Novel therapeutic delivery moieties and uses thereof

Abstract

This document provides compounds comprising a delivery portion for selectively delivering a therapeutic agent and / or oligonucleotide to the lungs. This document also provides compounds comprising a single delivery portion for selectively delivering a therapeutic agent and / or oligonucleotide to the lungs. This document also discloses novel α- v β x Integrin ligands, such as those with α v β5, α v β6 and / or α v Ligands that bind to β8 integrin.

Description

[0001] Reference to sequence lists

[0002] This application is filed concurrently with a sequence list in ST.26 XML format. This sequence list is provided as a file entitled “30619_WO”, created on October 15, 2024, and measuring 252 kilobytes in size. The sequence list information in ST.26 XML format is incorporated herein by reference in its entirety.

[0003] Invention Field

[0004] This invention relates to novel compounds comprising a novel delivery portion for delivering oligonucleotides to the lungs, which can be used to treat lung diseases. The invention also relates to novel delivery portions that can be linked to oligonucleotides to facilitate delivery of the oligonucleotides to the lungs and / or lung cells. Background of the Invention

[0006] Compounds containing oligonucleotides can target genes in a sequence-specific manner for the treatment of many different types of diseases involving gene dysregulation. For example, RNA interference (RNAi) compounds such as small interfering RNA (siRNA) and antisense oligonucleotides (ASO) can be used to knock down gene expression. Conversely, other compounds containing oligonucleotides can activate genes using oligonucleotides such as short activating RNA (saRNA). By delivering oligonucleotides to the desired patient tissue, gene expression can be downregulated, upregulated, or corrected.

[0007] Oligonucleotides can be delivered to desired tissues by linking a delivery moiety. For example, by linking N-acetylgalactosamine (GalNAc), RNAi compounds, such as oligonucleotides, can be selectively delivered to the liver to target the asialic acid glycoprotein receptor on hepatocytes. An exemplary compound containing GalNAc is givosiran, an FDA-approved siRNA that targets the ALAS1 gene to treat acute hepatic porphyria. Despite the existence of such compounds for liver delivery, there is still a need for oligonucleotides to be delivered to other tissues, such as the lungs.

[0008] While some delivery moieties have been proposed for the selective delivery of oligonucleotide-containing compounds to the lungs, such as those in U.S. Patent No. 11,597,701, there remains a need for delivery moieties capable of delivering oligonucleotide-containing compounds to the lungs. For example, the delivery moieties proposed in U.S. Patent No. 11,597,701 require three ligands to achieve adequate and selective delivery of oligonucleotide-containing compounds to the lungs, which could increase the costs associated with the synthesis, application, and storage of these delivery moieties. Therefore, there is a need for delivery moieties capable of selectively delivering oligonucleotide-containing compounds to the lungs using only a single ligand, thereby reducing the overall complexity of the molecule. Invention Overview

[0010] This article discloses compounds with the following formula:

[0011] Where: R1 is H or C 1- C6 alkyl; (C5-C) 14 aryl)-O- or (C5-C) heteroatoms having 1-5 N, O, and S atoms. 14 (Heteroaryl)-O-; L contains a linker or is absent; Z contains an oligonucleotide; and q is a whole number integer from 1 to 3; or a pharmaceutically acceptable salt thereof.

[0012] This article also discloses compounds with the following formula:

[0013] Where: R1 is H or C 1- C6 alkyl; (C5-C) 14 aryl)-O- or (C5-C) heteroatoms having 1-5 N, O, and S atoms. 14 (Heteroaryl)-O-; L contains a linker or is absent; and Z contains an oligonucleotide; or a pharmaceutically acceptable salt thereof.

[0014] This article also discloses compounds with the following formula:

[0015] Where: R1 is H or C 1- C6 alkyl; (C5-C) 14 aryl)-O- or (C5-C) heteroatoms having 1-5 N, O, and S atoms. 14 (Heteroaryl)-O-; L contains a linker or is absent; and Z contains an oligonucleotide; or a pharmaceutically acceptable salt thereof.

[0016] This article also discloses the use of the disclosed compounds in therapeutics, such as in the treatment of lung diseases.

[0017] This article also discloses compounds with the following formula:

[0018] Where R1 is H or C 1- C6 alkyl, and (C5-C) 14 aryl)-OH or (C5-C) heteroatoms having 1-5 N, O, and S atoms. 14 (heteroaryl)-OH. Invention Details

[0020] This invention relates to novel compounds, including novel delivery portions and novel compounds comprising the novel delivery portion and an oligonucleotide, for selectively delivering the oligonucleotide to the lungs, which can be used to treat lung diseases. The novel delivery portion may be linked to the oligonucleotide to facilitate delivery of the oligonucleotide to a desired site, such as the lungs or lung cells.

[0021] Surprisingly, it was found that by combining oligonucleotides with integrin α... v By linking the delivery portion of the β-ligand, oligonucleotides can be selectively delivered to lung cells or lung tissue. In particular, and surprisingly, it was found that by linking the oligonucleotide to a β-ligand containing integrin α... v The delivery portion of the β6 ligand is linked, allowing oligonucleotides to be selectively delivered to lung cells or lung tissue because α... v β6 integrin is highly expressed in lung cells.

[0022] Integrins are a family of cell surface receptor proteins that contribute to cell adhesion, cell signaling, and cell attachment to the extracellular matrix and other cells. Integrins are essential components of various biological processes, including tissue development, immune responses, wound healing, and cancer metastasis. Integrin heterodimers are formed by combinations of 18 distinct α subunits and 8 distinct β subunits. Furthermore, several integrins, including α... v β1, α v β3, α v β5, α v β6 and α v β8 is highly expressed in lung cells. In lung cells, several integrins, such as α... v β6 is expressed in lung epithelial cells and is thought to be involved in extracellular matrix remodeling and TGF-β activation, for example, during lung injury and repair.

[0023] While not wishing to be bound by theory, it is believed that the delivery component can be designed as one or more α... v β x , for example α v The ligand of β6 integrin plays a role, allowing the delivery moiety to be transported across the cell membrane of lung cells. Furthermore, although not wishing to be bound by theory, if the delivery moiety is linked to an oligonucleotide, as further described herein, selective delivery of the oligonucleotide to the lung could be permitted, since α... v β x Integrins, such as α v β6 is highly expressed in lung cells.

[0024] As further described in this paper, it was surprisingly found that, through a single α... v β x The ligand can selectively disclose the α v βx The ligand can selectively deliver oligonucleotides to the lungs. This can provide a simpler delivery motif that is easier to handle than the one containing a three-ligand delivery motif.

[0025]

[0026] Formula I.

[0027] This paper discloses a novel compound of formula I, wherein D is a delivery portion comprising α v β x Integrins, such as α v A β6 ligand, L being a linker or absent, and Z being an oligonucleotide. The oligonucleotide can be coupled to the linker. For example, the oligonucleotide and / or linker can be alkyne- or NHS-ester functionalized, which can facilitate subsequent coupling of the linker to the oligonucleotide via a coupling reaction. Compounds comprising an oligonucleotide and a delivery moiety can be coupling compounds. Pharmaceutically acceptable salts of compounds of formula I are also disclosed herein.

[0028]

[0029] Formula II.

[0030] This paper also discloses novel compounds of formula II, wherein D1 and D2, which can be independently selected from each other or are the same, are compounds containing α. v β x Integrins, such as α v The delivery moiety of the β6 ligand, L1 and L2, may be independently selected, identical, or absent, and Z is an oligonucleotide. The oligonucleotide can be linked to a linker via coupling. For example, the oligonucleotide and / or linker may be alkyne- or NHS-ester-functionalized, which can facilitate the subsequent linking of the linker to the oligonucleotide via a coupling reaction. Compounds comprising an oligonucleotide and a delivery moiety can be coupling compounds. Pharmaceutically acceptable salts of compounds of formula II are also disclosed herein.

[0031]

[0032] Formula III.

[0033] This document also discloses novel compounds of Formula III, wherein D is a delivery moiety comprising an αvβx integrin, such as an αvβ6 ligand, L is a linker or is absent, and Z is an oligonucleotide. The oligonucleotide can be coupled to the linker. For example, the oligonucleotide and / or linker can be alkyne- or NHS-ester-functionalized, which can facilitate subsequent coupling of the linker to the oligonucleotide via a coupling reaction. Compounds comprising an oligonucleotide and a delivery moiety can be coupling compounds. Pharmaceutically acceptable salts of compounds of Formula III are also disclosed.

[0034]

[0035] Formula IV.

[0036] This document also discloses novel compounds of formula IV, wherein D is a delivery moiety comprising an αvβx integrin, such as an αvβ6 ligand, L is a linker or is absent, and Z is an oligonucleotide. The oligonucleotide can be coupled to the linker. For example, the oligonucleotide and / or linker can be alkyne- or NHS-ester-functionalized, which can facilitate the subsequent coupling of the linker to the oligonucleotide via a coupling reaction. Compounds comprising an oligonucleotide and a delivery moiety can be coupling compounds. Pharmaceutically acceptable salts of compounds of formula IV are also disclosed.

[0037] Compounds of formulas I, II, III, and IV can be called conjugates because they include oligonucleotides and α-nucleotides. v β x Integrins, such as α v β6 ligand. Therefore, the compound may include a 3' or 5' delivery portion optionally linked to the oligonucleotide via a linker (Formula I). ​​Alternatively, the compound may include two delivery portions, each optionally linked to the 3' or 5' end of the oligonucleotide via a linker (Formula II). Additionally, the compound may include three delivery portions, all optionally linked to the 3' or 5' end of the oligonucleotide via linkers (Formulas III and IV).

[0038] Delivery section

[0039] This article discloses a novel α v β x Integrin ligands, represented by formulas V and VI. This document also discloses novel compounds comprising a delivery moiety comprising one or more novel α-ligands represented by formulas VII and VIII. v β x Integrin ligands.

[0040] In Formulas I-IV, the compounds provided herein include delivery moieties D, D1, and / or D2 for delivering oligonucleotides to a target site, such as the lung or lung cells. v β x Integrin ligands.

[0041] Other suitable delivery components for the selective delivery of oligonucleotides to the lungs include those that can be combined with α v β x Any delivery portion of integrin-bonded proteins, such as α v β6 integrin ligand. In formulas I-IV, suitable delivery portions D, D1, and / or D2 are shown in formulas V and VI below, which are not linked to oligonucleotides, and in formulas VII and VIII as shown in the conjugates where the ligands are linked to the remaining portions.

