directly to a delivery moiety comprising an αvβxintegrin, such as αvβ6integrin, ligand.

[0076] In some embodiments, the linker is a compound of Formula XII. m can be a whole number integer from 1 to 20, from 1 to 10, from 5 to 10, or from 1 to 5. C and D are connection points for either the oligonucleotide, the conjugation portion, and / or the delivery moiety. For example, connection point C can connect to an oligonucleotide and a conjugation portion (i.e. the oligonucleotide is conjugated to the linker) and connection point D can connect directly to a delivery moiety comprising an αvβxintegrin, such as αvβ6, integrin, ligand.

[0079] In some embodiments, the linker is a compound of Formula XIII, which is a combination of Formula XI and Formula XII. n can be a whole number integer from 1 to 20, from 6 to 20, from 10 to 20, from 12 to 18, or from 12 to 15. m can be a whole number integer from 1 to 20, from 1 to 10, from 5 to 10, or from 1 to 5. E and F are connection points for either the oligonucleotide, the conjugation portion, and / or the delivery moiety. For example, connection point E can connect to an oligonucleotide and a conjugation portion (i.e. the oligonucleotide is conjugated to the linker) and connection point F can connect directly to a delivery moiety comprising an αvβxintegrin, such as αvβ6integrin, ligand.

[0080]

[0081]

[0082] In some embodiments, the linker is a compound of Formula XIV-A. G and H are connection points for either the oligonucleotide, the conjugation portion, and / or the delivery moiety. For example, connection point G can connect to an oligonucleotide anda conjugation portion (i.e. the oligonucleotide is conjugated to the linker) and connection point H can connect directly to a delivery moiety comprising an αvβxintegrin, such as αvβ6integrin, ligand.

[0085] In some embodiments, the linker is a compound of Formula XIV-B, which is a combination of Formula XIV-A and Formula XII. n can be a whole number integer from 1 to 20, from 1 to 10, from 5 to 10, or from 1 to 5. m can be a whole number integer from 1 to 20, from 1 to 10, from 5 to 10, or from 1 to 5. J and K are connection points for either the oligonucleotide, the conjugation portion, and / or the delivery moiety. For example, connection point J can connect to an oligonucleotide and a conjugation portion (i.e. the oligonucleotide is conjugated to the linker) and connection point K can connect directly to a delivery moiety comprising an αvβxintegrin, such as αvβ6integrin, ligand.

[0086] In some embodiments, the linker includes a compound of Formula XI-XIV and a conjugation portion. Conjugation is the process of chemically joining two or more molecules or biomolecules by a covalent bond. A person of ordinary skill in the art would recognize that there are a variety of conjugation reagents that can be used to connect a portion of a molecule, such as the delivery moiety and / or the linker, as described herein, to a biomolecule, such as the oligonucleotide, as described herein. A conjugation portion is an additional compound introduced to connect the oligonucleotide to the linker and / or delivery moiety via conjugation reagents.

[0087] Suitable conjugation reagents for conjugating the oligonucleotide to the remaining portions of the conjugate, such as the delivery moiety and / or the linker, are dependent on the desired location of the covalent bond between the oligonucleotide and the linker and / or delivery. Once the location of the covalent bond is identified, a person of ordinary skill in the art can select from a well-known list of conjugation reagents to make the selective link. Examples of conjugation chemistries include, but are not limited to,Michael additions (i.e. thiosuccinamide), amidations, cycloadditions (i.e.1,3 dipolar cycloadditions), and / or disulfides. In each of the preceding, non-limiting examples, additional portions of the molecules, such as linear alkyl bridges, may be incorporated to more easily introduce the needed functional groups to selectively associate the oligonucleotide with the linker and delivery moiety (also known as the linked αvβxintegrin ligand). The oligonucleotide can be conjugated to the delivery moiety directly or through a linker. For example, the oligonucleotide can be conjugated to the connection point A in Formula XI and the delivery moiety comprising the αvβxintegrin, such as αvβ6integrin, ligand can be covalently bonded to connection point B. Alternatively, the oligonucleotide can be conjugated to the linker and the delivery moiety can also be conjugated to the linker. For example, the oligonucleotide can be conjugated to the connection point A in Formula XI and the delivery moiety comprising the αvβxintegrin ligand can be conjugated to connection point B. Alternatively, the oligonucleotide can be covalently connected to the linker without any conjugation portion or conjugation synthesis step and / or the delivery moiety can be covalently connected to the linker without any conjugation portion or conjugation synthesis step.

[0088] The delivery moiety and / or the linker may be connected to the 5’ or 3’ end of an oligonucleotide, or attached to one of the internal nucleotide or nucleoside bases. The delivery moiety and / or the linker may also be linked or conjugated to the 5’ or 3’ end of an oligonucleotide. If the oligonucleotide is a siRNA, the delivery moiety and / or the linker may be connected or conjugated to the 5’ or 3’ end of the sense strand or the antisense strand of the oligonucleotide.

[0089] One of skill in the art will also recognize that placement of a delivery moiety, whether via a linker or not, on the 5’ end of an antisense strand may need to overcome potential inefficient loading of Ago2 loading, or other hindrance of the RISC complex activity. In some embodiments, the delivery moiety is conjugated to the 3’ end of the sense strand. In a further embodiment, the delivery moiety is conjugated to the 3’ end of the sense strand via a linker.

[0090] Compounds Comprising Oligonucleotide(s) and a Delivery Moiety

[0091] Provided herein are compounds that can selectively deliver oligonucleotides to the lungs. Suitable compounds include the compounds described in Formula XV to XVIII. Other suitable compounds can include one or more oligonucleotides, a delivery moiety comprising one or more αvβxligand(s), and, optionally, a linker between the oligonucleotide and an αvβxligand. For example, a suitable compound may include an oligonucleotide, a delivery moiety comprising an αvβxligand, and a linker connecting the oligonucleotide to the deliver moiety. Additional possible components are further discussed below.

[0094] Also disclosed herein are novel compounds of Formula XV, wherein L is a linker or absent, Z is an oligonucleotide. R1can be H or C1to C6alkyl.can be (C5to C14aryl)-O-, or (C5to C14heteroaryl)-O- with from 1 to 5 heteroatoms selected from N, O, and S.can also be an aryl or heteroaryl shown in Formula IX and / or X. Also disclosed herein are pharmaceutically acceptable salts of compounds of Formula XV.

[0095]

[0097] Also disclosed herein are novel compounds of Formula XVI, wherein L is a linker or absent, and Z is an oligonucleotide. R1can be H or C1to C6alkyl. can be (C5toC14aryl)-O-, or (C5to C14heteroaryl)-O- with from 1 to 5 heteroatoms selected from N, O, and S.can also be an aryl or heteroaryl shown in Formula IX and / or X. Also disclosed herein are pharmaceutically acceptable salts of compounds of Formula XVI.

[0098]

[0100] Also disclosed herein are novel compounds of Formula XVII, wherein Z is an oligonucleotide. R1can be H or C1to C6alkyl.can be (C5to C14aryl)-O-, or (C5to C14heteroaryl)-O- with from 1 to 5 heteroatoms selected from N, O, and S. q can be a whole number integer from 1 to 3.can also be an aryl or heteroaryl shown in FormulaIX and / or X. Also disclosed herein are pharmaceutically acceptable salts of compounds of Formula XVII.

[0101]

[0103] Also disclosed herein are novel compounds of Formula XV to XVII, whereinis represented by one of the aryl or heteroaryl groups of Formula XVIII.

[0104] Method of Treatment

[0105] The compounds described herein, such as the compounds of Formula I to XVIII can be useful in therapy, for diseases of the lungs. One suitable embodiment can be a pharmaceutical composition for administering the compounds comprising Formula I to XVIII for use in therapy or treatment of disease. Another embodiment can be compounds comprising Formula I to XVIII or pharmaceutical compositions thereof, for use in therapy. A further embodiment is wherein the therapy is for diseases of the lungs. Another embodiment is a method of treatment of a lung disease comprising administering a compound disclosed herein, suitably a compound comprising Formula I to XVIII, suitably administered in an effective amount, or a pharmaceutical composition of any of the preceding. Another embodiment is a compound disclosed herein, suitably a compound comprising Formula I to XVIII, or a pharmaceutical composition thereof, for use in the manufacture of a medicament, suitably for the treatment of a lung disease.

[0106] Formulation

[0107] The compounds disclosed herein can be included in a pharmaceutical formulation. The pharmaceutical formulation can comprise one or more carriers, diluents, and excipients that are compatible with the compounds and other components of the composition or formulation and not deleterious to the patient. Examples of pharmaceutical compositions and processes for their preparation can be found in “Remington: The Science and Practice of Pharmacy”, Loyd, V., et al. Eds., 22nd Ed., Mack Publishing Co., 2012.

[0108] In some embodiments, the formulation can be designed to be suitable for use for pulmonary delivery. Pharmaceutically acceptable carriers for pulmonary delivery are known in the art and will vary depending on the desired location for deposition of the agent, e.g., upper or lower respiratory system, and the type of device to be used for delivery, e.g. sprayer, nebulizer, dry powder inhaler.

[0109] Definitions

[0110] As used herein, the term “αvβx” means an integrin heterodimer protein with a αv protein subunit non-covalently bonded to a β1, β2, β3, β4, β5, β6, β7, or β8protein subunit.

[0111] As used herein, the terms “a,” “an,” “the,” and similar terms used in the context of the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.

[0112] As used herein, the term “alkyl” means saturated linear or branched-chain monovalent hydrocarbon radical, containing the indicated number of carbon atoms. For example, “C1-C20alkyl” means a radical having 1-20 carbon atoms in a linear or branched arrangement.

[0113] As used herein, the term “C1-Cnalkoxy” refers to a straight, or branched chain saturated hydrocarbon containing 1 to n carbon atoms containing a terminal “O” in the chain, i.e., -O(alkyl). Examples of C1-C4alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy and butoxy.

[0114] As used, herein, the term “aryl” means a radical derived from an aromatic monocyclic ring, an aromatic polycyclic ring, or more than one aromatic monocyclic rings attached by a single covalent bond, with only carbon atoms in the monocyclic ring(s) or polycyclic ring. The aryl group can be unsubstituted or the aryl group can be substituted with from 1 to 5 suitable substituents, which are well known to a person of ordinary skill in the art. The aryl group can be referred to by the number of total carbon atoms in the monocyclic or polycyclic ring. For example, a C5 to C7aryl includes an aryl radical group with 5, 6, or 7 carbon atoms.

[0115] As used herein, the term “heteroaryl” means a radical derived from an aromatic monocyclic ring, an aromatic polycyclic ring, or more than one aromatic monocyclic rings attached by a single covalent bond, including one or more carbon atoms and one or more heteroatoms in the monocyclic or polycyclic ring. The heteroaryl group can be unsubstituted or the heteroaryl group can be substituted with from 1 to 5 suitable substituents, which are well known to person of ordinary skill in the art. The heteroaryl group can be referred to by the number of total atoms in the monocyclic or polycyclic ring. For example, a four member to seven member heteroaryl includes four, five, six, or seven members (including carbon atoms and heteroatoms).

[0116] As used herein, “antisense strand” means a single-stranded oligonucleotide that is complementary to a region of a target sequence. Likewise, and as used herein, “sensestrand” means a single-stranded oligonucleotide that is complementary to a region of an antisense strand.

[0117] As used herein, “complementary” means a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand, e.g., a hairpin) that permits the two nucleotides to form base pairs with one another. For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another. Complementary nucleotides can base pair in the Watson-Crick manner or in any other manner that allows for the formation of stable duplexes. Likewise, two nucleic acids may have regions of multiple nucleotides that are complementary with each other to form regions of complementarity, as described herein.

[0118] As used herein, “duplex,” in reference to nucleic acids or oligonucleotides, means a structure formed through complementary base pairing of two antiparallel sequences of nucleotides (i.e., in opposite directions), whether formed by two separate nucleic acid strands or by a single, folded strand (e.g., via a hairpin).

[0119] An “effective amount” refers to an amount necessary (for periods of time and for the means of administration) to achieve the desired therapeutic result. An effective amount of a RNAi agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the RNAi agent to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the RNAi agent are outweighed by the therapeutically beneficial effects.

[0120] The term “knockdown” or “expression knockdown” refers to reduced mRNA or protein expression of a gene after treatment of a reagent, e.g., a RNAi agent.

[0121] As used herein, “modified internucleotide linkage” means an internucleotide linkage having one or more chemical modifications when compared with a reference internucleotide linkage having a phosphodiester bond. A modified intemucleotide linkage can be a non-naturally occurring linkage. In some embodiments, the modified internucleotide linkage is phosphorothioate linkage.

[0122] As used herein, “modified nucleotide” refers to a nucleotide having one or more chemical modifications when compared with a corresponding reference nucleotideselected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. A modified nucleotide can have, for example, one or more chemical modification in its sugar, nucleobase, and / or phosphate group. Additionally, or alternatively, a modified nucleotide can have one or more chemical moieties conjugated to a corresponding reference nucleotide. In some embodiments, the modified nucleotide is a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide, or 2'-O-alkyl modified nucleotide. In some embodiments, the modified nucleotide has a phosphate analog, e.g., 5’-vinylphosphonate. In some embodiments, the modified nucleotide has an abasic moiety or inverted abasic moiety, e.g., a moiety shown in Table 1

[0123] Table 1 - Abasic or inverted abasic (iAb) moieties

[0124] As used herein, “nucleotide” means an organic compound having a nucleoside (a nucleobase, e.g., adenine, cytosine, guanine, thymine, or uracil, and a pentose sugar, e.g., ribose or 2'-deoxyribose) linked to a phosphate group. A “nucleotide” can serve as a monomeric unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0125] As used herein, “oligonucleotide” means a polymer of linked nucleotides, each of which can be modified or unmodified. An oligonucleotide is typically less than about 100 nucleotides in length.

[0126] As used herein, “overhang” means the unpaired nucleotide or nucleotides that protrude from the duplex structure of a double stranded oligonucleotide. An overhang may include one or more unpaired nucleotides extending from a duplex region at the 5’ terminus or 3’ terminus of a double stranded oligonucleotide. The overhang can be a 3’ or 5’ overhang on the antisense strand or sense strand of a double stranded oligonucleotide.

[0127] The term “patient”, as used herein, refers to a human patient.

[0128] As used herein, “phosphate analog” means a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, a phosphate analog is positioned at the 5’ terminal nucleotide of an oligonucleotide in place of a 5’-phosphate, which is often susceptible to enzymatic removal. A 5’ phosphate analog can include a phosphatase-resistant linkage. Examples of phosphate analogs include 5’ methylene phosphonate (5’-MP) and 5’-(E)-vinylphosphonate (5’-VP). In some embodiments, the phosphate analog is 5’-VP.

[0129] The term “% sequence identity” or “percentage sequence identity” with respect to a reference nucleic acid sequence is defined as the percentage of nucleotides, nucleosides, or nucleobases in a candidate sequence that are identical with the nucleotides, nucleosides, or nucleobases in the reference nucleic acid sequence, after optimally aligning the sequences and introducing gaps or overhangs, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software programs, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987, Supp.30, section 7.7.18, Table 7.7.1), and including BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), Clustal W2.0 or Clustal X2.0 software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Percentage of “sequence identity” can be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the nucleic acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage can becalculated by determining the number of positions at which the identical nucleotide, nucleoside, or nucleobase occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. The output is the percent identity of the subject sequence with respect to the query sequence.

[0130] As used herein, “RNAi,” “RNAi agent,” “iRNA,” “iRNA agent,” and “RNA interference agent” means an agent that mediates sequence-specific degradation of a target mRNA by RNA interference, e.g., via RNA-induced silencing complex (RISC) pathway. In some embodiments, the RNAi agent has a sense strand and an antisense strand, and the sense strand and the antisense strand form a duplex (e.g., a double stranded RNA). In some embodiments, the sense strand has a delivery moiety conjugated to the 3’ end of the sense strand or a nucleotide of the sense strand.

[0131] As used herein, “strand” refers to a single, contiguous sequence of nucleotides linked together through internucleotide linkages (e.g., phosphodiester linkages or phosphorothioate linkages). A strand can have two free ends (e.g., a 5’ end and a 3’ end).

[0132] As used herein, “subject” means a mammal, including cat, dog, mouse, rat, chimpanzee, ape, monkey, and human. Preferably the subject is a human.

[0133] As used herein, “treatment” or “treating” refers to all processes wherein there may be a slowing, controlling, delaying, or stopping of the progression of the disorders or disease disclosed herein, or ameliorating disorder or disease symptoms, but does not necessarily indicate a total elimination of all disorder or disease symptoms. Treatment includes administration of a protein or nucleic acid or vector or composition for treatment of a disease or condition in a patient, particularly in a human.EXAMPLES

[0134] Example 1. Synthesis of Integrin Targeting Ligands.

[0135] Certain abbreviations used in the following experimental details are defined as follows: “THF” refers to tetrahydrofuran; “TEMPO” refers to 2,2,6,6- tetramethylpiperidinyloxy; “NaClO” refers to sodium hypochlorite; “ACN” refers to acetonitrile; “EtOAc” refers to ethyl acetate; “[Rh(COD)Cl]2” refers to chloro(1,5- cyclooctadiene)rhodium(I) dimer; “(R)-BINAP” refers to (R)-(+)-2,2’- bis(diphenylphosphino)-1,1’-binaphthalene; “MeOH” refers to methanol; “DCE” refers to 1,2-dichloroethane; “TFA” refers to trifluoroacetic acid; “DCM” refers to dichloromethane; “DMAP” refers to 4-(dimethylamino)pyridine; “DIPEA” refers to N,N- diisopropylethylamine; “HBTU” refers to N,N,N’,N’-tetramethyl-O-(1H-benzotriazol-1- yl)uranium hexafluorophosphate; “HOBt” refers to 1-hydroxybenzotriazole; “DMF” refers to N,N-dimethylformamide; “HATU” refers to 1-[bis(dimethylamino)methylene]- 1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; “EDCI·HCl” refers to N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride; “NHS” refers to N- hydroxysuccinimide; “CuSO4·5H2O” refers to copper(II)sulfate pentahydrate; “THPTA” refers to tris(3-hydroxypropyltriazolylmethyl)amine; “Pd / C” refers to palladium on carbon; “LTQ” refers to linear ion trap; “DMSO” refers to dimethyl sulfoxide; “IPA” refers to isopropyl alcohol; “RBF” refers to round bottom flask; and “SFC” refers to supercritical fluid chromatography.

[0136] Scheme 1

[0137] Scheme 1, step A depicts a reduction of compound (1) using an appropriate reducing agent such as LiBH4in a suitable solvent such as THF to give compound (2). Step B depicts a TEMPO-catalyzed oxidation of compound (2) with an oxidizing agent such as NaClO in an appropriate solvent such as ACN to give compound (3).Scheme 2

[0138] Scheme 2, step A depicts a Wittig reaction of compound (4) with an appropriate reagent such as compound (5) in a suitable solvent such as EtOAc to give α,β-unsaturated ester compound (6).

[0139] Scheme 3

[0140] Scheme 3, step A depicts a Hayashi asymmetric 1,4-addition of arylboronic acid (7) to a crotonate ester (6) using an appropriate catalyst such as [Rh(COD)Cl]2, an appropriate chiral ligand such as (R)-BINAP and an appropriate base such as Cs2CO3in a suitable solvent such as dioxane to give compound (8). Step B depicts a Suzuki reaction of compounds (8) and boronic acid (9) using a catalyst such as 1,1'- bis(diphenylphosphino)ferrocene-palladium(II) dichloride, an appropriate base such as K2CO3, and a suitable solvent system such as a mixture of dioxane and water to give compound (10). Step C depicts a deprotection of a Boc-protecting group using an appropriate reagent such as HCl in a suitable solvent such as EtOAc to give compound (11). Step D depicts a reductive amination reaction of compounds (11) and (3) with areducing agent such as sodium triacetoxyborohydride in a suitable solvent such as MeOH to give compound (12). Scheme 4

[0141] Scheme 4, step A depicts a Hayashi asymmetric 1,4-addition of arylboronic acid (13) to a crotonate ester (6) using an appropriate catalyst such as [Rh(COD)Cl]2, an appropriate chiral ligand such as (R)-BINAP and an appropriate base such as Cs2CO3in a suitable solvent such as dioxane to give compound (14). Step B depicts a deprotection of a Boc-protecting group using an appropriate reagent such as HCl in a suitable solvent such as EtOAc to give compound (15). Step C depicts a reductive amination reaction of compounds (15) and (3) with a reducing agent such as sodium triacetoxyborohydride in a suitable solvent such as MeOH to give compound (16).Scheme 5

[0142] Scheme 5, step A depicts a Hayashi asymmetric 1,4-addition of arylboronic ester (17) to a crotonate ester (6) using an appropriate catalyst such as [Rh(COD)Cl]2, an appropriate chiral ligand such as (R)-BINAP and an appropriate base such as Cs2CO3in a suitable solvent such as dioxane to give compound (18). Step B depicts a Suzuki reaction of compounds (18) and boronic acid (9) using a catalyst such as 1,1'- bis(diphenylphosphino)ferrocene-palladium(II) dichloride, an appropriate base such as K2CO3, and a suitable solvent system such as a mixture of dioxane and water to give compound (19). Step C depicts a deprotection of a Boc-protecting group using an appropriate reagent such as HCl in a suitable solvent such as EtOAc to give compound (20). Step D depicts a reductive amination reaction of compounds (20) and (3) with a reducing agent such as sodium triacetoxyborohydride in a suitable solvent such as MeOH to give compound (21).Scheme 6

[0143] Scheme 6, step A depicts a Hayashi asymmetric 1,4-addition of arylboronic ester (22) to a crotonate ester (6) using an appropriate catalyst such as [Rh(COD)Cl]2, an appropriate chiral ligand such as (R)-BINAP and an appropriate base such as Cs2CO3in a suitable solvent such as dioxane to give compound (23). Step B depicts a demethylation reaction with a suitable Lewis acid such as BBr3in a suitable solvent such as DCE followed by step C which depicts an esterification reaction with SOCl2and MeOH to give compound (24). Step D depicts a reductive amination reaction of compounds (24) and (3) with a reducing agent such as sodium triacetoxyborohydride in a suitable solvent such as MeOH to give compound (25).Scheme 7

[0144] Scheme 7, step A depicts an ester hydrolysis of compound (26) using a base such as LiOH in a suitable solvent system such as THF and water to give compound (27a). Step B shows a deprotection of compound (27a) using a suitable acid such as TFA in a solvent such as DCM to give compound (27). X is C or N. R is OH or 4-phenol.Scheme 8

[0145] Scheme 8, step A depicts the tosylation of compound (28) with compound (29) using a suitable base such as triethylamine and a suitable catalyst such as DMAP in a solvent such as DCM to give compound (30).Scheme 9

[0146] Scheme 9, step A depicts a nucleophilic substitution reaction of compounds (31) and (30) using an appropriate base such as Cs2CO3in a suitable solvent such as DMF to give compound (32). Step B shows the ester hydrolysis of compound (32) with a base such as LiOH and in a solvent system such as MeOH and water to give compound (33a). Step C depicts a deprotection reaction using an acid such as aqueous HCl to give compound (33). X is C or N.Scheme 10

[0147] Scheme 10, step A depicts a nucleophilic substitution reaction of compounds (34) and (30) using an appropriate base such as Cs2CO3in a suitable solvent such as DMF to give compound (35). Step B shows the ester hydrolysis of compound (35) with a base such as LiOH and in a solvent system such as MeOH and water to give compound (36a). Step C depicts a deprotection reaction using an acid such as aqueous HCl to give compound (36). X is C or NScheme 11

[0148] Scheme 11, step A depicts an amide coupling between compounds (37) and di- tert-butyl (azanediylbis(ethane-2,1-diyl))dicarbamate using reagents such as HBTU and HOBt, a base such as DIPEA, and a solvent such as DMF to give compound (38). Step B shows the ester hydrolysis of compound (38) using a base such as NaOH and a solvent system such as THF and MeOH to give compound (39). Step C depicts an amide coupling between compounds (39) and ethyl 6-aminohexanoate hydrochloride using reagents such as HBTU and HOBt, a base such as DIPEA, and in a solvent such as DMF to give compound (40). Step D shows a deprotection of compound (40) using an acid such as HCl in a solvent such as diethyl ether to give compound (41). Step E depicts an amide coupling between compounds (41) and propargyl-PEG5-acid using a reagent such as HATU, a base such as DIPEA and in a solvent such as DMF to give compound (42). Step F shows an ester hydrolysis of compound (42) using a base such as NaOH in a solvent system such as THF and MeOH to give compound (43). Step G shows the coupling ofcompounds (43) and 1 -hydroxypyrrolidine-2, 5-dione using a suitable reagent such as EDCI·HCl and in a solvent such as DCM to give the succinate ester compound (44).Alternative Ligand Synthesis

[0149] The fully elaborated targeting ligand, as an NHS-ester, may be constructed before conjugation to the RNAi agent, as illustrated in Schemes 12 and 13. After conjugation of the ligand to the amine-functionalized sense strand, the methyl esters on the ligand are hydrolyzed with lithium hydroxide. The ligand-conjugated sense strand is then annealed to the antisense strand to give the RNAi agent. This approach avoids introduction of copper to the RNAi agent.Scheme 12

[0150] Scheme 11, step A depicts a copper-catalyzed azide-alkyne cycloaddition (CuAAC) of compounds (43) and (45) using a suitable copper catalyst such as CuSO4·5H2O, reagents such as THPTA and sodium ascorbate and in a solvent systemsuch as DMF and water to give compound (46). Step B shows a Boc-deprotection of compound (46) using an acid such as TFA and in a solvent such as DCM to give compound (47). Step C depicts coupling of compounds (47) and 1-hydroxypyrrolidine- 2,5-dione using a reagent such as EDCI HCl and in a solvent such as DCM to give the succinate ester compound (48).Scheme 13

[0151] Scheme 13, step A depicts a copper-catalyzed azide-alkyne cycloaddition (CuAAC) of compounds (45) and tert-butyl 4,7,10,13,16-pentaoxanonadec-18-ynoate using a suitable copper catalyst such as CuSO4·5H2O, reagents such as THPTA andsodium ascorbate and in a solvent system such as DMF and water to give compound (49). Step B shows the deprotection of compound (49) using an acid such as TFA and in a solvent such as DCM to give compound (50) and salts thereof. Step C depicts a coupling reaction of compounds (50) and 1-hydroxypyrrolidine-2,5-dione using a reagent such as EDCI·HCl and in a solvent such as DCM to give the succinate ester compound (51).