[0042] The wavy lines in Formulas VII and VIII show that, in the case of delivery fraction-oligonucleotide conjugates, D, D1, and / or D2 may be linked to a linker and / or an oligonucleotide, or in the case of agonist compounds, D, D1, and / or D2 may be linked to a leaving group or another suitable atom or molecule.

[0043]

[0044] Formula V.

[0045] In equation V, R1 can be H or C. 1- C6 alkyl. R2 can be OH, (C5-C7 aryl)-OW, or (C5-C7 heteroaryl)-OW having 1-3 heteroatoms selected from N, O, and S. Y1 and Y2 can be independently selected from N or CH. W can be H or C. 1- C6 alkyl.

[0046]

[0047] Formula VI.

[0048] In equation VI, R1 can be H or C. 1- C6 alkyl. It can be (C5-C) 14 aryl)-OH or (C5-C)-OH having 1-5 heteroatoms selected from N, O and S 14 (heteroaryl)-OH.

[0049]

[0050] Formula VII.

[0051] In equation VII, R1 can be H or C. 1-C6 alkyl. R2 can be O-, (C5-C7 aryl)-O-, or (C5-C7 heteroaryl)-O- having 1-3 heteroatoms selected from N, O, and S. Y1 and Y2 can be independently selected from N or CH.

[0052]

[0053] Formula VIII.

[0054] In equation VIII, R1 can be H or C. 1- C6 alkyl. It can be (C5-C) 14 aryl)-O- or (C5-C)- having 1-5 heteroatoms selected from N, O and S 14 (Heteroary aryl)-O-.

[0055] The IX and X symbols can be displayed by Examples of suitable portions of the compound.

[0056]

[0057] Formula IX.

[0058]

[0059] Formula X.

[0060] The novel compounds disclosed herein may have a total of 1-3 delivery portions linked to an oligonucleotide. The delivery portions may be linked to any one or both sides of the compound. For example, the compound may have a single delivery portion optionally linked via a linker at the 5' end, 3' end, or both ends of the oligonucleotide. Furthermore, the conjugate compound may have multiple delivery portions linked to the same end of the oligonucleotide via one or more optional linkers.

[0061] Oligonucleotides

[0062] One or more oligonucleotides can be selectively delivered to the lungs for diagnostic or therapeutic purposes using the delivery portion described herein. The one or more oligonucleotides may comprise DNA or RNA nucleotides, nucleosides, or combinations thereof, and may comprise one or more or all of modified nucleotides, nucleosides, or modified bonds.

[0063] Oligonucleotides can target specific DNA or RNA sequences in cells to regulate gene expression. In some implementations, oligonucleotides reduce the expression of target mRNA transcripts and / or further reduce the expression of target proteins. Suitable conjugates may have a target gene knockdown percentage of at least about 20%, at least about 40%, or at least about 50%. The reduction in expression may last for about 1 week, 3 weeks, and / or 4 weeks.

[0064] Those skilled in the art will recognize that one or more mismatches may exist between the oligonucleotide and the target nucleotide sequence, and it may still retain its function in regulating gene expression. Therefore, in one embodiment, the oligonucleotide has a 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, or 85% identity percentage with the target sequence. The oligonucleotide may also have an overhang of 1-10, 1-5, 1-3, 3, 2, or 1 residue at its 5' or 3' end. The oligonucleotide may also include an abasic or inverted abasic moiety or a modified phosphate group. Suitable modifications are known in the art.

[0065] In some embodiments, the oligonucleotide includes a 2′-modification of a sugar residue, such as ribose or deoxyribose. For example, some 2′-modifications of ribose or deoxyribose can increase the stability and half-life of RNA or DNA. Such 2′-modifications include 2′-fluorine, 2′-O-methyl (i.e., 2′-methoxy), 2′-O-alkyl, or 2′-O-methoxyethyl (2′-O-MOE). In some embodiments, the oligonucleotide includes modified internucleotide linkages, such as phosphate thioester (PS) linkages.

[0066] In some embodiments, one or more oligonucleotides are RNAi substances, such as small interfering RNA (siRNA), small (also called short) activating RNA (saRNA), microRNA (miRNA), short hairpin RNA (shRNA), or antisense oligonucleotides (ASO). In a suitable embodiment, the oligonucleotide is siRNA. In another suitable embodiment, the oligonucleotide is siRNA comprising a sense strand and an antisense strand.

[0067] In some implementations, the oligonucleotide is siRNA, which contains an antisense strand that targets the gene of interest in the lung.

[0068] Appropriate genes to focus on may include, but are not limited to, any gene whose regulation will improve lung cell function.

[0069] In some implementation schemes, the genes of interest are selected from α-ENaC, AGER, etc. (RAGE), AOC3, APLNR, β-ENaC, CCL24, CTGF, CXCR1, CXCR2, CXCR4, ENPP2, ET-A, ET-B, FGFR1, FG FR2, 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. Those skilled in the art can modify the antisense strand of siRNA to target different genes of interest.

[0070] In some implementations, the length of the sense strand and the antisense strand is each between 15 and 40 nucleotides, for example, 18 to 25 nucleotides. The sense strand and the antisense strand form a double helix, optionally having one or more 5' or 3' nucleotide overhangs.

[0071] In some embodiments, one or more nucleotides of the sense strand and / or antisense strand are independently modified nucleotides, meaning that the sense strand and 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'-fluorine-modified nucleotide, a 2'-O-methyl-modified nucleotide, or a 2'-O-alkyl-modified nucleotide. In some embodiments, each of the sense strand and antisense strand is independently a modified nucleotide, such as a 2'-fluorine-modified nucleotide, a 2'-O-methyl-modified nucleotide, or a 2'-O-alkyl-modified nucleotide.

[0072] In some implementations, the antisense chain includes a phosphate analog, such as 5'-vinylphosphonate (5'-VP), at the 5' end.

[0073] In some implementations, the justice chain has a base-free or reverse base-free portion at positions 9, 10, or 11.

[0074] In some embodiments, the sense and antisense strands have one or more modified nucleotide links. In some embodiments, the modified nucleotide links are phosphate thioester links. In some embodiments, the sense strand has 4 or 5 phosphate thioester links. In some embodiments, the antisense strand has 4 or 5 phosphate thioester links. In some embodiments, both the sense and antisense strands have 4 or 5 phosphate thioester links. In some embodiments, the sense strand has 4 phosphate thioester links, and the antisense strand has 4 phosphate thioester links.

[0075] In some implementations, Z may also include a branched portion, allowing multiple linkers and multiple delivery portions to be linked to the same site on the oligonucleotide, for example, in Figure 3 middle.

[0076] In some implementations, the branched portion can be linked to an oligonucleotide and / or one or more linkers via well-known coupling chemistry methods such as amidation.

[0077] connector

[0078] The present invention may also include linkers L, L1, and / or L2 to attach one or more oligonucleotides to a delivery moiety and / or α. v β x Integrins, such as α v β6 integrin ligand linkage. Suitable linkers include any molecule that can deliver the moiety and / or α. v β x Integrins, such as α v The β6 ligand is linked to one or more oligonucleotides. The linker can directly link to α... v β x Integrins, such as α v β6 integrin ligands and oligonucleotides may have other coupling heads.

[0079] Suitable connectors may include C 1- C 20 straight-chain alkyl, C 1- C 20 Branched alkyl, C 1- C 20 Alkenyl, cycloalkyl groups containing 3-7 carbon atoms, heterocyclic compounds containing 3-7 membered rings, aryl groups containing 3-7 carbon atoms on an aryl ring, -[OCH2CH2] n - where n is an integer from 1 to 20; and / or combinations thereof.

[0080]

[0081] Formula XI.

[0082] In some embodiments, the linker is a compound of formula XI. n can be an integer from 1 to 20, 6 to 20, 10 to 20, 12 to 18, or 12 to 15. A and B are connection points for any oligonucleotide, coupling moiety, and / or delivery moiety. For example, connection point A can be connected to both the oligonucleotide and the coupling moiety (i.e., the oligonucleotide is coupled to the linker), while connection point B can be directly connected to a compound containing α... v β x Integrins, such as α v The delivery portion of the β6 integrin ligand is linked.

[0083]

[0084] Formula XII.

[0085] In some embodiments, the linker is a compound of formula XII. m can be an integer from 1 to 20, 1 to 10, 5 to 10, or 1 to 5. C and D are connection points for any oligonucleotide, coupling moiety, and / or delivery moiety. For example, connection point C can be connected to both the oligonucleotide and the coupling moiety (i.e., the oligonucleotide is coupled to the linker), while connection point D can be directly connected to a compound containing α... v β x Integrins, such as α v The delivery portion of the β6 integrin ligand is linked.

[0086]

[0087] Formula XIII.

[0088] In some embodiments, the linker is a compound of formula XIII, which is a combination of formulas XI and XII. n can be an integer from 1 to 20, 6 to 20, 10 to 20, 12 to 18, or 12 to 15. m can be an integer from 1 to 20, 1 to 10, 5 to 10, or 1 to 5. E and F are connection points for any oligonucleotide, coupling portion, and / or delivery portion. For example, connection point E can be connected to both the oligonucleotide and the coupling portion (i.e., the oligonucleotide is coupled to the linker), while connection point F can be directly connected to a compound containing α... v β x Integrins, such as α v The delivery portion of the β6 integrin ligand is linked.

[0089]

[0090] Formula XIV-A.

[0091] In some embodiments, the linker is a compound of formula XIV-A. G and H are connection sites for any oligonucleotide, coupling moiety, and / or delivery moiety. For example, connection site G may be connected to both the oligonucleotide and the coupling moiety (i.e., the oligonucleotide is coupled to the linker), while connection site H may be directly connected to a compound containing α-... v β x Integrins, such as α v The delivery portion of the β6 integrin ligand is linked.

[0092]

[0093] Formula XIV-B.

[0094] 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 an integer from 1 to 20, 1 to 10, 5 to 10, or 1 to 5. m can be an integer from 1 to 20, 1 to 10, 5 to 10, or 1 to 5. J and K are connection points for any oligonucleotide, coupling portion, and / or delivery portion. For example, connection point J can be connected to both the oligonucleotide and the coupling portion (i.e., the oligonucleotide is coupled to the linker), while connection point K can be directly connected to a compound containing α... v β x Integrins, such as α v The delivery portion of the β6 integrin ligand is linked.