[0152] Scheme 14, step A depicts a nucleophilic substitution reaction of compounds (31) and (52) using an appropriate base such as Cs2CO3in a suitable solvent such as DMF to give compound (53). Step B shows the deprotection of compound (53) using an acid such as HCl in dioxane to give compound (54). X is C or N.Scheme 15

[0153] Scheme 15, step A depicts a nucleophilic substitution reaction of compounds (34) and (52) using an appropriate base such as Cs2CO3in a suitable solvent such as DMF to give compound (55). Step B shows the deprotection of compound (55) using an acid such as HCl in dioxane to give compound (56). X is C or N. Scheme 16

[0154] Scheme 16, step A depicts an esterification reaction of compounds (57) and (58) using an appropriate reagent such as thionyl chloride in a suitable solvent such as THF to give compound (59). Step B depicts an amide coupling reaction between compounds (59) and (39) using regents such as HBTU and HOBt, a base such asDIPEA, and a solvent such as DMF to give compound (60). Step C depicts a deprotection of compound (60) using an acid such as HC1 in dioxane to give compound (61).Scheme 17

[0155] Scheme 17, step A depicts an amide coupling reaction between compounds (54) and (61) using a suitable reagent such as HATU, a base such as DIPEA, and a solvent such as DMF to give compound (62). Step B depicts the hydrogenolysis of compound (62) with hydrogen gas, a suitable catalyst such as Pd / C and a suitable solvent such as MeOH to give compound (63). Step C shows coupling reaction of compounds (63)and 1 -hydroxypyrrolidine-2, 5-dione using a reagent such as EDCI HCl and in a solvent such as DCM to give the succinate ester compound (64). X is C or N.Scheme 18

[0156] Scheme 18, step A depicts an amide coupling reaction between compounds (56) and (61) using a suitable reagent such as HATU, a base such as DIPEA, and a solvent such as DMF to give compound (65). Step B depicts the hydrogenolysis of compound (65) with hydrogen gas, a suitable catalyst such as Pd / C and a suitable solvent such as MeOH to give compound (66). Step C shows coupling reaction of compounds (66) and 1-hydroxypyrrolidine-2,5-dione using a reagent such as EDCI·HCl and in a solvent such as DCM to give the succinate ester compound (67). X is C or N. Preparation 1

[0157] tert-Butyl 7-(4-hydroxybutyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate

[0158] To a solution of tert-butyl 7-(4-ethoxy-4-oxobutyl)-3,4-dihydro-1,8- naphthyridine-1(2H)-carboxylate (240 g, 688.8 mmol) in THF (1680 mL) was added LiBH4(2M in THF, 688.8 mL, 1378 mmol) at 0 °C. After addition, the mixture was stirred at ambient temperature for 16 hours. The mixture was cooled to 0 °C, quenched with saturated aqueous NH4Cl and extracted with EtOAc (1.5 L). The organic layer was washed with saturated aqueous sodium chloride solution (480 mL), dried over Na2SO4, filtered, and concentrated in vacuo to give the title compound as a colorless oil (204 g, 96.6%). ES / MS m / z 307.2 (M+H). Preparation 2

[0159] tert-Butyl 7-(4-oxobutyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate

[0160] A mixture of tert-butyl 7-(4-hydroxybutyl)-3,4-dihydro-1,8-naphthyridine-1(2H)- carboxylate (204 g, 666 mmol), TEMPO (20.9 g, 133 mmol) and NaHCO3(559 g, 6666mmol) in ACN (1500 mL) was degassed with N2. NaClO (820 mL, 1332 mmol) was added at 0 °C and the resulting mixture stirred at 20 °C for 2 hours. The mixture was diluted with water and extracted with EtOAc. The organic layer was washed with saturated aqueous sodium chloride solution, dried over Na2SO4, filtered, and concentrated in vacuo to give the title compound as a yellow oil (141 g, 69.4%). ES / MS m / z 323.1 (M+18). Preparation 3

[0161] Methyl (E)-4-((tert-butoxycarbonyl)(methyl)amino)but-2-enoate

[0162] To a 3-necked flask containing a solution of tert-butyl methyl(2- oxoethyl)carbamate (700 g, 4.04 mol) in EtOAc (4.9 L) was added methyl (triphenylphosphoranylidene)acetate (2.33 kg, 6.78 mol) in portions at 25 °C. The mixture was stirred for 1 hour under argon atmosphere and filtered. The filtrate was concentrated in vacuo to give a residue which was purified by silica gel flash chromatography eluting with 5-20% EtOAc in PE to give the title compound as a colorless oil (730 g, 78.8%).1H NMR (CDCl3) δ 6.85-6.74 (m, 1H), 5.80-5.76 (m, 1H), 3.90 (bs, 2H), 3.66 (s, 3H), 2.77 (s, 3H), 1.37 (s, 9H). Preparation 4

[0163] Methyl 3-(3-bromophenyl)-4-((tert-butoxycarbonyl)(methyl)amino)butanoate

[0164] To a solution of methyl (E)-4-((tert-butoxycarbonyl)(methyl)amino)but-2-enoate (55.0 g, 239.9 mmol) in 1,4-dioxane (385 mL) was added 3-bromophenylboronic acid (96.3 g, 479 mmol), (R)-BINAP (29.8 g, 23.9 mmol), [Rh(COD)Cl]2(2.37 g, 4.80 mmol), and Cs2CO3(152 g, 467 mmol). The mixture was stirred at 90 °C for 12 hours. The cooled mixture was quenched with water (100 mL) and extracted with EtOAc (330mL). The organic layer was washed with saturated aqueous sodium chloride solution (100 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue was purified by reverse phased flash chromatography to give the title compound as a yellow oil (77.0 g, 83.1%). ES / MS m / z 287.0 ([M-Boc]+H). Preparation 5

[0165] Methyl (S)-4-((tert-butoxycarbonyl)(methyl)amino)-3-(4'-hydroxy-[1,1'- biphenyl]-3-yl)butanoate (isomer 2)

[0166] To a mixture of methyl 3-(3-bromophenyl)-4-((tert- butoxycarbonyl)(methyl)amino)butanoate (75.0 g, 194 mmol) in 1,4-dioxane (400 mL) and water (100 mL) was added 1,1'-bis(diphenylphosphino)ferrocene- palladium(II) dichloride (14.0 g, 19.4 mmol), (4-hydroxyphenyl)boronic acid (40.1 g, 291 mmol), and K2CO3(53.6 g, 388 mmol). The mixture was stirred at 100 °C for 12 hours. The cooled mixture was diluted with water and extracted with EtOAc (300 mL). The organic layer was washed with saturated aqueous sodium chloride solution, dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue was purified by flash silica gel chromatography, eluting with 0-80% EtOAc in PE to give a yellow oil (75.0 g, 96.7%, 90% ee). This was further purified by chiral SFC (DAICEL Chiralpak IC 250 x 50mm column, 10um; 25% IPA / CO2, isocratic, retention time 1.86 minutes) to give the title compound as a yellow oil, as the second eluting isomer (45.0 g, 60.0%, 99.98% ee). ES / MS m / z 400.3 (M+H). While not wishing to be bound by theory it is believed that the compound has an S stereocenter based on literature precedent (J. Med. Chem.2018, 61, 8417-8443).

[0167] Preparation 6

[0168] Methyl (S)-3-(4'-hydroxy-[1,1'-biphenyl]-3-yl)-4-(methylamino)butanoate hydrochloride

[0169] To a solution of methyl (S)-4-((tert-butoxycarbonyl)(methyl)amino)-3-(4'- hydroxy-[1,1'-biphenyl]-3-yl)butanoate (isomer 2, 45.0 g, 112.6 mmol) in EtOAc (90 mL) was added HCl (4M in EtOAc, 360 mL, 1.44 mol). The mixture was stirred at 25 °C for 1 hour and concentrated in vacuo to give the title compound as a white solid (27 g, 71.3%). ES / MS m / z 300.1 ([M-HCl]+H). Preparation 7

[0170] tert-Butyl (S)-7-(4-((2-(4'-hydroxy-[1,1'-biphenyl]-3-yl)-4-methoxy-4- oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate

[0171] A mixture of methyl (S)-3-(4'-hydroxy-[1,1'-biphenyl]-3-yl)-4- (methylamino)butanoate hydrochloride (18.0 g, 53.6 mmol) and tert-butyl 7-(4- oxobutyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (22.8 g, 74.9 mmol) in MeOH (125 mL) was degassed with N2. The mixture was stirred for 30 minutes at 25 °C. Sodium triacetoxyborohydride (34.2 g, 161.4 mmol) was added in portions at 0 °C. After addition, the mixture was stirred at 25 °C for 1.5 hours. The mixture was poured into saturated aqueous NaHCO3(200 mL) and extracted with EtOAc (2x200 mL). The organic layer was washed with saturated aqueous sodium chloride solution, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash silica gelchromatography, eluting with 0-10% MeOH in EtOAc to give the title compound as a brown solid (20 g, 63.5%). ES / MS m / z 588.4 (M+H). Preparation 8

[0172] Methyl (S)-4-((tert-butoxycarbonyl)(methyl)amino)-3-(3- hydroxyphenyl)butanoate (isomer 2)The title compound was prepared from methyl (E)-4-((tert- butoxycarbonyl)(methyl)amino)but-2-enoate (80.0 g, 348.9 mmol) and (3- hydroxyphenyl)boronic acid (96.3 g, 698.2 mmol) in a manner essentially analogous to preparation 4 to give 100 g (88.6%) of yellow oil, 50 g of which was further purified by chiral SFC (DAICEL Chiralpak IC 50 x 250mm column, 10um; 20% IPA / CO2, 0.1% NH3H2O, isocratic, retention time 2.096 minutes) to give the title compound as a yellow oil, as the second eluting isomer (45 g, 90% , 99.52% ee). ES / MS m / z 224.2 ([M-Boc]+H). While not wishing to be bound by theory it is believed that the compound has an S stereocenter based on literature precedent (J. Med. Chem.2018, 61, 8417-8443).

[0173] Preparation 9

[0174] Methyl (S)-3-(3-hydroxyphenyl)-4-(methylamino)butanoate hydrochloride

[0175] The title compound was prepared from methyl (S)-4-((tert- butoxycarbonyl)(methyl)amino)-3-(3-hydroxyphenyl)butanoate (isomer 1, 45 g, 139.2 mmol) in a manner essentially analogous to preparation 6 to give the title compound as a yellow oil (34 g, 94.2%). ES / MS m / z 224.1 ([M-HCl]+H).Preparation 10

[0176] tert-Butyl (S)-7-(4-((2-(3-hydroxyphenyl)-4-methoxy-4- oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate

[0177] The title compound was prepared from methyl (S)-3-(3-hydroxyphenyl)-4- (methylamino)butanoate hydrochloride (18.0 g, 69.3 mmol) and tert-butyl 7-(4- oxobutyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (27.42 g, 90.1 mmol) in a manner essentially analogous to preparation 7 to give the title compound as a yellow, oily solid (20.0 g, 56.4%). ES / MS m / z 512.3 (M+H). Preparation 11

[0178] Methyl 3-(6-bromopyridin-2-yl)-4-((tert- butoxycarbonyl)(methyl)amino)butanoate

[0179] The title compound was prepared from methyl (E)-4-((tert- butoxycarbonyl)(methyl)amino)but-2-enoate (61.2 g, 266.9 mmol) and 2-bromo-6- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (152 g, 535.3 mmol) in a manner essentially analogous to preparation 4 to give the title compound as yellow oil (34.4 g, 33.3%). ES / MS m / z 389.2 (M+H).Preparation 12

[0180] Methyl (R)-4-((tert-butoxycarbonyl)(methyl)amino)-3-(6-(4- hydroxyphenyl)pyridin-2-yl)butanoate (isomer 2)The title compound was prepared from methyl 3-(6-bromopyridin-2-yl)-4-((tert- butoxycarbonyl)(methyl)amino)butanoate (34.4 g, 88.8 mmol) and (4- hydroxyphenyl)boronic acid (24.5 g, 177.7 mmol) in a manner essentially analogous to preparation 5 to give the title compound as a brown oil, as the second eluting isomer (27 g, 75.8%, 99.8% ee). ES / MS m / z 401.3 (M+H). While not wishing to be bound by theory it is believed that the compound has an R stereocenter based on literature precedent (J. Med. Chem.2018, 61, 8417-8443). Preparation 13

[0181] Methyl (R)-3-(6-(4-hydroxyphenyl)pyridin-2-yl)-4-(methylamino)butanoate hydrochlorideThe title compound was prepared from methyl (R)-4-((tert- butoxycarbonyl)(methyl)amino)-3-(6-(4-hydroxyphenyl)pyridin-2-yl)butanoate (isomer 2, 27.0 g, 67.5 mmol) in a manner essentially analogous to preparation 6 to give the title compound as a yellow solid (24.5 g, 99+%). ES / MS m / z 301.0 ([M- HCl]+H).Preparation 14 tert-Butyl (R)-7-(4-((2-(6-(4-hydroxyphenyl)pyridin-2-yl)-4-methoxy-4- oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylateThe title compound was prepared from methyl (R)-3-(6-(4-hydroxyphenyl)pyridin-2- yl)-4-(methylamino)butanoate hydrochloride (24.5 g, 72.7 mmol) and tert-butyl 7-(4- oxobutyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (41.1 g, 135 mmol) in a manner essentially analogous to preparation 7 to give the title compound as a brown solid (17.4 g, 40.6%). ES / MS m / z 589.4 (M+H). Preparation 15 Methyl (S)-4-((tert-butoxycarbonyl)(methyl)amino)-3-(5-methoxypyridin-3- yl)butanoate (isomer 2)The title compound was prepared from methyl (E)-4-((tert- butoxycarbonyl)(methyl)amino)but-2-enoate (60 g, 261.7 mmol) and 3-methoxy-5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (123 g, 523.2 mmol) in a manner essentially analogous to preparation 4 to give 46 g (51.9%, 90% ee) of yellow oil which was further purified by chiral SFC (DAICEL Chiralpak IC 50 x 250mm column, 10um; 20% MeOH / CO2, 0.1% NH3H2O, isocratic, retention time 2.43 minutes) to give the title compound as a yellow oil, as the second eluting isomer (38 g, 82.6%, 97.8% ee). ES / MS m / z 339.2 (M+H). While not wishing to be bound by theory it is believed that the compound has an S stereocenter based on literature precedent (J. Med. Chem.2018, 61, 8417-8443).Preparations 16 & 17 Methyl (S)-3-(5-hydroxypyridin-3-yl)-4-(methylamino)butanoate hydrochlorideTo a solution of methyl (S)-4-((tert-butoxycarbonyl)(methyl)amino)-3-(5- methoxypyridin-3-yl)butanoate (isomer 2, 38 g, 112.2 mmol) in DCE (210 mL) was added BBr3(63.9 mL, 673.2 mmol). The mixture was stirred at 25 °C for 12 hours and quenched by addition of MeOH (210 mL). The mixture was concentrated in vacuo to give a mixture of desired product and acid byproduct (hydrolysis of the methyl ester). This mixture was dissolved in MeOH (105 mL) and degassed with N2. To the mixture was added SOCl2(31.2 ml, 430 mmol) dropwise. The reaction mixture was stirred at 45 °C for 1 hour under N2atmosphere and concentrated in vacuo to give the title compound as a purple solid (30 g, 99+%). ES / MS m / z 225.2 ([M-HCl]+H). Preparation 18 tert-Butyl (S)-7-(4-((2-(5-hydroxypyridin-3-yl)-4-methoxy-4- oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylateThe title compound was prepared from methyl (S)-3-(5-hydroxypyridin-3-yl)-4- (methylamino)butanoate hydrochloride (24 g, 92.1 mmol) and tert-butyl 7-(4- oxobutyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (22.41 g, 73.62 mmol) in a manner essentially analogous to preparation 7 to give the title compound as a brown, oily solid (20.3 g, 43%). ES / MS m / z 513.3 (M+H).Preparation 19 (S)-3-(4'-Hydroxy-[1,1'-biphenyl]-3-yl)-4-(methyl(4-(5,6,7,8-tetrahydro-1,8- naphthyridin-2-yl)butyl)amino)butanoic acidTo tert-butyl (S)-7-(4-((2-(4'-hydroxy-[1,1'-biphenyl]-3-yl)-4-methoxy-4- oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (59 mg, 0.10 mmol) in THF (1 mL) was added aqueous lithium hydroxide (1N, 0.30 mL, 0.30 mmol). The mixture was stirred at ambient temperature for 16 hours, then concentrated in vacuo. The residue was suspended in DCM (1 mL) and TFA (0.77 mL, 10 mmol) was added. The reaction mixture was stirred for 16 hours and concentrated in vacuo. The crude residue was purified by high pH preparative reversed phase chromatography, eluting with 5-100% 10mM NH4HCO3 / 5%MeOH in ACN, to give the title compound as a white powder (15.8 mg, 33%). ES / MS m / z 474.2 (M+H). Preparation 20 (S)-3-(3-Hydroxyphenyl)-4-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl)amino)butanoic acidThe title compound was prepared in a manner essentially analogous to preparation 19 using tert-butyl (S)-7-(4-((2-3-hydroxyphenyl)-4-methoxy-4- oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (100 mg, 0.195 mmol) to give the title compound as a white solid (55.4 mg, 71.3%). ES / MS m / z 398.2 (M+H).Preparation 21 (R)-3-(6-(4-Hydroxyphenyl)pyridin-2-yl)-4-(methyl(4-(5,6,7,8-tetrahydro-1,8- naphthyridin-2-yl)butyl)amino)butanoic acidThe title compound was prepared in a manner essentially analogous to preparation 19 using tert-butyl (R)-7-(4-((2-(6-(4-hydroxyphenyl)pyridin-2-yl)-4-methoxy-4- oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (50 mg, 0.085 mmol) to give the title compound as a white solid (11 mg, 27%). ES / MS m / z 475.2 (M+H). Preparation 22 (S)-3-(5-Hydroxypyridin-3-yl)-4-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl)amino)butanoic acidThe title compound was prepared in a manner essentially analogous to preparation 19 using tert-butyl (S)-7-(4-((2-(5-hydroxypyridin-3-yl)-4-methoxy-4- oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (50 mg, 0.098 mmol) to give the title compound as a white solid (20 mg, 51%). ES / MS m / z 399.2 (M+H).Preparation 23 14-Azido-3,6,9,12-tetraoxatetradecyl 4-methylbenzenesulfonateTo a stirred solution of 14-azido-3,6,9,12-tetraoxatetradecan-1-ol (69 g, 262 mmol) in DCM (500 mL) was added triethylamine (54.8 mL, 393 mmol) and DMAP (3.2 g, 26.2 mmol). The mixture was cooled to 0 °C and p-toluenesulfonyl chloride (60 g, 314 mmol) was added in portions. After 10 min, the ice bath was removed, and the mixture was stirred at ambient temperature for 16 hours. The mixture was quenched with saturated aqueous NH4Cl and stirred for 5 minutes. The layers were separated. The organic layer was washed with saturated aqueous sodium chloride solution, dried over MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with 30-60% EtOAc in cyclohexane to give the title compound as a yellow liquid (80.44 g, 73.5%). ES / MS m / z 435.4 (M+18). Preparation 24 tert-Butyl (S)-7-(4-((2-(4'-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-[1,1'-biphenyl]- 3-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine- 1(2H)-carboxylateTo a vial was added tert-butyl (S)-7-(4-((2-(4'-hydroxy-[1,1'-biphenyl]-3-yl)-4- methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)- carboxylate (700 mg, 1.19 mmol); 14-azido-3,6,9,12-tetraoxatetradecyl 4-methylbenzenesulfonate (746 mg, 1.79 mmol), cesium carbonate (582 mg, 1.79 mmol), and DMF (6 mL). The mixture was degassed with N2 and stirred at 40 °C for 3 hours. The cooled mixture was diluted with water and extracted three times with EtOAc. The organic layer was washed with water, saturated aqueous sodium chloride solution, dried over MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with 0-100% acetone in hexanes to give the title compound as a viscous, yellow oil (777 mg, 78.3%). ES / MS m / z 834.4 (M+H). Preparation 25 (S)-3-(4'-((14-Azido-3,6,9,12-tetraoxatetradecyl)oxy)-[1,1'-biphenyl]-3-yl)-4- (methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)butyl)amino)butanoic acidTo tert-butyl (S)-7-(4-((2-(4'-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-[1,1'- biphenyl]-3-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8- naphthyridine-1(2H)-carboxylate (775 mg, 0.93 mmol) was added MeOH (3 mL) and aqueous lithium hydroxide (1N, 2.79 mL, 2.79 mmol). The mixture was stirred at ambient temperature for 16 hours, then concentrated in vacuo. The residue was dissolved in water, acidified with 1N aq. HCl, and concentrated in vacuo to remove the Boc-group. The crude residue was purified by high pH reversed phase chromatography, eluting with 0-100% 10mM NH4HCO3 / 5% MeOH in ACN to give the title compound as a viscous, yellow oil (415 mg, 62.1%). ES / MS m / z 720.0 (M+H).Preparation 26 (S)-3-(3-((14-Azido-3,6,9,12-tetraoxatetradecyl)oxy)phenyl)-4-(methyl(4-(5,6,7,8- tetrahydro-1,8-naphthyridin-2-yl)butyl)amino)butanoic acidThe title compound was prepared in a manner essentially analogous to preparations 24 and 25 using tert-butyl (S)-7-(4-((2-(3-((14-azido-3,6,9,12- tetraoxatetradecyl)oxy)phenyl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4- dihydro-1,8-naphthyridine-1(2H)-carboxylate (1.29 g, 2.52 mmol) and 14-azido- 3,6,9,12-tetraoxatetradecyl 4-methylbenzenesulfonate (1.58 g, 3.78 mmol) to give the title compound as a viscous, yellow oil (711 mg, 43.9% over two steps). ES / MS m / z 644.2 (M+H). Preparation 27 (R)-3-(6-(4-((14-Azido-3,6,9,12-tetraoxatetradecyl)oxy)phenyl)pyridin-2-yl)-4- (methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)butyl)amino)butanoic acidThe title compound was prepared in a manner essentially analogous to preparations 24 and 25 using tert-butyl (R)-7-(4-((2-(6-(4-hydroxyphenyl)pyridin-2-yl)-4-methoxy-4- oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (300 mg, 0.510 mmol) and 14-azido-3,6,9,12-tetraoxatetradecyl 4- methylbenzenesulfonate (319 mg, 0.764 mmol) to give the title compound as a viscous, yellow oil (246 mg, 67% over two steps). ES / MS m / z 721.4 (M+H).Preparation 28 (S)-3-(5-((14-Azido-3,6,9,12-tetraoxatetradecyl)oxy)pyridin-3-yl)-4-(methyl(4- (5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)butyl)amino)butanoic acidThe title compound was prepared in a manner essentially analogous to preparations 24 and 25, using tert-butyl (S)-7-(4-((2-(5-hydroxypyridin-3-yl)-4-methoxy-4- oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (1.00 g, 1.95 mmol) and 14-azido-3,6,9,12-tetraoxatetradecyl 4- methylbenzenesulfonate (1.22 g, 2.93 mmol), to give the title compound as a viscous, yellow oil (548 mg, 43.6% over two steps). ES / MS m / z 645.2 (M+H). Preparation 29 Methyl N2-(tert-butoxycarbonyl)-N5,N5-bis(2-((tert-butoxycarbonyl)amino)ethyl)-L- glutaminateTo a mixture of Boc-Glu-OMe (11.0 g, 42.1 mmol), HBTU (18.4 g, 48.4 mmol), and HOBt (6.54 g, 48.4 mmol) in DMF (300 mL) was added DIPEA (21.8 mL, 126 mmol). The mixture was stirred at ambient temperature for 5 min, then di- tert-butyl (azanediylbis(ethane-2,1-diyl))dicarbamate (14.1 g, 46.3 mmol) was added. The reaction mixture was stirred under N2 atmosphere for 14 hours. The mixture was diluted with EtOAc and washed with water and saturated aqueous sodium chloridesolution. The organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography, eluting with 0-20% MeOH in DCM to give the title compound as an off-white foam (17.64 g, 76.6%). ES / MS m / z 447.4 ([M-Boc]+H). Preparation 30 N2-(tert-Butoxycarbonyl)-N5,N5-bis(2-((tert-butoxycarbonyl)amino)ethyl)-L- glutamineMethyl N2-(tert-butoxycarbonyl)-N5,N5-bis(2-((tert-butoxycarbonyl)amino)ethyl)-L- glutaminate (17.6 g, 32.2 mmol) was dissolved in THF (100 mL) and MeOH (100 mL). Aqueous sodium hydroxide (1N, 96.6 mL, 96.6 mmol) was added and the mixture was stirred at ambient temperature for 1 hour. The mixture was concentrated in vacuo to remove the organics. The residue was acidified to pH 3 with 5N HCl and extracted three times with DCM. The organic layer was washed with saturated aqueous sodium chloride solution, dried over MgSO4, filtered, and concentrated in vacuo to give the title compound as an off-white foam (14.54 g, 84.8%). ES / MS m / z 433.4 ([M-Boc]+H).Preparation 31 Ethyl (S)-12-((tert-butoxycarbonyl)amino)-8-(2-((tert-butoxycarbonyl)amino)ethyl)- 2,2-dimethyl-4,9,13-trioxo-3-oxa-5,8,14-triazaicosan-20-oateThe title compound was prepared in a manner essentially analogous to preparation 29, using N2-(tert-butoxycarbonyl)-N5,N5-bis(2-((tert-butoxycarbonyl)amino)ethyl)-L- glutamine (14.5 g, 27.2 mmol) and ethyl 6-aminohexanoate hydrochloride (5.86 g, 29.9 mmol), to give the title compound as an off-white foam (9.16 g, 49.9%). ES / MS m / z 674.4 (M+H). Preparation 32 Ethyl (S)-6-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanamido)hexanoate trihydrochlorideHCl (4N in 1,4-dioxane) (51 mL, 200 mol) was added to ethyl (S)-12-((tert- butoxycarbonyl)amino)-8-(2-((tert-butoxycarbonyl)amino)ethyl)-2,2-dimethyl-4,9,13- trioxo-3-oxa-5,8,14-triazaicosan-20-oate (9.1 g, 14 mmol) and the mixture was stirred at ambient temperature. After 10 min the mixture became a thick suspension. Added 50 mL of diethyl ether to make suspension easier to stir. After 1 hour, the mixture was concentrated in vacuo to give the title compound as an off-white solid (6.12 g, 94%). ES / MS m / z 374.4 ([M-3HCl]+H).Preparation 33 Ethyl (S)-27-(4,7,10,13,16-pentaoxanonadec-18-ynamido)-19,24,28-trioxo-23-(4- oxo-7,10,13,16,19-pentaoxa-3-azadocos-21-yn-1-yl)-4,7,10,13,16-pentaoxa-20,23,29- triazapentatriacont-1-yn-35-oateTo a mixture of ethyl (S)-6-(2-amino-5-(bis(2-aminoethyl)amino)-5- oxopentanamido)hexanoate trihydrochloride (6.12 g, 12.7 mmol) and propargyl- PEG5-acid (13.5 g, 44.4 mmol) in DMF (180 mL) was added HATU (16.9 g, 44.4 mmol) and DIPEA (26.3 mL, 152 mmol). The mixture was stirred at ambient temperature for 16 hours. The mixture was diluted with water and extracted three times with DCM. The organic layer was washed successively with water, 1N HCl, saturated aqueous NaHCO3, and saturated aqueous sodium chloride solution. The organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with 0-100% EtOAc in hexanes then 0-20% MeOH in DCM to give the title compound as a viscous, yellow oil (12.03 g, 77%). ES / MS m / z 625.5 ([M+18] / 2).Preparation 34 (S)-27-(4,7,10,13,16-Pentaoxanonadec-18-ynamido)-19,24,28-trioxo-23-(4-oxo- 7,10,13,16,19-pentaoxa-3-azadocos-21-yn-1-yl)-4,7,10,13,16-pentaoxa-20,23,29- triazapentatriacont-1-yn-35-oic acidThe title compound was prepared in a manner essentially analogous to preparation 30, using ethyl (S)-27-(4,7,10,13,16-pentaoxanonadec-18-ynamido)-19,24,28-trioxo-23- (4-oxo-7,10,13,16,19-pentaoxa-3-azadocos-21-yn-1-yl)-4,7,10,13,16-pentaoxa- 20,23,29-triazapentatriacont-1-yn-35-oate (12.0 g, 9.74 mmol) and aqueous LiOH (1N, 48.7 mL, 48.7 mmol), to give the title compound as a viscous, tan oil (11.4 g, 97.1%). ES / MS m / z 602.9 (M / 2+H).Preparation 35 2,5-Dioxopyrrolidin-1-yl (S)-27-(4,7,10,13,16-pentaoxanonadec-18-ynamido)- 19,24,28-trioxo-23-(4-oxo-7,10,13,16,19-pentaoxa-3-azadocos-21-yn-1-yl)- 4,7,10,13,16-pentaoxa-20,23,29-triazapentatriacont-1-yn-35-oateA mixture of (S)-27-(4,7,10,13,16-pentaoxanonadec-18-ynamido)-19,24,28-trioxo- 23-(4-oxo-7,10,13,16,19-pentaoxa-3-azadocos-21-yn-1-yl)-4,7,10,13,16-pentaoxa- 20,23,29-triazapentatriacont-1-yn-35-oic acid (300 mg, 0.249 mmol), 1- hydroxypyrrolidine-2,5-dione (57.3 mg, 0.498 mmol) and