[0095] In some embodiments, the linker comprises a compound of formula XI-XIV and a coupling moiety. Coupling is a process of chemically linking two or more molecules or biomolecules by covalent bonds. Those skilled in the art will recognize that various coupling agents exist for linking a portion of a molecule, such as a delivery moiety as described herein, and / or a linker, to a biomolecule, such as an oligonucleotide as described herein. The coupling moiety is an additional compound introduced by the coupling agent to link the nucleotide to the linker and / or delivery moiety.

[0096] Suitable coupling agents for coupling oligonucleotides to the remainder of the conjugate, such as the delivery moiety and / or linker, depend on the desired position of the covalent bond between the oligonucleotide and the linker and / or delivery. Once the position of the covalent bond is determined, those skilled in the art can select from a well-known list of coupling agents to form a selective link. Examples of coupling chemistry include, but are not limited to, Michael addition (i.e., thiosuccinamide), amidation, cycloaddition (e.g., 1,3-dipolar cycloaddition), and / or disulfide bonds. In each of the above, as a non-limiting example, additional portions of the molecule, such as straight-chain alkyl bridges, can be incorporated to more readily introduce the desired functional groups to selectively link the oligonucleotide to the linker and delivery moiety (also referred to as the α-linker). v β x(Integrin ligand) binding. Oligonucleotides can be coupled to the delivery site directly or via a linker. For example, an oligonucleotide can be coupled to linker site A in formula XI and contains α v β x Integrins, such as α v The delivery portion of the β6 integrin ligand can be covalently bonded to linker site B. Alternatively, the oligonucleotide can be coupled to the linker, and the delivery portion can also be coupled to the linker. For example, the oligonucleotide can be coupled to linker site A in formula XI and contains α v β x The delivery portion of the integrin ligand can be coupled to linker site B. Alternatively, the oligonucleotide can be covalently linked to the linker without any coupling portion or coupling step, and / or the delivery portion can be covalently linked to the linker without any coupling portion or coupling step.

[0097] The delivery portion and / or adapter may be attached to the 5' or 3' end of the oligonucleotide, or to one of the internal nucleotide or nucleoside bases. The delivery portion and / or adapter may also be attached to or coupled to the 5' or 3' end of the oligonucleotide. If the oligonucleotide is siRNA, the delivery portion and / or adapter may be attached to or coupled to the 5' or 3' end of either the sense or antisense strand of the oligonucleotide.

[0098] Those skilled in the art will also recognize that placing the delivery portion at the 5' end of the antisense strand, whether or not via a connector, may require overcoming potentially inefficient loading of the Ago2 load or other barriers to RISC complex activity. In other embodiments, the delivery portion is coupled to the 3' end of the sense strand. In yet another embodiment, the delivery portion is coupled to the 3' end of the sense strand via a connector.

[0099] Compounds containing oligonucleotides and delivery moieties

[0100] This document provides compounds that can selectively deliver oligonucleotides to the lungs. Suitable compounds include those described in formulas XV-XVIII. Other suitable compounds may include one or more oligonucleotides, comprising one or more α-nucleotides. v β x The ligand delivery moiety and optional oligonucleotides with α v β x Linkages between ligands. For example, suitable compounds may include oligonucleotides, containing α-ligands... v β x The delivery portion of the ligand and the linker that connects the oligonucleotide to the delivery portion. Other possible components are described below.

[0101]

[0102] Formula XV.

[0103] This paper also discloses novel compounds of formula XV, where L is a linker or is absent, and Z is an oligonucleotide. R1 can be H or C. 1- C6 alkyl. It can be (C5-C) 14 aryl)-O- or (C5-C)- having 1-5 heteroatoms selected from N, O and S 14 (Heteroary aryl)-O-. It can also be an aryl or heteroaryl group as shown in Formula IX and / or X. Pharmaceutically acceptable salts of compounds of Formula XV are also disclosed herein.

[0104]

[0105] Formula XVI.

[0106] This paper also discloses novel compounds of formula XVI, where L is a linker or is absent, and Z is an oligonucleotide. R1 can be H or C. 1- C6 alkyl. It can be (C5-C) 14 aryl)-O- or (C5-C)- having 1-5 heteroatoms selected from N, O and S 14 (Heteroary aryl)-O-. It can also be an aryl or heteroaryl group as shown in Formula IX and / or X. Pharmaceutically acceptable salts of compounds of Formula XVI are also disclosed herein.

[0107]

[0108] Formula XVII.

[0109] This paper also discloses novel compounds of formula XVII, wherein Z is an oligonucleotide. R1 can be H or C. 1- C6 alkyl. It can be (C5-C) 14 aryl)-O- or (C5-C)- having 1-5 heteroatoms selected from N, O and S 14 (Heteroaryl)-O-. q can be an integer from 1 to 3. It can also be an aryl or heteroaryl group as shown in Formula IX and / or X. Pharmaceutically acceptable salts of compounds of Formula XVII are also disclosed herein.

[0110]

[0111] Formula XVIII.

[0112] This article also discloses novel compounds of formulas XV-XVII, in which It is represented by one of the aryl or heteroaryl groups of formula XVIII.

[0113] Treatment

[0114] The compounds described herein, such as compounds of formula I-XVIII, can be used as a therapy for lung diseases. A suitable embodiment may be a pharmaceutical composition for administering a compound containing formula I-XVIII, used as a therapy or to treat a disease. Another embodiment may be a compound containing formula I-XVIII or a pharmaceutical composition thereof, used as a therapy. Another embodiment is wherein the therapy is for lung diseases. Another embodiment is a method of treating lung diseases comprising administering a compound disclosed herein, suitably a compound containing formula I-XVIII, suitably in an effective amount, or a composition of any of the foregoing. Another embodiment is a compound disclosed herein, suitably a compound containing formula I-XVIII or a pharmaceutical composition thereof, used to prepare a medicament suitable for treating lung diseases.

[0115] preparation

[0116] The compounds disclosed herein may be included in pharmaceutical formulations. Pharmaceutical formulations may contain one or more carriers, diluents, and excipients that are compatible with the compounds and other components of the composition or formulation and are harmless to patients. Examples of pharmaceutical compositions and methods of their preparation can be found in *Remington: The Science and Practice of Pharmacy*, edited by Loyd, V. et al., 22nd edition, Mack Publishing Co., 2012.

[0117] In some embodiments, the formulation may be designed for pulmonary delivery. Pharmaceutically acceptable carriers for pulmonary delivery are known in the art and vary depending on the desired location of active agent deposition, such as the upper or lower respiratory system, and the type of delivery device, such as a nebulizer, a sprayer, or a dry powder inhaler.

[0118] definition

[0119] As used in this article, the term "α" v β x "" refers to an integrative heterodimer protein, which has an α-molecule that is non-covalently bonded to the β1, β2, β3, β4, β5, β6, β7, or β8 protein subunits. v Protein subunits.

[0120] As used herein, the terms “a,” “an,” “the,” and similar terms used in the context of this disclosure (especially in the context of the claims) shall be interpreted to cover both the singular and the plural, unless otherwise indicated herein or there is a clear contradiction in the context.

[0121] As used herein, the term "alkyl" refers to a saturated straight-chain or branched monovalent hydrocarbon group containing an indicated number of carbon atoms. For example, "C1-C 20 "Alkyl" refers to a group having 1-20 carbon atoms arranged in a straight or branched chain.

[0122] As used in this article, the term "C1-C" n "Alkoxy" refers to a straight-chain or branched saturated hydrocarbon containing 1 to n carbon atoms and ending with an "O" in the chain, i.e., -O (alkyl). Examples of C1-C4 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, and butoxy.

[0123] As used herein, the term "aryl" refers to a group derived from an aromatic monocyclic, aromatic polycyclic, or one or more aromatic monocyclic rings linked by a single covalent bond, wherein the monocyclic or polycyclic ring contains only carbon atoms. An aryl group can be unsubstituted or substituted with 1 to 5 suitable substituents, which are well known to those skilled in the art. Aryl groups can be named by the total number of carbon atoms on the monocyclic or polycyclic ring. For example, C5-C7 aryl groups include aryl groups having 5, 6, or 7 carbon atoms.

[0124] As used herein, the term "heteroaryl" refers to a group derived from an aromatic monocyclic, aromatic polycyclic, or monocovalently linked aromatic monocyclic group, comprising one or more carbon atoms and one or more heteroatoms on the monocyclic or polycyclic group. Heteroaryls can be unsubstituted or substituted with 1 to 5 suitable substituents well known to those skilled in the art. Heteroaryls can be named by the total number of atoms in the monocyclic or polycyclic group. For example, 4- to 7-membered heteroaryls include 4, 5, 6, or 7 members (including carbon atoms and heteroatoms).

[0125] As used herein, “antisense strand” refers to a single-stranded oligonucleotide complementary to the target sequence region. Similarly, and as used herein, “sense strand” refers to a single-stranded oligonucleotide complementary to the antisense strand region.

[0126] As used herein, “complementary” refers to a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand, such as a hairpin) that allows the two nucleotides to form base pairs with each other. For example, a purine nucleotide in one nucleic acid may be complementary to a pyrimidine nucleotide in another opposing nucleic acid, and the two can pair bases by forming hydrogen bonds with each other. Complementary nucleotides can pair bases in a Watson-Crick manner or any other manner that allows for the formation of a stable duplex. Similarly, as described herein, two nucleic acids can have complementary polynucleotide regions to form complementary regions.

[0127] As used herein, a “double strand” involving nucleic acids or oligonucleotides refers to a structure formed by complementary base pairing (i.e., in opposite directions) of two antiparallel nucleotide sequences, whether formed by two separate nucleic acid chains or by a single folded single strand (e.g., by a hairpin).

[0128] "Effective dose" refers to the amount (for a given time period and method of administration) necessary to achieve the desired therapeutic effect. The effective dose of an RNAi substance can vary depending on a variety of factors, such as an individual's disease state, age, sex, and weight, as well as the RNAi substance's ability to elicit the desired response in the individual. The effective dose is also the amount in which any toxic or adverse effects of the RNAi substance are offset by the beneficial therapeutic effects.

[0129] The term "knockdown" or "expression knockdown" refers to the reduction of gene mRNA or protein expression after treatment with substances such as RNAi.

[0130] As used herein, "modified internucleotide link" refers to an internucleotide link that has one or more chemical modifications compared to a reference internucleotide link having a phosphodiester bond. The modified internucleotide link can be a non-naturally occurring bond. In some embodiments, the modified internucleotide link is a phosphate thioester link.

[0131] As used herein, a “modified nucleotide” refers to a nucleotide having one or more chemical modifications compared to a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymine deoxyribonucleotide. For example, a modified nucleotide may have one or more chemical modifications on its sugar, nucleobase, and / or phosphate ester groups. Alternatively, a modified nucleotide may have one or more chemical moieties coupled to a corresponding reference nucleotide. In some embodiments, the modified nucleotide is a 2'-fluoromodified nucleotide, a 2'-O-methylmodified nucleotide, or a 2'-O-alkylmodified nucleotide. In some embodiments, the modified nucleotide has a phosphate ester analog, such as 5'-vinylphosphonate. In some embodiments, the modified nucleotide has a base-free moiety or a reverse base-free moiety, as shown in Table 1, for example.

[0132] Table 1 - Base-free or reverse base-free (iAb) section

[0133]

[0134] “5’” and “3’” indicate the 5’ to 3’ direction of the sequence.

[0135] As used herein, "nucleotide" refers to an organic compound having a nucleoside (nucleobase, such as adenine, cytosine, guanine, thymine, or uracil, linked to a phosphate group, and a pentose, such as ribose or 2'-deoxyribose) attached to it. "Nucleotide" can be used as a monomeric unit for nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0136] As used herein, “oligonucleotide” refers to a polymer of linked nucleotides, each of which may be modified or unmodified. Oligonucleotides are typically less than about 100 nucleotides in length.

[0137] As used herein, a "protrusion" refers to one or more unpaired nucleotides that protrude from the double-stranded structure of a double-stranded oligonucleotide. A protrusion may include one or more unpaired nucleotides extending from the double-stranded region at the 5' or 3' end of the double-stranded oligonucleotide. A protrusion may be a 3' or 5' protrusion on the antisense or sense strand of the double-stranded oligonucleotide.

[0138] As used in this article, the term "patient" refers to a human patient.

[0139] As used herein, a "phosphate ester analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate ester group. In some embodiments, the phosphate ester analog is located on the 5' terminal nucleotide of the oligonucleotide to replace the 5'-phosphate, which is typically enzymatically removable. The 5' phosphate ester analog may include a phosphatase-resistant linker. Examples of phosphate ester analogs include 5'-methylenephosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP). In some embodiments, the phosphate ester analog is 5'-VP.

[0140] The term "% sequence identity" or "percentage of sequence identity" relative to a reference nucleic acid sequence is defined as the percentage of nucleotides, nucleosides, or nucleobases in a candidate sequence that are identical to those in the reference nucleic acid sequence after optimal alignment and the introduction of gaps or overhangs (if necessary) to achieve maximum percentage sequence identity. Sequence alignment used to determine the percentage of nucleic acid sequence identity can be performed in various ways within the scope of those skilled in the art, such as using publicly available computer software programs, for example, those described in Current Protocols in Molecular Biology (edited by Ausubel et al., 1987, Supplement 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 sequence alignment, including any algorithms required to achieve maximum sequence alignment across the full length of the compared sequences. The percentage of "sequence identity" is determined by comparing two best-aligned sequences within a comparison window. This comparison window may include additions or deletions (e.g., gaps or protrusions) compared to the reference sequence (excluding additions or deletions) to achieve optimal sequence alignment. The percentage is calculated as follows: determine the number of positions in both sequences where the same nucleotide, nucleoside, or nucleotide base appears, thus obtaining the number of matching positions; divide this number of matching positions by the total number of positions in the comparison window; multiply the result by 100 to obtain the sequence identity percentage. The output is the percentage of identity between the subject sequence and the query sequence.

[0141] As used herein, “RNAi,” “RNAi substance,” “iRNA,” “iRNA material,” and “RNA interfering agent” refer to substances that mediate the sequence-specific degradation of target mRNA through RNA interference, such as via the RNA-induced silencing complex (RISC) pathway. In some embodiments, the RNAi substance has a sense strand and an antisense strand, and the sense strand and antisense strand form a double strand (e.g., double-stranded RNA). In some embodiments, the sense strand has a delivery portion coupled to the 3' end of the sense strand or to a nucleotide of the sense strand.

[0142] As used herein, a “chain” refers to a single, continuous sequence of nucleotides linked together by internucleotide linkages (e.g., phosphodiester linkages or thiophosphate linkages). A chain may have two free ends (e.g., a 5' end and a 3' end).

[0143] As used herein, “subject” refers to mammals, including cats, dogs, mice, rats, chimpanzees, apes, monkeys, and humans. Humans are preferred as subjects.

[0144] As used herein, “treatment” means the entire process by which the progression of the disorder or disease disclosed herein may be slowed, controlled, delayed, or terminated, or the symptoms of the disorder or disease may be improved, but does not necessarily indicate the complete elimination of all disorders or symptoms. Treatment includes the administration of proteins or nucleic acids or carriers or compositions for the treatment of a patient, particularly a human, disease or condition.

[0145] Example

[0146] Example 1. Synthesis of integrin-targeting ligands

[0147] The following abbreviations are defined as follows: "THF" refers to tetrahydrofuran; "TEMPO" refers to 2,2,6,6-tetramethylpiperidinoxy; "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'-binaphthyl; "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)ureon 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-oxide 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 isopropanol; "RBF" refers to round-bottom flask; and "SFC" refers to supercritical fluid chromatography.

[0148] Option 1

[0149]

[0150] Scheme 1, step A, describes the reduction of compound (1) in a suitable solvent, such as THF, using a suitable reducing agent, such as LiBH4, to obtain compound (2). Step B describes the TEMPO-catalyzed oxidation of compound (2) in a suitable solvent, such as ACN, using an oxidizing agent, such as NaClO, to obtain compound (3).

[0151] Option 2

[0152]

[0153] Scheme 2, step A describes the Wittig reaction of compound (4) with a suitable reagent, such as compound (5), in a suitable solvent, such as EtOAc, to give α,β-unsaturated ester compound (6).

[0154] Option 3

[0155]

[0156] Scheme 3, step A describes the use of a suitable catalyst, such as [Rh(COD)Cl]2, and a suitable chiral ligand, such as ( R The Hayashi asymmetric 1,4-addition of arylboronic acid (7) to crotonate (6) with arylboronic acid (7) and a suitable base such as Cs2CO3 in a suitable solvent such as dioxane yields compound (8). Step B describes the Suzuki reaction of compound (8) with boric acid (9) using a catalyst such as 1,1'-bis(diphenylphosphino)ferrocene-palladium dichloride (II), a suitable base such as K2CO3, and a suitable solvent system such as a mixture of dioxane and water to yield compound (10). Step C describes the deprotection of the Boc-protecting group using a suitable reagent such as HCl in a suitable solvent such as EtOAc to yield compound (11). Step D describes the reductive amination reaction of compounds (11) and (3) with a reducing agent such as sodium triacetoxyborohydride in a suitable solvent such as MeOH to yield compound (12).

[0157] Option 4

[0158]

[0159] Scheme 4, step A describes the use of a suitable catalyst, such as [Rh(COD)Cl]2, and a suitable chiral ligand, such as ( RThe Hayashi asymmetric 1,4-addition of arylboronic acid (13) to p-crotonic acid (6) with a suitable base such as Cs₂CO₃ in a suitable solvent such as dioxane yields compound (14). Step B describes the deprotection of the Boc-protecting group using a suitable reagent such as HCl in a suitable solvent such as EtOAc to yield compound (15). Step C describes the reductive amination reaction of compounds (15) and (3) with a reducing agent such as sodium triacetoxyborohydride in a suitable solvent such as MeOH to yield compound (16).

[0160] Option 5

[0161]

[0162] Scheme 5, step A describes the use of a suitable catalyst, such as [Rh(COD)Cl]2, and a suitable chiral ligand, such as ( R The Hayashi asymmetric 1,4-addition of crotonate (6) to aryl borate (17) with aryl borate (17) in a suitable solvent, such as dioxane, yields compound (18). Step B describes the Suzuki reaction of compound (18) with boric acid (9) using a catalyst such as 1,1'-bis(diphenylphosphino)ferrocene-palladium dichloride (II), a suitable base such as K2CO3, and a suitable solvent system such as a mixture of dioxane and water, to yield compound (19). Step C describes the deprotection of the Boc-protecting group using a suitable reagent such as HCl in a suitable solvent such as EtOAc to yield compound (20). Step D describes the reductive amination reaction of compounds (20) and (3) with a reducing agent such as sodium triacetoxyborohydride in a suitable solvent such as MeOH to yield compound (21).

[0163] Option 6

[0164]

[0165] Scheme 6, step A describes the use of a suitable catalyst, such as [Rh(COD)Cl]2, and a suitable chiral ligand, such as ( RThe Hayashi asymmetric 1,4-addition of arylboronic acid ester (22) to crotonate (6) with arylboronic acid ester (22) in a suitable solvent such as dioxane yields compound (23). Step B describes the demethylation reaction with a suitable Lewis acid such as BBr3 in a suitable solvent such as DCE, followed by step C, which describes the esterification reaction with SOCl2 and MeOH, yielding compound (24). Step D describes the reductive amination reaction of compounds (24) and (3) with a reducing agent such as sodium triacetoxyborohydride in a suitable solvent such as MeOH, yielding compound (25).

[0166] Option 7

[0167]

[0168] Scheme 7, step A, describes the 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 the 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.

[0169] Option 8

[0170]

[0171] Scheme 8, step A describes the toluenesulfonation of compound (28) with compound (29) in a solvent such as DCM using a suitable base such as triethylamine and a suitable catalyst such as DMAP to obtain compound (30).

[0172] Option 9

[0173]

[0174] Scheme 9, step A, describes the nucleophilic substitution reaction of compounds (31) and (30) using a suitable base, such as Cs₂CO₃, in a suitable solvent, such as DMF, to give compound (32). Step B shows the ester hydrolysis of compound (32) using a base, such as LiOH, in a solvent system, such as MeOH and water, to give compound (33a). Step C describes the deprotection reaction using an acid, such as an aqueous solution of HCl, to give compound (33). X is C or N.