EDCI-HCl (95.5 mg, 0.498 mmol) in DCM (3 mL) was stirred at ambient temperature for 3 hours. The mixture was purified by silica gel flash chromatography, eluting with 0-20% MeOH in DCM to give the title compound as a colorless oil (321 mg, 79%). ES / MS m / z 651.5 (M / 2+H).Preparation 36 (S)-1-(1-(14-((3'-((S)-1-((4-(8-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-1,8- naphthyridin-2-yl)butyl)(methyl)amino)-4-methoxy-4-oxobutan-2-yl)-[1,1'-biphenyl]- 4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-21-(1-(1-(14-((3'-((S)-1- ((4-(8-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl)(methyl)amino)-4-methoxy-4-oxobutan-2-yl)-[1,1'-biphenyl]-4-yl)oxy)- 3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-17-oxo-2,5,8,11,14-pentaoxa-18- azaicosan-20-yl)-25-(1-(1-(14-((3'-((S)-1-((4-(8-(tert-butoxycarbonyl)-5,6,7,8- tetrahydro-1,8-naphthyridin-2-yl)butyl)(methyl)amino)-4-methoxy-4-oxobutan-2-yl)- [1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)- 2,5,8,11,14-pentaoxaheptadecan-17-amido)-17,22,26-trioxo-2,5,8,11,14-pentaoxa- 18,21,27-triazatritriacontan-33-oic acidTo (S)-27-(4,7,10,13,16-pentaoxanonadec-18-ynamido)-19,24,28-trioxo-23-(4-oxo- 7,10,13,16,19-pentaoxa-3-azadocos-21-yn-1-yl)-4,7,10,13,16-pentaoxa-20,23,29- triazapentatriacont-1-yn-35-oic acid (5.60 g, 4.65 mmol) and tert-butyl (S)-7-(4-((2- (4'-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-[1,1'-biphenyl]-3-yl)-4-methoxy-4- oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate(12.4 g, 14.9 mmol) was added DMF (80 mL). A solution of sodium ascorbate (553 mg, 2.79 mmol) and THPTA (1.21 g, 2.79 mmol) in water (6 mL) was added, followed by a solution of copper(II) sulfate pentahydrate (348 mg, 1.39 mmol) in water (4 mL). The mixture was stirred at ambient temperature. After 30 minutes, the mixture was diluted with water and extracted 3x with DCM. The organic layer was washed twice with saturated aqueous sodium chloride solution and twice with a mixture of saturated aqueous sodium chloride solution and 50mM EDTA, dried over MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by flash silica gel chromatography, eluting with 0-30% MeOH in DCM, to give the title compound as a viscous, yellow oil (12.69 g, 68%). ES / MS m / z 741.6 (M / 7+H).Preparation 37 (S)-1-(1-(14-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl)amino)-4-oxobutan-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12- tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-21-(1-(1-(14-((3'-((S)-4-methoxy-1- (methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)butyl)amino)-4-oxobutan-2-yl)- [1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-17-oxo- 2,5,8,11,14-pentaoxa-18-azaicosan-20-yl)-25-(1-(1-(14-((3'-((S)-4-methoxy-1- (methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)butyl)amino)-4-oxobutan-2-yl)- [1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)- 2,5,8,11,14-pentaoxaheptadecan-17-amido)-17,22,26-trioxo-2,5,8,11,14-pentaoxa- 18,21,27-triazatritriacontan-33-oic acid(S)-1-(1-(14-((3'-((S)-1-((4-(8-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-1,8- naphthyridin-2-yl)butyl)(methyl)amino)-4-methoxy-4-oxobutan-2-yl)-[1,1'-biphenyl]- 4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-21-(1-(1-(14-((3'-((S)-1- ((4-(8-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl)(methyl)amino)-4-methoxy-4-oxobutan-2-yl)-[1,1'-biphenyl]-4-yl)oxy)- 3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-17-oxo-2,5,8,11,14-pentaoxa-18-azaicosan-20-yl)-25-(1-(1-(14-((3'-((S)-1-((4-(8-(tert-butoxycarbonyl)-5,6,7,8- tetrahydro-1,8-naphthyridin-2-yl)butyl)(methyl)amino)-4-methoxy-4-oxobutan-2-yl)- [1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)- 2,5,8,11,14-pentaoxaheptadecan-17-amido)-17,22,26-trioxo-2,5,8,11,14-pentaoxa- 18,21,27-triazatritriacontan-33-oic acid (11.7 g, 3.16 mmol) was dissolved in DCM (50 mL) and TFA (24.3 mL, 316 mmol) was added. The mixture was stirred at ambient temperature under N2atmosphere. After 14 hours, the mixture was concentrated in vacuo. The residue was diluted with DCM and washed twice with water. The organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by low pH flash reversed phase chromatography. Appropriate fractions were pooled and concentrated in vacuo to remove the organics. The aqueous layer was diluted with saturated aqueous sodium chloride solution and extracted 3x with DCM. The organic layer was dried over MgSO4, filtered, and concentrated in vacuo to give the title compound as a yellow foam (8.23 g, 76.6%). ES / MS m / z 681.6 (M / 6+H).Preparation 38 2,5-Dioxopyrrolidin-1-yl (S)-1-(1-(14-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8- tetrahydro-1,8-naphthyridin-2-yl)butyl)amino)-4-oxobutan-2-yl)-[1,1'-biphenyl]-4- yl)oxy)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-21-(1-(1-(14-((3'-((S)-4- methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)butyl)amino)-4- oxobutan-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3- triazol-4-yl)-17-oxo-2,5,8,11,14-pentaoxa-18-azaicosan-20-yl)-25-(1-(1-(14-((3'-((S)- 4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)butyl)amino)-4- oxobutan-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3- triazol-4-yl)-2,5,8,11,14-pentaoxaheptadecan-17-amido)-17,22,26-trioxo-2,5,8,11,14- pentaoxa-18,21,27-triazatritriacontan-33-oateTo (S)-1-(1-(14-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8- naphthyridin-2-yl)butyl)amino)-4-oxobutan-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12- tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-21-(1-(1-(14-((3'-((S)-4-methoxy-1- (methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)butyl)amino)-4-oxobutan-2-yl)- [1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-17-oxo- 2,5,8,11,14-pentaoxa-18-azaicosan-20-yl)-25-(1-(1-(14-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)butyl)amino)-4-oxobutan-2-yl)- [1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)- 2,5,8,11,14-pentaoxaheptadecan-17-amido)-17,22,26-trioxo-2,5,8,11,14-pentaoxa- 18,21,27-triazatritriacontan-33-oic acid (8.23 g, 2.42 mmol) was added 1- hydroxypyrrolidine-2,5-dione (835 mg, 7.25 mmol), EDCI (1.39 g, 7.25 mmol), and DCM (60 mL). The mixture was stirred at ambient temperature under N2 atmosphere. After 5 hours, the mixture was purified by flash silica gel chromatography eluting with 0-50% MeOH in DCM. The product was dissolved in DCM and washed 3x with saturated aqueous sodium chloride solution, dried over MgSO4, filtered, and concentrated in vacuo to give the title compound as a pale, yellow foam (6.72 g, 79.4%). ES / MS m / z 584.4 (M / 6+H). Preparation 39 tert-Butyl (S)-7-(4-((2-(4'-((14-(4-(19,19-dimethyl-17-oxo-2,5,8,11,14,18- hexaoxaicosyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12-tetraoxatetradecyl)oxy)-[1,1'- biphenyl]-3-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8- naphthyridine-1(2H)-carboxylatetert-Butyl (S)-7-(4-((2-(4'-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-[1,1'-biphenyl]- 3-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine- 1(2H)-carboxylate (8.4 g, 10 mmol) and tert-butyl 4,7,10,13,16-pentaoxanonadec-18- ynoate (3.8 g, 11 mmol) were dissolved in DMF (50 mL). A solution of sodium ascorbate (0.40 g, 2.0 mmol) and THPTA (0.88 g, 2.0 mmol) in water (4 mL) was added, followed by a solution of copper(II) sulfate pentahydrate (0.25 g, 1.0 mmol) in water (1 mL). The mixture was stirred at ambient temperature under N2 atmosphere. After 30 minutes, the mixture was diluted with water and extracted twice with DCM. The organic layer was washed three times with water (added saturatedaqueous sodium chloride solution to break emulsion), dried over MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with 0-20% MeOH in DCM to give the title compound as a viscous, yellow oil (9.69 g, 81%). ES / MS m / z 597.9 (M / 2+H). Preparation 40 (S)-1-(1-(14-((3'-(4-Methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl)amino)-4-oxobutan-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12- tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-2,5,8,11,14-pentaoxaheptadecan-17-oic acidtert-Butyl (S)-7-(4-((2-(4'-((14-(4-(19,19-dimethyl-17-oxo-2,5,8,11,14,18- hexaoxaicosyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12-tetraoxatetradecyl)oxy)-[1,1'- biphenyl]-3-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8- naphthyridine-1(2H)-carboxylate (9.6 g, 8.0 mmol) was dissolved in DCM (50 mL) and TFA (25 mL, 0.32 mol) was added. The mixture was stirred at ambient temperature under N2 atmosphere. After 14 hours, the mixture was concentrated in vacuo. The residue was diluted with DCM and washed twice with water. The organic layer was dried over MgSO4, filtered, and concentrated in vacuo to give the title compound as a viscous, yellow oil (10.6 g, 99+%, 87% purity). ES / MS m / z 519.5 (M / 2+H).Preparation 41 2,5-Dioxopyrrolidin-1-yl (S)-1-(1-(14-((3'-(4-methoxy-1-(methyl(4-(5,6,7,8- tetrahydro-1,8-naphthyridin-2-yl)butyl)amino)-4-oxobutan-2-yl)-[1,1'-biphenyl]-4- yl)oxy)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-2,5,8,11,14- pentaoxaheptadecan-17-oateTo (S)-1-(1-(14-((3'-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl)amino)-4-oxobutan-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12- tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-2,5,8,11,14-pentaoxaheptadecan-17-oic acid (8.3 g, 8.0 mmol) was added 1-hydroxypyrrolidine-2,5-dione (1.8 g, 16 mmol), EDCI (3.1 g, 16 mmol), and DCM (30 mL). The mixture was stirred at ambient temperature for 4 hours. The mixture was diluted with DCM and washed three times with saturated aqueous sodium chloride solution, dried over MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with 0-40% MeOH in DCM to give the title compound as a viscous, yellow oil (7.25 g, 64%). ES / MS m / z 568.0 (M / 2+H). Preparation 42 tert-Butyl (S)-7-(4-((2-(4'-((17,17-dimethyl-15-oxo-3,6,9,12,16- pentaoxaoctadecyl)oxy)-[1,1'-biphenyl]-3-yl)-4-methoxy-4- oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylateTo tert-butyl (S)-7-(4-((2-(4'-((17,17-dimethyl-15-oxo-3,6,9,12,16- pentaoxaoctadecyl)oxy)-[1,1'-biphenyl]-3-yl)-4-methoxy-4- oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (4.0 g, 6.8 mmol) and tert-butyl 1-(tosyloxy)-3,6,9,12-tetraoxapentadecan-15-oate (4.9 g, 10 mmol) in DMF (40 mL) was added Cs2CO3. The mixture was stirred at 40 °C under N2atmosphere for 3 hours. The cooled mixture was diluted with water and extracted three times with EtOAc. The organic layer was washed with water, brine, dried over MgSO4, filtered and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography eluting with 0-100% acetone in DCM to give the title compound as a viscous, yellow oil (5.09 g, 84%). ES / MS m / z 893.4 (M+H). Preparation 43 (S)-1-((3'-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl)amino)-4-oxobutan-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12- tetraoxapentadecan-15-oic acid hydrochlorideTo tert-butyl (S)-7-(4-((2-(4'-((17,17-dimethyl-15-oxo-3,6,9,12,16- pentaoxaoctadecyl)oxy)-[1,1'-biphenyl]-3-yl)-4-methoxy-4- oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate was added HCl (4 M in dioxane, 28 mL, 110 mmol). The mixture was stirred at room temperature. After 20 hours, the mixture was concentrated under reduced pressure. The residue was diluted with dioxane and concentrated under reduced pressure to give the title compound as a foam (4.8 g, quantitative yield, 90% purity). ES / MS m / z 736.4 ([M-HCl]+H). Preparation 44 (R)-1-(4-(6-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl)amino)-4-oxobutan-2-yl)pyridin-2-yl)phenoxy)-3,6,9,12-tetraoxapentadecan- 15-oic acid hydrochlorideThe title compound was prepared in a manner essentially analogous to preparations 42 and 43 using tert-butyl (R)-7-(4-((2-(6-(4-hydroxyphenyl)pyridin-2-yl)-4-methoxy-4- oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (3.0 g, 5.1 mmol) and tert-butyl 1-(tosyloxy)-3,6,9,12-tetraoxapentadecan-15-oate (2.7 g, 5.6 mmol) to give the title compound as a foam (3.18 g, 81% over two steps). ES / MS m / z 737.4 ([M-HCl]+H). Preparation 45 (S)-1-(3-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl)amino)-4-oxobutan-2-yl)phenoxy)-3,6,9,12-tetraoxapentadecan-15-oic acid hydrochlorideThe title compound was prepared in a manner essentially analogous to preparations 42 and 43 using tert-butyl (S)-7-(4-((2-(3-hydroxyphenyl)-4-methoxy-4- oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (3.4 g, 6.6 mmol) and tert-butyl 1-(tosyloxy)-3,6,9,12-tetraoxapentadecan-15-oate (3.5 g, 7.3 mmol) to give the title compound (3.11 g, 67% over two steps). ES / MS m / z 660.4 ([M-HCl]+H). Preparation 46 (S)-1-((5-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl)amino)-4-oxobutan-2-yl)pyridin-3-yl)oxy)-3,6,9,12-tetraoxapentadecan-15- oic acid hydrochlorideThe title compound was prepared in a manner essentially analogous to preparations 42 and 43 using tert-butyl (S)-7-(4-((2-(5-hydroxypyridin-3-yl)-4-methoxy-4- oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (9.0 g, 18 mmol) and tert-butyl 1-(tosyloxy)-3,6,9,12-tetraoxapentadecan-15-oate (9.2 g, 19 mmol) to give the title compound as yellow solid (8.04 g, 66% over two steps). ES / MS m / z 661.4 ([M-HCl]+H). Preparation 47 benzyl 6-aminohexanoate hydrochloride6-Aminohexanoic acid (20 g, 150 mmol) was suspended in THF (150 mL) and benzyl alcohol (190 mL, 1830 mmol) was added. The mixture was cooled to 0 °C and thionyl chloride (33 mL, 460 mol) was added as slow stream. The mixture was stirred in the ice bath while warming to rt. After 3 days, diethyl ether (1 L) was added to the mixture, stirred for 5 min and kept in -20 °C freezer for 4 h. The precipitate was collected by filtration, washed with diethyl ether (2 L) and dried under N2 to give the title compound as off-white powder (30.9 g, 79% yield). ES / MS m / z 222.4 ([M- HCl]+H). Preparation 48 benzyl (S)-12-((tert-butoxycarbonyl)amino)-8-(2-((tert-butoxycarbonyl)amino)ethyl)- 2,2-dimethyl-4,9,13-trioxo-3-oxa-5,8,14-triazaicosan-20-oateTo a RBF containing HOBt (8.23 g, 60.9 mmol) and HBTU (23.1 g, 60.9 mmol) was added a solution of N2-(tert-butoxycarbonyl)-N5,N5-bis(2-((tert- butoxycarbonyl)amino)ethyl)-L-glutamine (28.2 g, 52.9 mmol) in DMF (265 mL). DIPEA (36.6 mL, 212 mmol) was added and the mixture was stirred for 5 min, then benzyl 6-aminohexanoate hydrochloride (15.0 g, 58.2 mmol) was added. The mixture was stirred overnight at room temperature. The mixture was diluted with DCM (500 mL) and washed with water (3x500mL), brine (3x500 