[0175] Option 10

[0176]

[0177] Scheme 10, step A, describes the nucleophilic substitution reaction of compounds (34) and (30) using a suitable base, such as Cs₂CO₃, in 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 describes the deprotection reaction using an acid, such as an aqueous solution of HCl, to give compound (36). X is C or N.

[0178] Option 11

[0179]

[0180]

[0181] Scheme 11, step A describes the amide coupling of compound (37) with di-tert-butyl(azanediylbis(ethane-2,1-diyl))dicarbamate using reagents such as HBTU and HOBt, bases such as DIPEA, and solvents such as DMF to give compound (38). Step B shows the ester hydrolysis of compound (38) using bases such as NaOH and solvent systems such as THF and MeOH to give compound (39). Step C describes the amide coupling of compound (39) with ethyl 6-aminohexanoate hydrochloride using reagents such as HBTU and HOBt, bases such as DIPEA, and solvents such as DMF to give compound (40). Step D shows the deprotection of compound (40) using an acid such as HCl in a solvent such as diethyl ether to give compound (41). Step E describes the amide coupling of compound (41) with propargyl-PEG5-acid using a reagent such as HATU, a base such as DIPEA, and a solvent such as DMF to give compound (42). Step F shows the 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 of compound (43) with 1-hydroxypyrrolidine-2,5-dione using a suitable reagent such as EDCI·HCl and a solvent such as DCM to give succinate compound (44).

[0182] Optional ligand synthesis

[0183] As illustrated in schemes 12 and 13, fully formulated targeting ligands, such as NHS esters, can be constructed prior to conjugation with the RNAi substance. After conjugation of the ligand with an amine-functionalized sense strand, the methyl ester on the ligand is hydrolyzed with lithium hydroxide. The ligand-conjugated sense strand is then annealed to an antisense strand to obtain the RNAi substance. This method avoids introducing copper into the RNAi substance.

[0184] Option 12

[0185]

[0186]

[0187] Scheme 11, step A describes the copper-catalyzed cycloaddition (CuAAC) of compounds (43) and (45) to compound (46) using a suitable copper catalyst, such as CuSO4·5H2O, a reagent such as THPTA, and sodium ascorbate in a solvent system such as DMF and water. Step B shows the Boc-deprotection of compound (46) using an acid such as TFA and in a solvent such as DCM to give compound (47). Step C describes the coupling of compound (47) with 1-hydroxypyrrolidine-2,5-dione using a reagent such as EDCI·HCl and in a solvent such as DCM to give succinate compound (48).

[0188] Option 13

[0189]

[0190] Scheme 13, step A describes the copper-catalyzed cycloaddition (CuAAC) of compound (45) and tert-butyl 4,7,10,13,16-pentaenoadecan-18-acetylic acid using a suitable copper catalyst such as CuSO4·5H2O, reagents such as THPTA and sodium ascorbate 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 its salt. Step C describes the coupling reaction of compound (50) and 1-hydroxypyrrolidine-2,5-dione using a reagent such as EDCI·HCl and in a solvent such as DCM to give succinate compound (51).

[0191] Option 14

[0192]

[0193] Scheme 14, step A, describes the nucleophilic substitution reaction of compounds (31) and (52) using a suitable base, such as Cs₂CO₃, in a suitable solvent, such as DMF, to give compound (53). Step B shows the deprotection of compound (53) using a dioxane solution of an acid, such as HCl, to give compound (54). X is C or N.

[0194] Option 15

[0195]

[0196] Scheme 15, step A, describes the nucleophilic substitution reaction of compounds (34) and (52) using a suitable base, such as Cs₂CO₃, in a suitable solvent, such as DMF, to give compound (55). Step B shows the deprotection of compound (55) using a dioxane solution of an acid, such as HCl, to give compound (56). X is C or N.

[0197] Option 16

[0198]

[0199] Scheme 16, step A, describes the esterification of compounds (57) and (58) using a suitable reagent, such as thionyl chloride, in a suitable solvent, such as THF, to give compound (59). Step B describes the amidation coupling reaction of compounds (59) and (39) using reagents such as HBTU and HOBt, a base such as DIPEA, and a solvent such as DMF, to give compound (60). Step C describes the deprotection of compound (60) using a dioxane solution of an acid such as HCl to give compound (61).

[0200] Option 17

[0201]

[0202] Scheme 17, step A describes the amidation coupling reaction of 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 describes the hydrogenolysis of compound (62) using hydrogen, a suitable catalyst such as Pd / C, and a suitable solvent such as MeOH to give compound (63). Step C shows the coupling reaction of compound (63) and 1-hydroxypyrrolidine-2,5-dione using a reagent such as EDCI·HCl and a solvent such as DCM to give succinate compound (64). X is C or N.

[0203] Option 18

[0204]

[0205] Scheme 18, step A describes the amide coupling reaction of 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 describes the hydrogenolysis of compound (65) using hydrogen, a suitable catalyst such as Pd / C, and a suitable solvent such as MeOH to give compound (66). Step C shows the coupling reaction of compound (66) and 1-hydroxypyrrolidine-2,5-dione using a reagent such as EDCI·HCl and a solvent such as DCM to give succinate compound (67). X is C or N.

[0206] Preparation Example 1

[0207] 7-(4-hydroxybutyl)-3,4-dihydro-1,8-naphthyl-1(2H)-carboxylic acid tert-butyl ester

[0208]

[0209] LiBH4 (2M, 688.8 mL, 1378 mmol in THF) was added to a solution of tert-butyl 7-(4-ethoxy-4-oxobutyl)-3,4-dihydro-1,8-naphthidine-1(2H)-carboxylate (240 g, 688.8 mmol) in THF (1680 mL) at 0 °C. After addition, the mixture was stirred at ambient temperature for 16 h. The mixture was cooled to 0 °C, quenched with aqueous NH4Cl solution, 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 under vacuum to give the title compound as a colorless oil (204 g, 96.6%). ES / MS m / z 307.2 (M+H).

[0210] Preparation Example 2

[0211] 7-(4-O-butyl)-3,4-dihydro-1,8-naphthyl-1(2H)-carboxylic acid tert-butyl ester

[0212]

[0213] A mixture of tert-butyl 7-(4-hydroxybutyl)-3,4-dihydro-1,8-naphthyl-1(2H)-carboxylate (204 g, 666 mmol), TEMPO (20.9 g, 133 mmol), and NaHCO3 (559 g, 6666 mmol) in ACN (1500 mL) was degassed with N2. NaClO (820 mL, 1332 mmol) was added at 0 °C, and the resulting mixture was stirred at 20 °C for 2 h. The mixture was diluted with water and extracted with EtOAc. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over Na2SO4, filtered, and concentrated under vacuum to give the title compound (141 g, 69.4%) as a yellow oil. ES / MS m / z 323.1 (M+18).

[0214] Preparation Example 3

[0215] (E)-4-((tert-butoxycarbonyl)(methyl)amino)but-2-enoic acid methyl ester

[0216]

[0217] In a 3-necked flask containing a solution of methyl(triphenylphosphoranylidene)acetate (700 g, 4.04 mol) in EtOAc (4.9 L), methyl(triphenylphosphoranylidene)acetate (2.33 kg, 6.78 mol) was added in portions. The mixture was stirred for 1 hour under an argon atmosphere and filtered. The filtrate was concentrated under vacuum to obtain a residue, which was purified by silica gel chromatography using PE solution of 5–20% EtOAc to give the title compound as a colorless oil (730 g, 78.8%). 1 H 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).

[0218] Preparation Example 4

[0219] Methyl 3-(3-bromophenyl)-4-((tert-butoxycarbonyl)(methyl)amino)butyrate

[0220]

[0221] 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), 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) were added. The mixture was stirred at 90 °C for 12 hours. The cooled mixture was quenched with water (100 mL) and extracted with EtOAc (330 mL). The organic layer was washed with a saturated aqueous sodium chloride solution (100 mL), dried over Na₂SO₄, filtered, and concentrated under vacuum. The crude residue was purified by reversed-phase rapid chromatography to give the title compound (77.0 g, 83.1%) as a yellow oil. ES / MS m / z 287.0 ([M-Boc]+H).

[0222] Preparation Example 5

[0223] (S)-4-((tert-butoxycarbonyl)(methyl)amino)-3-(4'-hydroxy-[1,1'-biphenyl]-3-yl)methyl butyrate (isomer 2)

[0224]

[0225] To a mixture of methyl 3-(3-bromophenyl)-4-((tert-butoxycarbonyl)(methyl)amino)butyrate (75.0 g, 194 mmol) in 1,4-dioxane (400 mL) and water (100 mL), 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) were added. 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 a saturated aqueous sodium chloride solution, dried over Na₂SO₄, filtered, and concentrated under vacuum. The crude residue was purified by silica gel rapid chromatography, eluting with PE solution containing 0-80% EtOAc, to give a yellow oil (75.0 g, 96.7%, 90% ee). Further purification was performed by chiral SFC (DAICEL Chiralpak IC 250 x 50 mm column, 10 μm; 25% IPA / CO2, isocratic, retention time 1.86 min) to give the title compound, the 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, based on literature precedent, it is assumed that this compound possesses an S stereocenter ( J. Med. Chem. 2018, 61, 8417-8443).

[0226] Preparation Example 6

[0227] (S)-3-(4'-hydroxy-[1,1'-biphenyl]-3-yl)-4-(methylamino)butyrate methyl hydrochloride

[0228]

[0229] HCl (4 M, 360 mL, 1.44 mol in EtOAc, tert-butoxycarbonyl)(methyl)amino) was added to a solution of methyl (S)-4-((tert-butoxycarbonyl)(methyl)amino)-3-(4'-hydroxy-[1,1'-biphenyl]-3-yl)butyrate (isomer 2, 45.0 g, 112.6 mmol) in EtOAc (90 mL). The mixture was stirred at 25 °C for 1 h and concentrated under vacuum. The title compound (27 g, 71.3%) was given as a white solid. ES / MS m / z 300.1 ([M-HCl]+H).

[0230] Preparation Example 7

[0231] (S)-7-(4-((2-(4'-hydroxy-[1,1'-biphenyl]-3-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyl-1(2H)-tert-butyl formate

[0232]

[0233] A mixture of (S)-3-(4'-hydroxy-[1,1'-biphenyl]-3-yl)-4-(methylamino)butyrate methyl hydrochloride (18.0 g, 53.6 mmol) and 7-(4-oxobutyl)-3,4-dihydro-1,8-naphthyl-1(2H)-carboxylate tert-butyl ester (22.8 g, 74.9 mmol) in MeOH (125 mL) was degassed with N2. The mixture was stirred at 25 °C for 30 min. 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 h. The mixture was poured into a saturated aqueous solution of NaHCO3 (200 mL) and extracted with EtOAc (2 x 200 mL). The organic layer was washed with a saturated aqueous solution of sodium chloride, dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by silica gel rapid chromatography, eluting with EtOAc solution containing 0-10% MeOH, to give the title compound as a brown solid (20 g, 63.5%). ES / MS m / z 588.4 (M+H).

[0234] Preparation Example 8

[0235] (S)-4-((tert-butoxycarbonyl)(methyl)amino)-3-(3-hydroxyphenyl)butyrate methyl ester (isomer 2)

[0236]

[0237] Following a method substantially similar to that used in Preparation Example 4, 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) to give 100 g (88.6%) of a yellow oil. 50 g of this oil was then purified by chiral SFC (DAICEL Chiralpak IC 50 x 250 mm column, 10 μm; 20% IPA / CO2, 0.1% NH3H2O, isocratic, retention time 2.096 min) to give the title compound as a yellow oil, the second eluting isomer (45 g, 90%, 99.52% ee). ES / MS m / z 224.2 ([M-Boc]+H). Although we do not wish to be bound by theory, based on prior literature, we believe that this compound possesses an S stereocenter ( J. Med. Chem. 2018, 61, 8417-8443).

[0238] Preparation Example 9

[0239] (S)-3-(3-hydroxyphenyl)-4-(methylamino)butyrate methyl hydrochloride

[0240]

[0241] The title compound was prepared from methyl (S)-4-((tert-butoxycarbonyl)(methyl)amino)-3-(3-hydroxyphenyl)butyrate (isomer 1, 45 g, 139.2 mmol) in a manner substantially similar to that used in Preparation Example 6, yielding the title compound (34 g, 94.2%) as a yellow oil. ES / MS m / z 224.1 ([M-HCl]+H).

[0242] Preparation Example 10

[0243] (S)-7-(4-((2-(3-hydroxyphenyl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyl-1(2H)-carboxylic acid tert-butyl ester

[0244]

[0245] The title compound was prepared in a manner substantially similar to that used in Preparation Example 7, from methyl (S)-3-(3-hydroxyphenyl)-4-(methylamino)butyrate hydrochloride (18.0 g, 69.3 mmol) and tert-butyl 7-(4-oxobutyl)-3,4-dihydro-1,8-naphthidine-1(2H)-carboxylate (27.42 g, 90.1 mmol), yielding a yellow, oily solid title compound (20.0 g, 56.4%). ES / MS m / z 512.3 (M+H).

[0246] Preparation Example 11

[0247] Methyl 3-(6-bromopyridin-2-yl)-4-((tert-butoxycarbonyl)(methyl)amino)butyrate

[0248]

[0249] The title compound was prepared in a manner substantially similar to that used in Preparation Example 4, 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-dioxaborhexacyclopentan-2-yl)pyridine (152 g, 535.3 mmol), yielding the title compound (34.4 g, 33.3%) as a yellow oil. ES / MS m / z 389.2 (M+H).

[0250] Preparation Example 12

[0251] (R)-4-((tert-butoxycarbonyl)(methyl)amino)-3-(6-(4-hydroxyphenyl)pyridin-2-yl)butyrate (isomer 2)

[0252]

[0253] The title compound was prepared in a manner substantially similar to that used in Preparation Example 5, from methyl 3-(6-bromopyridin-2-yl)-4-((tert-butoxycarbonyl)(methyl)amino)butyrate (34.4 g, 88.8 mmol) and (4-hydroxyphenyl)boronic acid (24.5 g, 177.7 mmol), yielding the title compound as a brown oil, the second eluting isomer (27 g, 75.8%, 99.8% ee). ES / MS m / z 401.3 (M+H). Although not wishing to be bound by theory, based on prior literature, this compound is considered to possess an R stereocenter ( J. Med. Chem. 2018, 61, 8417-8443).

[0254] Preparation Example 13

[0255] (R)-3-(6-(4-hydroxyphenyl)pyridin-2-yl)-4-(methylamino)butyrate methyl hydrochloride

[0256]

[0257] The title compound was prepared from (R)-4-((tert-butoxycarbonyl)(methyl)amino)-3-(6-(4-hydroxyphenyl)pyridin-2-yl)butyrate (isomer 2, 27.0 g, 67.5 mmol) in a manner substantially similar to that used in Example 6, yielding the title compound as a yellow solid (24.5 g, 99+%). ES / MS m / z 301.0 ([M-HCl]+H).

[0258] Preparation Example 14

[0259] (R)-7-(4-((2-(6-(4-hydroxyphenyl)pyridin-2-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyl-1(2H)-carboxylic acid tert-butyl ester

[0260]

[0261] The title compound was prepared in a manner substantially similar to that used in Preparation Example 7, from methyl (R)-3-(6-(4-hydroxyphenyl)pyridin-2-yl)-4-(methylamino)butyrate hydrochloride (24.5 g, 72.7 mmol) and tert-butyl 7-(4-oxobutyl)-3,4-dihydro-1,8-naphthyl-1(2H)-carboxylate (41.1 g, 135 mmol), yielding the title compound as a brown solid (17.4 g, 40.6%). ES / MS m / z 589.4 (M+H).

[0262] Preparation Example 15

[0263] (S)-4-((tert-butoxycarbonyl)(methyl)amino)-3-(5-methoxypyridin-3-yl)butyrate methyl ester (isomer 2)

[0264]

[0265] Following a method substantially similar to that used in Preparation Example 4, 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-dioxaborhexacyclopentan-2-yl)pyridine (123 g, 523.2 mmol), yielding 46 g of a yellow oil (51.9%, 90% ee). This oil was then purified by chiral SFC (DAICEL Chiralpak IC 50 x 250 mm column, 10 μm; 20% MeOH / CO2, 0.1% NH3H2O, isocratic, retention time 2.43 min) to give the title compound as a yellow oil, 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, based on prior literature, it is assumed that this compound possesses an S stereocenter. J. Med. Chem. 2018, 61, 8417-8443).

[0266] Preparation Examples 16 & 17

[0267] (S)-3-(5-hydroxypyridin-3-yl)-4-(methylamino)butyrate methyl hydrochloride

[0268]

[0269] BBr3 (63.9 mL, 673.2 mmol) was added to a solution of (S)-4-((tert-butoxycarbonyl)(methyl)amino)-3-(5-methoxypyridin-3-yl)butyrate (isomer 2, 38 g, 112.2 mmol) in DCE (210 mL). The mixture was stirred at 25 °C for 12 h and quenched by adding MeOH (210 mL). The mixture was concentrated under vacuum to give a mixture of the desired product and an acidic byproduct (hydrolysis of methyl ester).

[0270] The mixture was dissolved in MeOH (105 mL) and degassed with N2. SOCl2 (31.2 mL, 430 mmol) was added dropwise to the mixture. The reaction mixture was stirred at 45 °C under N2 atmosphere for 1 hour and concentrated under vacuum to give the title compound as a purple solid (30 g, 99+%). ES / MS m / z 225.2 ([M-HCl]+H).

[0271] Preparation Example 18

[0272] (S)-7-(4-((2-(5-hydroxypyridin-3-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyl-1(2H)-tert-butyl carboxylate

[0273]

[0274] The title compound was prepared in a manner substantially similar to that used in Preparation Example 7, from methyl (S)-3-(5-hydroxypyridin-3-yl)-4-(methylamino)butyrate hydrochloride (24 g, 92.1 mmol) and tert-butyl 7-(4-oxobutyl)-3,4-dihydro-1,8-naphthyl-1(2H)-carboxylate (22.41 g, 73.62 mmol), yielding the title compound (20.3 g, 43%) as a brown oily solid. ES / MS m / z 513.3 (M+H).

[0275] Preparation Example 19

[0276] (S)-3-(4'-hydroxy-[1,1'-biphenyl]-3-yl)-4-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)butyric acid

[0277]

[0278] To (S)-7-(4-((2-(4'-hydroxy-[1,1'-biphenyl]-3-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyl-1(2H)-carboxylic acid tert-butyl ester (59 mg, 0.10 mmol) in THF (1 mL), an aqueous solution of lithium hydroxide (1N, 0.30 mL, 0.30 mmol) was added. The mixture was stirred at ambient temperature for 16 hours and then concentrated under vacuum. 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 under vacuum. The crude residue was purified by high-pH preparative reversed-phase chromatography, eluting with ACN solution of 5-100% 10 mM NH4HCO3 / 5% MeOH, to give the title compound as a white powder (15.8 mg, 33%). ES / MSm / z 474.2 (M+H).

[0279] Preparation Example 20

[0280] (S)-3-(3-hydroxyphenyl)-4-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)butyric acid

[0281]

[0282] The title compound was prepared in a manner substantially similar to that used in Preparation Example 19, using (S)-7-(4-((2-3-hydroxyphenyl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthidine-1(2H)-carboxylic acid tert-butyl ester (100 mg, 0.195 mmol), yielding the title compound as a white solid (55.4 mg, 71.3%). ES / MS m / z 398.2 (M+H).

[0283] Preparation Example 21

[0284] (R)-3-(6-(4-hydroxyphenyl)pyridin-2-yl)-4-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)butyric acid

[0285]

[0286] The title compound was prepared in a manner substantially similar to that used in Preparation Example 19, using (R)-7-(4-((2-(6-(4-hydroxyphenyl)pyridin-2-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthidine-1(2H)-carboxylic acid tert-butyl ester (50 mg, 0.085 mmol), yielding the title compound as a white solid (11 mg, 27%). ES / MS m / z 475.2 (M+H).

[0287] Preparation Example 22

[0288] (S)-3-(5-hydroxypyridin-3-yl)-4-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)butyric acid

[0289]

[0290] The title compound was prepared in a manner substantially similar to that used in Preparation Example 19, using (S)-7-(4-((2-(5-hydroxypyridin-3-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthidine-1(2H)-carboxylic acid tert-butyl ester (50 mg, 0.098 mmol), yielding the title compound as a white solid (20 mg, 51%). ES / MS m / z 399.2 (M+H).