mL), dried over MgSO4, filtered and concentrated in vacuo. The crude residue was purified by silica gel chromatography, eluting with 0-80% ethyl acetate in hexanes, then 0-20% MeOH in DCM to give the title compound as off-white foam (23 g, 59% yield). ES / MS m / z 736.2 (M+H). Preparation 49benzyl (S)-6-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanamido)hexanoate trihydrochlorideTo benzyl (S)-12-((tert-butoxycarbonyl)amino)-8-(2-((tert- butoxycarbonyl)amino)ethyl)-2,2-dimethyl-4,9,13-trioxo-3-oxa-5,8,14-triazaicosan- 20-oate (22.0 g, 29.9 mmol) was added HCl (4 N in 1,4-dioxane) (112 mL, 448 mmol) and the mixture was stirred at room temperature. After 10 minutes, the mixture became a thick suspension. Added dioxane (20 mL) and continued stirring. After 30 minutes, the suspension was diluted with diethyl ether (100 mL), stirred for 5 minutes and filtered. The solid was washed with diethyl ether (500 mL) and dried under N2to give the title compound as off-white solid (16.3 g, 100% yield). ES / MS m / z 436.4 ([M-3HCl]+H). Preparation 50 benzyl 6-[[(2S)-5-[bis[2-[3-[2-[2-[2-[2-[4-[3-[(1S*)-3-methoxy-1-[[methyl-[4- (5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)butyl]amino]methyl]-3-oxo- propyl]phenyl]phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]ethyl]amino]- 2-[3-[2-[2-[2-[2-[4-[3-[(1S*)-3-methoxy-1-[[methyl-[4-(5,6,7,8-tetrahydro-1,8- naphthyridin-2-yl)butyl]amino]methyl]-3-oxo- propyl]phenyl]phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-5-oxo- pentanoyl]amino]hexanoateBenzyl (S)-6-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanamido)hexanoate trihydrochloride (70.0 mg, 0.128 mmol) and (S)-1-((3'-(4-methoxy-1-(methyl(4- (5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)butyl)amino)-4-oxobutan-2-yl)-[1,1'- biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxapentadecan-15-oic acid hydrochloride (298 mg, 0.385 mmol) were dissolved in DMF (5 mL). HATU (171 mg, 0.450 mmol) was added, followed by DIPEA (266 uL, 1.54 mmol). The mixture was stirred at room temperature. After 4 hours, the mixture was diluted with water and extracted twice with DCM. The organic layer was washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The crude residue was purified by reverse phase chromatography (high pH then switched to low pH) to give the title compound as thick, yellow oil (113 mg, 34% yield). ES / MS m / z 648.2 (M / 4+H).Preparation 51 (S)-1-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl)amino)-4-oxobutan-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-19-(1-((3'-((S)-4- methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)butyl)amino)-4- oxobutan-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-15-oxo-3,6,9,12-tetraoxa-16-azaoctadecan- 18-yl)-23-(1-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl)amino)-4-oxobutan-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12- tetraoxapentadecan-15-amido)-15,20,24-trioxo-3,6,9,12-tetraoxa-16,19,25- triazahentriacontan-31-oic acidbenzyl 6-[[(2S)-5-[bis[2-[3-[2-[2-[2-[2-[4-[3-[(1S*)-3-methoxy-1-[[methyl-[4- (5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)butyl]amino]methyl]-3-oxo-propyl]phenyl]phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]ethyl]amino]- 2-[3-[2-[2-[2-[2-[4-[3-[(1S*)-3-methoxy-1-[[methyl-[4-(5,6,7,8-tetrahydro-1,8- naphthyridin-2-yl)butyl]amino]methyl]-3-oxo- propyl]phenyl]phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-5-oxo- pentanoyl]amino]hexanoate (110 mg, 0.0425 mmol) was dissolved in MeOH (12 mL) and added to a pressure bottle containing 10% Pd / C (50% wet) (31.6 mg, 0.0297 mmol). The bottle was sealed, and the mixture purged three times with N2, then three times with H2gas. The mixture was stirred under 40 psi of H2gas for 6 hours, filtered over celite and washed with MeOH. The filtrate was concentrated under reduced pressure to give the title compound which was used without further purification (100 mg, 94.2% yield). ES / MS m / z 625.8 (M / 4+H). Preparation 52 Dimethyl (3S*,3'S*)-3,3'-{[19-(N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}- N~2~-[15-({3'-[(2S*)-4-methoxy-1-{methyl[4-(5,6,7,8-tetrahydro-1,8-naphthyridin- 2-yl)butyl]amino}-4-oxobutan-2-yl][1,1'-biphenyl]-4-yl}oxy)-4,7,10,13- tetraoxapentadecanan-1-oyl]-l-alpha-glutaminyl)-15,23-dioxo-3,6,9,12,26,29,32,35- octaoxa-16,19,22-triazaheptatriacontane-1,37-diyl]bis(oxy[1,1'-biphenyl]-4',3- diyl)}bis(4-{methyl[4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl]amino}butanoate)(S)-1-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl)amino)-4-oxobutan-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-19-(1-((3'-((S)-4- methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)butyl)amino)-4- oxobutan-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-15-oxo-3,6,9,12-tetraoxa-16-azaoctadecan- 18-yl)-23-(1-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl)amino)-4-oxobutan-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12- tetraoxapentadecan-15-amido)-15,20,24-trioxo-3,6,9,12-tetraoxa-16,19,25- triazahentriacontan-31-oic acid (100 mg, 0.040 mmol), N-hydroxysuccinimide (9.21 mg, 0.080 mmol) and EDCI·HCl (15.3 mg, 0.080 mmol) were dissolved in DCM (1 mL). The mixture was stirred at room temperature for 12 hours and loaded directly onto a 4 g silica column and purified by flash chromatography, eluting with 0-60%MeOH in DCM to give the title compound (33 mg, 32% yield). ES / MS m / z 650.0 (M / 4+H). Preparation 53 Dimethyl (3R,3'R)-3,3'-{[19-(N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}- N~2~-[15-(4-{6-[(2R)-4-methoxy-1-{methyl[4-(5,6,7,8-tetrahydro-1,8-naphthyridin- 2-yl)butyl]amino}-4-oxobutan-2-yl]pyridin-2-yl}phenoxy)-4,7,10,13- tetraoxapentadecanan-1-oyl]-l-alpha-glutaminyl)-15,23-dioxo-3,6,9,12,26,29,32,35- octaoxa-16,19,22-triazaheptatriacontane-1,37-diyl]bis(oxy-4,1-phenylenepyridine- 6,2-diyl)}bis(4-{methyl[4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl]amino}butanoate)The title compound was prepared in a manner essentially analogous to preparations 50, 51 and 52 using (R)-1-(4-(6-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8- naphthyridin-2-yl)butyl)amino)-4-oxobutan-2-yl)pyridin-2-yl)phenoxy)-3,6,9,12- tetraoxapentadecan-15-oic acid hydrochloride (3.18 g, 4.12 mmol) and benzyl (S)-6- (2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanamido)hexanoate trihydrochloride (680 mg, 1.25 mmol) to give the title compound as white solid (2.53 g, 57.5% over three steps). ES / MS m / z 650.8 (M / 4+H). Preparation 54 Dimethyl (3S,3'S)-3,3'-[{19-[N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}- N~2~-(15-{3-[(2S)-4-methoxy-1-{methyl[4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl]amino}-4-oxobutan-2-yl]phenoxy}-4,7,10,13-tetraoxapentadecanan-1-oyl)-l- alpha-glutaminyl]-15,23-dioxo-3,6,9,12,26,29,32,35-octaoxa-16,19,22- triazaheptatriacontane-1,37-diyl}bis(oxy-3,1-phenylene)]bis(4-{methyl[4-(5,6,7,8- tetrahydro-1,8-naphthyridin-2-yl)butyl]amino}butanoate)The title compound was prepared in a manner essentially analogous to preparations 50, 51 and 52 using (S)-1-(3-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8- naphthyridin-2-yl)butyl)amino)-4-oxobutan-2-yl)phenoxy)-3,6,9,12- tetraoxapentadecan-15-oic acid hydrochloride (3.08 g.4.42 mmol) and benzyl (S)-6- (2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanamido)hexanoate trihydrochloride(730 mg, 1.34 mmol) to give the title compound as off-white solid (550 mg, 17.3% over three steps). ES / MS m / z 593.0 (M / 4+H). Preparation 55 Dimethyl (3S,3'S)-3,3'-{[19-(N-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}- N~2~-[15-({5-[(2S)-4-methoxy-1-{methyl[4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2- yl)butyl]amino}-4-oxobutan-2-yl]pyridin-3-yl}oxy)-4,7,10,13-tetraoxapentadecanan- 1-oyl]-l-alpha-glutaminyl)-15,23-dioxo-3,6,9,12,26,29,32,35-octaoxa-16,19,22- triazaheptatriacontane-1,37-diyl]bis(oxypyridine-5,3-diyl)}bis(4-{methyl[4-(5,6,7,8- tetrahydro-1,8-naphthyridin-2-yl)butyl]amino}butanoate)The title compound was prepared in a manner essentially analogous to preparations 50, 51 and 52 using (S)-1-((5-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8- naphthyridin-2-yl)butyl)amino)-4-oxobutan-2-yl)pyridin-3-yl)oxy)-3,6,9,12- tetraoxapentadecan-15-oic acid hydrochloride (7.9 g, 11 mmol) and benzyl (S)-6-(2- amino-5-(bis(2-aminoethyl)amino)-5-oxopentanamido)hexanoate trihydrochloride (2.0 g, 3.7 mmol) to give the title compound as thick, yellow oil (1.38 g, 15.9% over three steps). ES / MS m / z 593.6 (M / 4+H). Example 2. Synthesis of Integrin Targeting Ligands, RNAi Agents and Conjugation of RNAi Agents to Integrin LigandsCertain abbreviations used in the following experimental details are defined as follows: “AEX” refers to anion exchange; “MPA” refers to mobile phase A; “MPB” refers to mobile phase B; “MWCO” refers to molecular weight cut-off; “CPG” refers to crystal pore glass; “LC-MS” refers to liquid chromatography-mass spectrometry; “RNA” refers to ribonucleic acid; “DNA” refers to deoxyribonucleic acid; “UV” refers to ultraviolet; “PVDF” refers to polyvinylidene difluoride; “EDTA” refer to ethylenediaminetetraacetic acid disodium salt dihydrate; “PCR” refers to polymerase chain reaction; “SCNN1A” refers to sodium channel epithelial 1 subunit alpha; “BCP” refers to 1-bromo-3-chloropropane; “mRNA” refers to messenger RNA; “cDNA” refers to complementary DNA; “UPLC” refers to ultra-performance liquid chromatography; “DEA” refers to diethylamine; “NaOAc” refers to sodium acetate; “RNAi” refers to RNA interference; and “MLE12 Cells” refers to mouse lung epithelial cells. Preparation 56 Synthesis of sense strands The single sense strands were synthesized on solid support with a K&A H8-SE DNA / RNA synthesizer (K&A Laborgeraete GbR). The oligonucleotides were synthesized via phosphoramidite chemistry at either 10, 50 or 100 µmol scales. The solid support was 3'-PT-Amino-Modifier C6 CPG (Chemgenes, Catalog No. N-8217- 05) or Universal UnyLinker CPG (Chemgenes, Catalog No. N-4000-05) and was purchased commercially. Standard reagents were used in the oligo synthesis (Table 4), where 0.1M xanthane hydride in pyridine was used as the sulfurization reagent and 20% DEA in ACN was used as an auxiliary wash post synthesis. All monomers (Table 5) were made at 0.1M in ACN and contained a molecular sieves trap bag. A TFA-hexylaminolinker phosphoramidite (CAS#: 133975-85-6) was coupled onto the 5’ end to produce a C6 amino terminal linker. The oligonucleotides sense strands were cleaved and deprotected (C / D) using 28-30% ammonia hydroxide: 40% methyl amine (1:1) at ambient temperature for 2 hours. The CPG was filtered via 0.45 um PVDF syringeless filter, 0.22 um PVDF Steriflip® vacuum filtration or 0.22 um PVDF Stericup® Quick release. The filtrate was partially concentrated viaGenevac™. After concentration, the crude oligonucleotides were diluted with RNAse free water and filtered a second time before purification on AKTA™ Pure purification system using either anion-exchange (AEX): (Conditions: ES Industry Source™ 15Q column with MPA: 20mM NaH2PO4, 15% ACN, pH 7.4 and MPB: 20 mM NaH2PO4, 1M^NaBr, 15% ACN, pH 7.4) or reversed phase chromatography: (Conditions: ES Industry SourceTM15Q column with MPA: 20mM NaOAc, 15% ACN, MPB: 80% ACN in water) . Fractions which contained a mass purity greater than 85% without individual impurities >5% were pooled. The purified material was desalted using a 3K MWCO centrifugal spin tubes or tangential flow filtration to give the title compounds as an aqueous solution shown in Tables 1 and 2. The solution absorbance at 260 nm was measured with a Thermo Scientific Nanodrop One C UV-Vis spectrophotometer. The concentration was then calculated using the measured absorbance and calculated extinction coefficient. The product purity was analyzed by UPLC and LC-MS. Table 2 – SCNN1A sense strandsPreparation 57 Synthesis of antisense strands The antisense strands were prepared according to the protocol described in Preparation 56 using standard CPG (mC: Chemgenes Catalog#: N-7905-05 or mG: Chemgenes Catalog#: N-7912-05). The title compounds were provided as an aqueous solution shown in Table 3. Table 3 – SCNN1A antisense strandsTable 4 – Oligonucleotide Synthesis ReagentsTable 5 – PhosphoramiditesPreparation 58 Conjugation of Amine-functionalized Sense Strand An aqueous solution of the amine-functionalized sense strands from Preparation 56 were buffered with phosphate buffer (0.5M, pH 7.5, 10% v / v of sense strand). A solution of NHS-ester (50mM in ACN, 5.0 eq per C6Am) was added. The mixture was shaken at 1000 rpm for 4-16 hours at ambient temperature. For preparations 58-4, 58-5, 58-6, 58-7, 58-8, 58-9, 58-10 and 58-11, aqueous LiOH (200 eq, 1N) was addedand shaken for an additional 1-2 hours. The conjugate was purified by either anion exchange or reverse phase chromatography. The purified material was desalted using a 3K MWCO centrifugal spin tubes to give the title compounds as an aqueous solution as shown in Table 6.Preparation 59 Annealing of Sense and Antisense Strands Equimolar amounts of sense strands and complimentary antisense strands, as a solution in RNAse-free water, were mixed and vortexed for 20 seconds (Table 7). Unconjugated duplex: the solution was shaken at 90 °C for 2 min and allowed to sit and cool to 25 °C over 45 minutes. The solution was lyophilized and reconstituted in 1X PBS. Targeting Ligand and Alkyne-functionalized duplex: the solution was lyophilized or concentrated on Genevac evaporator and reconstituted in RNAse-free water to a concentration ≥50 mg / mL. Duplex concentration was calculated from the measured solution absorbance (260 nm) and extinction coefficient, corrected by a factor of 0.85. Purity was analyzed by UPLC and LC-MS. For in vivo samples, endotoxin levels were measured with Charles River Endosafe® Device (upper limit 2 EU / mg). Table 7 – Annealing of Sense and Antisense StrandsPreparation 60 Conjugation of Targeting Ligands to Alkyne-Functionalized Duplex The alkyne-functionalized duplexes (Table 7, preparations 59-2; 59-3 and 59-4) were conjugated to the targeting ligands (Table 8) via a copper-catalyzed Click reaction (CuAAC) as follows: stock solutions of copper(II) sulfate pentahydrate (0.5M), THPTA (0.5 M), and sodium ascorbate (2M) were prepared in RNAse-free water. A 0.1M solution of targeting ligand in DMSO and 50 mg / mL solution of duplex in RNAse-free water were prepared. To a falcon tube containing the alkyne- functionalized duplex was added DMSO (0.1M, ~8 eq per alkyne). A solution of the targeting ligand in DMSO (2 eq per alkyne) was added and vortexed. Borate buffer (50mM, pH 8.5, 2 eq per alkyne) was added and vortexed. The solution was placed in a shaker cooled to 10 °C. A 4:1 v / v mixture of THPTA and copper(II) sulfate pentahydrate (2 eq per alkyne) was added and vortexed. Sodium ascorbate (16 eq per alkyne) was added immediately, and the reaction mixture shaken at 10 °C until completion (30-100 minutes). After completion, as determined by IP-RP LCMS, EDTA (0.05M, 3.5 eq per alkyne) was added and purified by non-denaturing anion exchange chromatography or reversed phase chromatography. The purified material was desalted using a 3K MWCO centrifugal spin tubes or tangential flow filtration to give the title duplex RNAi agents as an aqueous solution. The duplexes were lyophilized and reconstituted in 1X PBS and ultrafiltered with 100K MWCO filter units to give duplex RNAi agents shown in Table 9. For in vivo samples, endotoxin levels were measured with Charles River Endosafe® Device (upper limit 2 EU / mg). Table 8 – Conjugation of Targeting Ligands to Alkyne-Functionalized DuplexesTable 9 – Duplex RNAi AgentsIn Vivo Intranasal Administration of SCNN1A RNAi Agents in Mice The efficacy of the RNAi agents was studied in wildtype male C57BL / 6 mice. Four mice were dosed per group. Prior to the start of study, animals were randomized based on body weight. The mice were anesthetized under isoflurane and given 100 µL + 100 µL (200 µL total, 5-10min in between 100 uL doses) of the dosing solution intranasally (0.75 mg / kg). Animals were held in a vertical position for 1 minute following administration. Afterwards, they were placed back in home cage and monitored for recovery. Clinical Observation and body weight were noted on day 1 and day of necropsy. On necropsy day (Day 8 or Day 15) animals were sacrificed, and lung tissues collected (right / left lung weighed separately and snap frozen).RNA Extraction and TaqMan Analysis The day before tissue processing, Lysing Matrix A tubes (MP BioMedicals, 116920500) were filled with 1 mL each of TRIzol (Invitrogen, 15596026) and cooled overnight at 4 °C. On the day of tissue processing, removed box of tissue samples from -80 °C and packed in dry ice in an ice tub to keep frozen during the processing procedure. For processing, the upper right lung pieces (~50-100 mg each) were quickly diced into 3-4 pieces before placing them into the Lysing Matrix A tubes and capping. Once all samples were added to their individual tubes, the tissue was then homogenized using a FastPrep-96 (MP BioMedicals, 116010500) at 1800RPM for 1 minute. After homogenization, 300 µL of BCP (Sigma-Aldrich, B9673) was added to each tube. The tubes were capped and shaken vigorously to mix contents. The tubes were spun down in a microfuge at 4 °C for 15 minutes at 15000xg. After centrifugation, removed 300 µL of aqueous solution from each tube and transferred to a 1.2 mL deep-well block. Added equal amounts (300 µL) of 100% EtOH to each well and mixed thoroughly. Once mixed, transferred the full 600 µL of solution to the silicone plate provided with the Quick-RNA 96 isolation kit (Zymo Research, R1053). Isolated the total RNA per the instructions provided with the kit, using 50 µL of RNase-free water for the final elution. The concentration of the isolated RNA was measured with a Nanodrop 8000. Diluted the isolated RNA to 100ng / µL using RNase-free water and aliquoted 10 µL from the dilution plate for cDNA synthesis. Performed cDNA synthesis using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (4368814) per the provided instructions. After synthesis was complete, diluted the cDNA to 20ng / µL with RNase-free water. Using 5 µL of diluted cDNA (100ng total), performed a TaqMan Assay using 20 µL total volume per instructions, using probes from ThermoFisher Scientic for SCNN1A (Mm00803386_m1) and HPRT (Mm03024075) as an endogenous control. The assay plate was run on an Applied Biosystems QuantStudio 6. Data analysis was done using the ∆∆Ct method and graphed using GraphPad Prism.Example 3. In Vivo Intranasal Administration of Unconjugated and Ligand- Conjugated SCNN1A RNAi Agents in Mice.Mice were dosed on day 1 with 0.75 mg / kg of RNAi agent and sacrificed on day 8 (Table 10).Table 10 - Average Relative SCNN1A mRNA Knockdown (%KD) at Sacrifice (Day 8)As shown in Table 10, each of the SCNN1A RNAi agents showed a reduction in mRNA expression in mice compared to control. In general, integrin ligand conjugates showed better potency compared to unconjugated RNAi agent. For instance, tridentate integrin ligand conjugates 60-1a, 60-2a and 60-3a showed 56% knockdown, 56% knockdown and 53% knockdown, respectively, compared to unconjugated RNAi agent 59-1 which showed 41% knockdown. Interestingly, bidentate integrin ligand conjugates also showed improved potency over unconjugated RNAi. For example, bidentate conjugates 60-1b, 60-3b and 60-4b showed 52% knockdown, 52%knockdown and 58% knockdown respectively. Furthermore, monodentate integrin ligand conjugates also showed improved potency compared to the unconjugated agent. For instance, RNAi agents 60-1c and 60-4c showed 54% and 51% knockdown respectively, compared to unconjugated agent 59-1 (41% knockdown). The data shown in Table 10 demonstrated the benefit of employing an integrin ligand to assist in the uptake of the RNAi agent as they showed up to 17% improved knockdown of the target mRNA compared to the unconjugated agent.Example 4. In Vivo Intranasal Administration of Unconjugated and Ligand- Conjugated SCNN1A RNAi Agents in Mice.Mice were dosed on day 1 with 0.75 mg / kg of RNAi agent and sacrificed on day 15 (Table 11).Table 11 - Average Relative SCNN1A mRNA Knockdown (%KD) at Sacrifice (Day 15)As shown in Table 11, each of the SCNN1 A RNAi agents showed a reduction in mRNA expression in mice compared to control. Except for the bidentate conjugate 60-2b and monodentate conjugate 60-2c, all other integrin ligand conjugates showed improved knockdown at 2 weeks post RNAi intranasal dosing. For instance, tridentateintegrin ligand conjugates 60-1a and 60-2a showed 58% and 55% knockdown respectively compared to unconjugated agent 59-1 (47% knockdown). Bidentate integrin ligand conjugate 60-1b showed an even higher mRNA knockdown of 65%, an 18% improved knockdown compared to 59-1 (47%). The data in Table 11 demonstrated the benefit of employing an integrin ligand to assist in the uptake of the RNAi agent compared to unconjugated RNAi agent. Example 5. In Vivo Intranasal Administration of Unconjugated and Ligand- Conjugated SCNN1A RNAi Agents in Mice. Mice were dosed on day 1 with 0.75 mg / kg of RNAi agent and sacrificed on day 15 (Table 12). Table 12 – Average Relative SCNN1A mRNA Knockdown (%KD) at Sacrifice (Day 8)As shown in Table 12, each of the SCNN1A RNAi agents showed a reduction in mRNA expression in mice compared to control. In general, integrin ligand conjugates showed better potency compared to unconjugated RNAi agent. For instance, tridentate integrin ligand conjugates 59-9, 59-10, 59-11 and 59-12 showed 46% knockdown, 44% knockdown, 49% knockdown and 52% knockdown, respectively, compared to unconjugated RNAi agent 59-1 which showed 25% knockdown. Example 6. In Vivo Intranasal Administration of Unconjugated and Ligand- Conjugated SCNN1A RNAi Agents in Mice.Mice were dosed on day 1 with 0.75 mg / kg, 0.5 mg / kg, 0.25 mg / kg or 0.01 mg / kg of RNAi agent and sacrificed on day 8 or 15 (Table 13). Table 13 – Average Relative SCNN1A mRNA Knockdown (%KD) at SacrificeAs shown in Table 13, two of the unconjugated RNAi agents (59-5 and 59-8) showed very good reduction in target mRNA expression at all doses tested with no dose response observed. These knockdowns were comparable to their integrin ligand conjugated analogs (59-9 and 59-12). The benefit of ligand conjugation was only observed at very low dose of 0.001 mg / kg (59-5 vs 59-9) albeit very low percent mRNA reduction. Titration of dose of RNAi agent 59-5 showed very tight curve (data not shown) which could explain why both unconjugated and conjugated agents had comparable potencies. Example 7. Binding Activity of Integrin Targeting Ligands The potency and selectivity of the integrin targeting ligands against a variety of integrins were experimentally measured as follows (Table 14): αvβ6 and αvβ8 binding assay – 96 well plates were coated over night at 4 °C with LAP (TGF-β) (0.4 µg / ml), (Acros Biosystems) dissolved in coating buffer (15mMNa2CO3, 35mM NaHCO3, pH 9.6). Plate was washed with wash buffer (PBS, 0.1% Tween20) and then blocked at ambient temperature with blocking buffer (Superblock TBS, Thermofisher Scientific) for 1 hour. Ligands serially diluted in blocking buffer were added to the plate along with purified αvβ6 (0.4 µg / mL, R&D) or αvβ8 (0.4 µg / mL, R&D) and incubated at ambient temperature for 1 hour. Plate was washed with wash buffer and then incubated with primary antibody (anti-αv mouse anti- human Millipore) for 1 hour at ambient temperature followed by a secondary antibody (Anti-Mouse IgG (H+L), HRP Conjugate, Promega) for 1 hour at ambient temperature. The plate was developed using Pierce 1 step Ultra TMB substrate solution (ThermoFisher Scientific) and then imaged using a Biotek Neos2 microplate reader after stopping the reaction with 2M H2SO4. IC50 values were calculated by plotting test compound concentration versus % binding using a 3-parameter logistic curve fitting equation. αvβ3, αvβ5 and αvβ1 binding assay – 96 well plates were coated over night at 4 °C with fibronectin (2 µg / mL, R&D) or vitronectin (2 µg / mL, R&D) dissolved in coating buffer (15mmol / L Na2CO3, 35mmol / L NaHCO3, 7.7mmol / L NaN₃, pH 9.6, with 1mmol / L CaCl2and 1mmol / L MgCl2). Plate was washed with wash buffer (0.05% Tween-20 in TBS, pH7.4, with 1mmol / L MnCl2) and then blocked at 37 °C with blocking buffer (2% BSA in wash buffer, pH 7.4) for 1.5 hours. Ligands serially diluted in sample dilution buffer (0.5% BSA in wash buffer, pH7.4) were added to the plate along with purified αvβ3 (1.2 µg / mL, Acros Biosystems) or αvβ5 (0.8 µg / mL, Acros Biosystems) or αvβ1 protein (4 µg / mL, Acros Biosystems) and incubated at 37 °C for 1 hour. Plate was washed with wash buffer and then incubated with primary antibody (2 µg / mL Human Integrin β3 Biotinylated Antibody, R&D; 2 ng / Ml human integrin β1 biotinylated antibody, R&D) for 1 hour at 37 °C. Plate was washed with wash buffer and then incubated with HRP conjugated anti-His tag antibody (Acros Biosystems; for αvβ5) or HRP conjugated streptavidin (ThermoFisher Scientific; for αvβ1 and αvβ3) for 1 hour at 37 °C. The plate was developed using Pierce 1 step Ultra TMB substrate solution (ThermoFisher Scientific) and then imaged using a Biotek Neos2 microplate reader after stopping the reaction with 2M H2SO4.IC50values were calculated by plotting test compound concentration versus % binding using a 3-parameter logistic curve fitting equation. Table 14 – Integrin Solid Phase BindingAs shown in Table 14, compounds from preparations 19, 20, 21 and 22 showed potent binding to integrins αvβ6, αvβ8, αvβ5 and αvβ3, with all compounds showing a particular preference for binding to αvβ6 and αvβ5. For instance, compound of preparation 19 showed potent binding to integrins αvβ6, αvβ8 and αvβ5 (IC50 = 5.0 nM, 12.9 nM and 8.9 nM respectively) compared to reduced binding for αvβ3 (284 nM) and αvβ1 (114.5 nM). The compounds of preparation 20, preparation 21, and preparation 22 all show higher preference for binding to integrin αvβ5 compared to compound preparation 19. However, the compound of preparation 19 showed better binding to integrin αvβ6 and αvβ8 when compared to the compounds of preparation 20, 21, and 22. Example 8. In vitro Activity of SCNN1A RNAi Agents In Mouse Lung Epithelial Cells MLE-12 cells were plated in 96 well plates (10,000 MLE12 cells / well; Falcon, 353072) and incubated overnight at 37 °C. Cells were transfected with siRNAs serial diluted 1:5 at a starting concentration of 100nM in Opti-Mem media (Gibco, 31985- 070) and delivered to cells with lipofectamine (Invitrogen, 13778-150) according to the RNAiMax Transfection protocol by Invitrogen. Cells were incubated for 24 hours at 37 °C. The media was aspirated from the 96 well plate and cells were lysed using 150 µL lysis buffer (Zymo, R1053) per well. The 96 well plate was frozen over nightat -80 °C. RNA was purified using a Total RNA Purification Kit (Zymo, R1053). The purified RNA was reverse transcribed to cDNA per the manufacturer instructions for the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, 4368813). The cDNA was diluted 2.5x using RNase-free water, plated in a 384 well plate, and run on the QuantStudio7 Flex (Applied Biosystems) per the manufacturer instructions. The TaqMan PCR assay was run using TaqMan probes for SCNN1A (Mm00803386_m1) and GAPDH as an endogenous control (Mm99999915_g1) (ThermoFisher Scientific). The Delta CT, Delta-Delta CT, and fold change values were calculated and graphed with GraphPad (Tables 15, 16 and 17). Table 15 – In Vitro Activity of RNAi Agents in Mouse Lung Epithelial CellsTable 16 – In Vitro Activity of RNAi Agents in Mouse Lung Epithelial CellsTable 17 – In Vitro Activity of RNAi Agents in Mouse Lung Epithelial CellsExample 9. In Vivo Intranasal Administration of Unconjugated and Ligand- Conjugated Muc5B RNAi Agents in Mice. Mice were dosed on day 1 with 3 mg / kg or 10 mg / kg of RNAi agent and sacrificed on day 15 (Table 18). Table 18 – In Vitro and In Vivo Activities of Muc5B RNAi AgentsAs shown in Table 18, unconjugated RNAi agent (59-17) showed no knockdown of target gene at a dose of 3mpk compared to a modest but significant 32% knockdown achieved with integrin ligand, thus exhibiting the benefit of employing integrin ligand to facilitate uptake of the RNAi agent. Example 10. In Vivo Intranasal Administration of Unconjugated and Ligand- Conjugated RAGE RNAi Agents in Mice. Mice were dosed on day 1 with 0.5 mg / kg of RNAi agent and sacrificed on day 15 (Table 19). Table 19 - Average Relative RAGE mRNA Knockdown (%KD) at SacrificeAs shown in Table 19, each of the RAGE RNAi agents showed a reduction in mRNA expression in mice compared to control. The integrin ligand conjugate (59-20) showed better potency compared to unconjugated RNAi agent (5-19), thus exhibiting the benefit of using integrin ligand to facilitate uptake of RNAi agent.