[0291] Preparation Example 23

[0292] 14-Azide-3,6,9,12-Tetraoxatetradecyl ester of 4-methylbenzenesulfonic acid

[0293]

[0294] Triethylamine (54.8 mL, 393 mmol) and DMAP (3.2 g, 26.2 mmol) were added to a stirred solution of 14-azido-3,6,9,12-tetraoxatetradecane-1-ol (69 g, 262 mmol) in DCM (500 mL). 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 h. The mixture was quenched with a saturated aqueous NH4Cl solution and stirred for 5 min. The layers were separated. The organic layer was washed with a saturated aqueous sodium chloride solution, dried over MgSO4, filtered, and concentrated under vacuum. The crude residue was purified by silica gel chromatography using a cyclohexane solution of 30–60% EtOAc to give the title compound as a yellow liquid (80.44 g, 73.5%). ES / MS m / z 435.4 (M+18).

[0295] Preparation Example 24

[0296] (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-naphthyl-1(2H)-tert-butyl formate

[0297]

[0298] Add (S)-7-(4-((2-(4'-hydroxy-[1,1'-biphenyl]-3-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyl-1(2H)-carboxylic acid tert-butyl ester (700 mg, 1.19 mmol), 14-azido-3,6,9,12-tetraoxatetradecyl ester of 4-methylbenzenesulfonic acid (746 mg, 1.79 mmol), cesium carbonate (582 mg, 1.79 mmol), and DMF (6 mL) to a vial. Degas the mixture with N2 and stir at 40 °C for 3 hours. Dilute the cooled mixture with water and extract three times with EtOAc. Wash the organic layer with water and saturated sodium chloride aqueous solution, dry with MgSO4, filter, and concentrate under vacuum. The crude residue was purified by silica gel rapid chromatography, eluting with a hexane solution of 0-100% acetone, to give the title compound as a viscous yellow oil (777 mg, 78.3%). ES / MS m / z 834.4 (M+H).

[0299] Preparation Example 25

[0300] (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-naphthidin-2-yl)butyl)amino)butyric acid

[0301]

[0302] MeOH (3 mL) and an aqueous solution of lithium hydroxide (1 N, 2.79 mL, 2.79 mmol) were added to (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-naphthyl-1(2H)-carboxylic acid tert-butyl ester (775 mg, 0.93 mmol). The mixture was stirred at ambient temperature for 16 hours and then concentrated under vacuum. The residue was dissolved in water, acidified with an aqueous solution of 1 N HCl, and concentrated under vacuum to remove the Boc- group. The crude residue was purified by high-pH reversed-phase chromatography using an ACN solution of 0-100% 10mM NH4HCO3 / 5% MeOH to give the title compound as a viscous yellow oil (415 mg, 62.1%). ES / MS m / z 720.0 (M+H).

[0303] Preparation Example 26

[0304] (S)-3-(3-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)phenyl)-4-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)butyric acid

[0305]

[0306] Following a manner substantially similar to that used in Preparation Examples 24 and 25, the title compound was prepared using (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-naphthyl-1(2H)-carboxylic acid tert-butyl ester (1.29 g, 2.52 mmol) and 14-azido-3,6,9,12-tetraoxatetradecyl ester of 4-methylbenzenesulfonic acid (1.58 g, 3.78 mmol) to give the title compound as a viscous yellow oil (711 mg, 43.9%, obtained in 2 steps). ES / MS m / z 644.2 (M+H).

[0307] Preparation Example 27

[0308] (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-naphthidin-2-yl)butyl)amino)butyric acid

[0309]

[0310] Following a manner substantially similar to that used in Preparation Examples 24 and 25, the title compound was prepared using (R)-7-(4-((2-(6-(4-hydroxyphenyl)pyridin-2-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyl-1(2H)-carboxylic acid tert-butyl ester (300 mg, 0.510 mmol) and 14-azido-3,6,9,12-tetraoxatetradecyl ester of 4-methylbenzenesulfonic acid (319 mg, 0.764 mmol), yielding the title compound as a viscous yellow oil (246 mg, 67%, obtained in 2 steps). ES / MS m / z 721.4 (M+H).

[0311] Preparation Example 28

[0312] (S)-3-(5-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)pyridin-3-yl)-4-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)butyric acid

[0313]

[0314] The title compound was prepared in a manner substantially similar to that used in Preparation Examples 24 and 25, using (S)-7-(4-((2-(5-hydroxypyridin-3-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthidine-1(2H)-carboxylic acid tert-butyl ester (1.00 g, 1.95 mmol) and 14-azido-3,6,9,12-tetraoxatetradecyl ester of 4-methylbenzenesulfonic acid (1.22 g, 2.93 mmol), yielding the title compound as a viscous yellow oil (548 mg, 43.6%, obtained via 2 steps). ES / MS m / z 645.2 (M+H).

[0315] Preparation Example 29

[0316] N2-(tert-butoxycarbonyl)-N5,N5-bis(2-((tert-butoxycarbonyl) ) (amino)ethyl)-L-glutamic acid methyl ester

[0317]

[0318] DIPEA (21.8 mL, 126 mmol) was added to 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). The mixture was stirred at ambient temperature for 5 min, and then di-tert-butyl (azadimethylbis(ethyl-2,1-dimethyl))dicarbamate (14.1 g, 46.3 mmol) was added. The reaction mixture was stirred in a nitrogen atmosphere for 14 h. The mixture was diluted with EtOAc and washed with water and a saturated aqueous sodium chloride solution. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The crude residue was purified by silica gel chromatography, eluting with DCM solution of 0–20% MeOH, to give the title compound as a milky white foam (17.64 g, 76.6%). ES / MS m / z 447.4 ([M-Boc]+H).

[0319] Preparation Example 30

[0320] N2-(tert-butoxycarbonyl)-N5,N5-bis(2-((tert-butoxycarbonyl) ) (amino)ethyl)-L-glutamine

[0321]

[0322] N2-(tert-butoxycarbonyl)-N5,N5-bis(2-((tert-butoxycarbonyl) ) (Amino)ethyl)-L-glutamic acid methyl ester (17.6 g, 32.2 mmol) was dissolved in THF (100 mL) and MeOH (100 mL). Sodium hydroxide aqueous solution (1 N, 96.6 mL, 96.6 mmol) was added, and the mixture was stirred at ambient temperature for 1 hour. The mixture was concentrated under vacuum to remove the organic layer. The residue was acidified to pH 3 with 5 N HCl and extracted three times with DCM. The organic layer was washed with saturated sodium chloride aqueous solution, dried over MgSO4, filtered, and concentrated under vacuum to give the title compound (14.54 g, 84.8%) as a milky white foam. ES / MS m / z 433.4 ([M-Boc]+H).

[0323] Preparation Example 31

[0324] (S)-12-((tert-butoxycarbonyl) ) (amino)-8-(2-((tert-butoxycarbonyl) ) (Amino)ethyl)-2,2-dimethyl-4,9,13-trioxo-3-oxa-5,8,14-triazaeicosano-20-olate

[0325]

[0326] Following a method substantially similar to that used in Preparation Example 29, N2-(tert-butoxycarbonyl)-N5,N5-bis(2-((tert-butoxycarbonyl) ) The title compound was prepared from (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 (9.16 g, 49.9%) as a milky white foam. ES / MS m / z 674.4 (M+H).

[0327] Preparation Example 32

[0328] (S)-6-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanoylamino)ethyl hexanoate trihydrochloride

[0329]

[0330] HCl (4N in 1,4-dioxane) (51 mL, 200 mol) was added to (S)-12-((tert-butoxycarbonyl) ) (amino)-8-(2-((tert-butoxycarbonyl) ) The mixture was stirred at ambient temperature in ethyl (aminoethyl)-2,2-dimethyl-4,9,13-trioxo-3-oxa-5,8,14-triazaeicosano-20-oate (9.1 g, 14 mmol). After 10 min, the mixture became a thick suspension. 50 mL of diethyl ether was added to prepare a more easily stirred suspension. After 1 hour, the mixture was concentrated under vacuum to give the title compound as a creamy white solid (6.12 g, 94%). ES / MS m / z 374.4 ([M-3HCl]+H).

[0331] Preparation Example 33

[0332] (S)-27-(4,7,10,13,16-pentoxanonadecan-18-ynylamino)-19,24,28-trioxo-23-(4-oxo-7,10,13,16,19-pentoxa-3-azatetracosane-21-yn-1-yl)-4,7,10,13,16-pentoxa-20,23,29-triazapentadecane-1-yn-35-ethyl ester

[0333]

[0334] To a mixture of (S)-6-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanoylamino)hexanoic acid ethyl ester trihydrochloride (6.12 g, 12.7 mmol) and propargyl-PEG5-acid (13.5 g, 44.4 mmol) in DMF (180 mL), HATU (16.9 g, 44.4 mmol) and DIPEA (26.3 mL, 152 mmol) were added. 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 NaHCO3 aqueous solution, and saturated sodium chloride aqueous solution. The organic layer was dried with MgSO4, filtered, and concentrated under vacuum. The crude residue was purified by silica gel rapid chromatography, eluting with a hexane solution of 0-100% EtOAc and then with a DCM solution of 0-20% MeOH, to give the title compound as a viscous yellow oil (12.03 g, 77%). ES / MS m / z 625.5 ([M+18] / 2).

[0335] Preparation Example 34

[0336] (S)-27-(4,7,10,13,16-pentoxanonadecan-18-ynylamino)-19,24,28-trioxo-23-(4-oxo-7,10,13,16,19-pentoxa-3-aza-tetracosane-21-yn-1-yl)-4,7,10,13,16-pentoxa-20,23,29-triazapentadecane-1-yn-35-acid

[0337]

[0338] The title compound was prepared in a manner substantially similar to that used in Preparation Example 30, using ethyl (S)-27-(4,7,10,13,16-pentaenodecane-18-ynylamino)-19,24,28-trioxo-23-(4-oxo-7,10,13,16,19-pentaoxo-3-aza-tetradecane-21-yn-1-yl)-4,7,10,13,16-pentaoxo-20,23,29-triazapentadecane-1-yn-35-olate (12.0 g, 9.74 mmol) and an aqueous solution of LiOH (1N, 48.7 mL, 48.7 mmol), yielding the title compound (11.4 g, 97.1%) as a viscous brown oil. ES / MS m / z 602.9 (M / 2+H).