Claims

CLAIMS We claim:

1. A compound of the formula:Wherein: R1is H or C1to C6alkyl,is (C5to C14aryl)-O-, or (C5to C14heteroaryl)-O- with from 1 to 5 heteroatoms selected from N, O, and S L comprises a linker or is absent, Z comprises an oligonucleotide, and q is a whole number integer from 1 to 3, or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein the compound is of the formula:.

3. The compound of claim 1, wherein the compound is of the formula:.

4. The compound of any one of claims 1 to 3, whereinis selected from:.

5. The compound of any one of claims 1 to 4, whereinis of the formula:.

6. The compound of any one of claims 1 to 4, whereinis of the formula:

7. The compound of any one of claims 1 to 4, whereinis of the formula:

8. The compound of any one of claims 1 to 4, whereinis of the formula:

9. The compound of any one of claims 1 to 8, wherein L comprises a linker of the formula:wherein each side of L can be connected toor Z, and n and m are independently selected whole number integers from 1 to 10.

10. The compound of any one of claims 1 to 9, wherein L comprises a linker of the formula:wherein each side of L can be connected toor Z, and n and m are independently selected whole number integers from 1 to 10.

11. The compound of any one of claims 1, 2, or 4 to 10, wherein the compound is of the formula:, or a pharmaceutically acceptable salt thereof.

12. The compound of any one of claims 1, 2, or 4 to 10, wherein the compound is of the formula:, or a pharmaceutically acceptable salt thereof.

13. The compound of any one of claims 1, 2, or 4 to 10, wherein the compound is of the formula:, or a pharmaceutically acceptable salt thereof.

14. The compound of any one of claims 1, 2, or 4 to 10, wherein the compound is of the formula:, or a pharmaceutically acceptable salt thereof.

15. The compound of any one of claims 1, 2, or 4 to 10, wherein the compound is of the formula:, or a pharmaceutically acceptable salt thereof.

16. The compound of any one of claims 1, 2, or 4 to 10, wherein the compound is of the formula:, or a pharmaceutically acceptable salt thereof.

17. The compound of any one of claims 1, 2, or 4 to 10, wherein the compound is of the formula:, or a pharmaceutically acceptable salt thereof.

18. The compound of any one of claims 1, 2, or 4 to 10, wherein the compound is of the formula:, or a pharmaceutically acceptable salt thereof.

19. The compound of any one of claims 1 to 9, wherein L comprises a linker of the formula:, wherein each side of L can be connected toor Z, m is a whole number integer from 1 to 10.

20. The compound of claim 19, wherein m is 5.

21. The compound of claim 19 or 20, wherein the compound is of the formula:, or a pharmaceutically acceptable salt thereof.

22. The compound of claim 19 or 20, wherein the compound is of the formula:, or a pharmaceutically acceptable salt thereof.

23. The compound of claim 19 or 20, wherein the compound is of the formula:, or a pharmaceutically acceptable salt thereof.

24. The compound of claim 19 or 20, wherein the compound is of the formula:, or a pharmaceutically acceptable salt thereof.

25. The compound of any one of claims 1 to 24, wherein the oligonucleotide comprises a sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ IDNO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ IDNO. 20, SEQ ID NO. 21, SEQ ID NO. 22, or SEQ ID NO. 23.

26. A compound according to any one of claims 1 to 25, or a pharmaceutically acceptable salt thereof, for use in therapy.

27. The compound of claim 26, wherein the compound is used in the treatment of a lung disease.

28. Use of a compound according to any one of claims 1 to 25, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament.

29. Use of a compound according to any one of claims 1 to 25, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament in the treatment of a lung disease.

30. A method of treatment of a lung disease comprising administration of a composition comprising a compound of any one of claims 1 to 25 to a patient in the need of treatment.

31. A compound of the formula:, wherein R1is H or C1to C6alkyl andis (C5to C14aryl)-OH, or (C5to C14heteroaryl)-OH with from 1 to 5 heteroatoms selected from N, O, and S.

32. The compound of claim 31, whereinis selected from:.

33. The compound of claim 31, wherein is selected from:

34. The compound of claim 31, wherein the compound is of the formula:

35. The compound of claim 31, wherein the compound is of the formula:

36. The compound of claim 31, wherein the compound is of the formula:

37. The compound of claim 31, wherein the compound is of the formula:

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