[0339] Preparation Example 35

[0340] (S)-27-(4,7,10,13,16-pentoxanonadecan-18-ynylamino)-19,24,28-trioxo-23-(4-oxo-7,10,13,16,19-pentoxa-3-azatetracosane-21-yn-1-yl)-4,7,10,13,16-pentoxa-20,23,29-triazapentadecane-1-yn-35-acid 2,5-dioxopyrrolidine-1-yl ester

[0341]

[0342] The mixture of (S)-27-(4,7,10,13,16-pentaenodecane-18-ynylamino)-19,24,28-trioxo-23-(4-oxo-7,10,13,16,19-pentaoxo-3-azatetradecane-21-yn-1-yl)-4,7,10,13,16-pentaoxo-20,23,29-triazapentadecane-1-yn-35-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 rapid chromatography, eluting with a DCM solution of 0–20% MeOH, to give the title compound as a colorless oil (321 mg, 79%). ES / MS m / z 651.5 (M / 2+H).

[0343] Preparation Example 36

[0344] (S)-1-(1-(14-((3'-((S)-1-((4-(8-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)(methyl)amino)-4-methoxy-4-oxobut-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-naphthidin-2-yl)butyl)(methyl)amino)-4-methoxy-4-oxobut-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-azaeicosano-20-yl)-25-(1-(1-(14-((3'-((S)-1-((4-(8-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)(methyl)amino)-4-methoxy-4-oxo Butyl-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-pentaheptadecane-17-acylamino)-17,22,26-trioxo-2,5,8,11,14-pentaoxa-18,21,27-triazatritriacontane-33-acid

[0345]

[0346] To (S)-27-(4,7,10,13,16-pentaenodecane-18-ynylamino)-19,24,28-trioxo-23-(4-oxo-7,10,13,16,19-pentaoxo-3-azatetracosane-21-yn-1-yl)-4,7,10,13,16-pentaoxo-20,23,29-triazapentadecane-1-yn-35-acid (5.60 g, 4.65 g) DMF (80 mL) was added to (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-naphthyl-1(2H)-carboxylic acid tert-butyl ester (12.4 g, 14.9 mmol). 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 a saturated aqueous sodium chloride solution, and twice with a mixture of saturated aqueous sodium chloride solution and 50 mM EDTA. The mixture was dried over MgSO4, filtered, and concentrated under vacuum. The crude residue was purified by silica gel chromatography using a 0–30% MeOH solution eluted with DCM to give the title compound as a viscous yellow oil (12.69 g, 68%). ES / MS m / z 741.6 (M / 7+H).

[0347] Preparation Example 37

[0348] (S)-1-(1-(14-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-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-naphthidin-2-yl)butyl)amino)-4-oxobut-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-azaeicosano-20-yl)-25-(1-(1-(14-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-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-pentaoxepachitagane-17-acylamino)-17,22,26-trioxo-2,5,8,11,14-pentaoxa-18,21,27-triazatritriacontane-33-acid

[0349]

[0350] (S)-1-(1-(14-((3'-((S)-1-((4-(8-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)(methyl)amino)-4-methoxy-4-oxobut-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-tri (-4-yl)-21-(1-(1-(14-((3'-((S)-1-((4-(8-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-1,8-naphthid-2-yl)butyl)(methyl)amino)-4-methoxy-4-oxobut-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-azaeicosano-20-yl)-25-(1-(1-(14-((3'-((S)-1-((4-(8-(tert-butoxycarbonyl)-5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)(methyl)amino)-4-methoxy-4-oxobut-2- (11.7 g, 3.16 mmol) of (1,1'-biphenyl)-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-2,5,8,11,14-pentaheptadecane-17-acylamino)-17,22,26-trioxo-2,5,8,11,14-pentaoxa-18,21,27-triazatritriacontane-33-acid (50 mL) was dissolved in DCM, and TFA (24.3 mL, 316 mmol) was added. The mixture was stirred at ambient temperature under a N2 atmosphere. After 14 hours, the mixture was concentrated under vacuum. The residue was diluted with DCM and washed twice with water. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The crude residue was purified by low-pH rapid reversed-phase chromatography. The appropriate fractions were collected, concentrated under vacuum, and the organic layer was removed. The aqueous layer was diluted with a saturated sodium chloride solution and extracted 3x with DCM. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum to give the title compound as a yellow foam (8.23 g, 76.6%). ES / MS m / z 681.6 (M / 6+H).

[0351] Preparation Example 38

[0352] (S)-1-(1-(14-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl) -21-(1-(14-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobutyl-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-azaeicosano-20-yl)-25-(1-(1-(14-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-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-pentaheptadecane-17-acylamino)-17,22,26-trioxo-2,5,8,11,14-pentaoxa-18,21,27-triazatritriacontane-33-acid 2,5-dioxopyrrolidine-1-yl ester

[0353]

[0354] (S)-1-(1-(14-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthid-2-yl)butyl)amino)-4-oxobut-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-21-( ... (-4-yl)-17-oxo-2,5,8,11,14-pentaoxa-18-azaeicosano-20-yl)-25-(1-(1-(14-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthid-2-yl)butyl)amino)-4-oxobutyl-2-yl)-[1,1'- 1-Hydroxypyrrolidine-2,5-dione (835 mg, 7.25 mmol), EDCI (1.39 g, 7.25 mmol), and DCM (60 mL) were added to a mixture of biphenyl-4-yloxy-3,6,9,12-tetraoxatetradecylyl-1H-1,2,3-triazol-4-yl)-2,5,8,11,14-pentaoxa-18,21,27-triazatritriacontane-33-acid (8.23 g, 2.42 mmol). The mixture was stirred at ambient temperature under a nitrogen atmosphere. After 5 hours, the mixture was purified by silica gel rapid chromatography, eluting with a DCM solution of 0-50% MeOH. The product was dissolved in DCM, washed 3x with saturated sodium chloride aqueous solution, dried over MgSO4, filtered, and concentrated under vacuum to give the title compound as a pale yellow foam (6.72 g, 79.4%). ES / MS m / z 584.4 (M / 6+H).

[0355] Preparation Example 39

[0356] (S)-7-(4-((2-(4'-((14-(4-(19,19-dimethyl-17-oxo-2,5,8,11,14,18-hexaoxaneeicosyl)-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-naphthyl-1(2H)-tert-butyl formate

[0357]

[0358] (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-naphthyl-1(2H)-carboxylic acid tert-butyl ester (8.4 g, 10 mmol) and 4,7,10,13,16-pentaenodecane-18-alkynyl acid tert-butyl ester (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 a nitrogen atmosphere. After 30 minutes, the mixture was diluted with water and extracted twice with DCM. The organic layer was washed three times with water (with the addition of saturated sodium chloride aqueous solution for demulsification), dried over MgSO4, filtered, and concentrated under vacuum. The crude residue was purified by silica gel chromatography by elution with DCM solution of 0–20% MeOH to give the title compound as a viscous yellow oil (9.69 g, 81%). ES / MS m / z 597.9 (M / 2+H).

[0359] Preparation Example 40

[0360] (S)-1-(1-(14-((3'-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-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-pentaheptadecane-17-acid

[0361]

[0362] (S)-7-(4-((2-(4'-((14-(4-(19,19-dimethyl-17-oxo-2,5,8,11,14,18-hexaoxaecoyl)-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-naphthyl-1(2H)-formate tert-butyl ester (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 a N2 atmosphere. After 14 hours, the mixture was concentrated under vacuum. The residue was diluted with DCM and washed with water. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum 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).

[0363] Preparation Example 41

[0364] (S)-1-(1-(14-((3'-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-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-pentaheptadecane-17-acid 2,5-dioxopyrrolidine-1-yl ester

[0365]

[0366] Add 1-hydroxypyrrolidine-2,5-dione (1.8 g, 16 mmol), EDCI (3.1 g, 16 mmol), and DCM (30 mL) to (S)-1-(1-(14-((3'-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-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-pentaheptadecane-17-acid (8.3 g, 8.0 mmol). Stir the mixture at ambient temperature for 4 hours. Dilute the mixture with DCM, wash three times with saturated sodium chloride aqueous solution, dry with MgSO4, filter, and concentrate under vacuum. The crude residue was purified by silica gel rapid chromatography using DCM solution of 0-40% MeOH to give the title compound as a viscous yellow oil (7.25 g, 64%). ES / MS m / z 568.0 (M / 2+H).

[0367] Preparation Example 42

[0368] (S)-7-(4-((2-(4'-((17,17-dimethyl-15-oxo-3,6,9,12,16-pentaoctadecyl)oxy)-[1,1'-biphenyl]-3-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyl-1(2H)-tert-butyl formate

[0369]

[0370] Cs₂CO₃ was added to (S)-7-(4-((2-(4'-((17,17-dimethyl-15-oxo-3,6,9,12,16-pentaoctadecyl)oxy)-[1,1'-biphenyl]-3-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyl-1(2H)-carboxylic acid tert-butyl ester (4.0 g, 6.8 mmol) and 1-(toluenesulfonyloxy)-3,6,9,12-tetraoxapentadecan-15-oic acid tert-butyl ester (4.9 g, 10 mmol) in DMF (40 mL). The mixture was stirred at 40 °C under a N₂ atmosphere for 3 hours. The cooled mixture was diluted with water and extracted three times with EtOAc. The organic layer was washed with water and brine, dried over MgSO₄, filtered, and then concentrated under vacuum. The crude residue was purified by silica gel rapid chromatography using a DCM solution of 0-100% acetone to give the title compound as a viscous yellow oil (5.09 g, 84%). ES / MS m / z 893.4 (M+H).

[0371] Preparation Example 43

[0372] (S)-1-((3'-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxapentadecan-15-acid salt

[0373]

[0374] HCl (4 M, in dioxane, 28 mL, 110 mmol) was added to (S)-7-(4-((2-(4'-((17,17-dimethyl-15-oxo-3,6,9,12,16-pentaoctadecyl)oxy)-[1,1'-biphenyl]-3-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyl-1(2H)-carboxylic acid tert-butyl ester. The mixture was stirred at room temperature, and after 20 hours, it 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).

[0375] Preparation Example 44

[0376] (R)-1-(4-(6-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-2-yl)pyridin-2-yl)phenoxy)-3,6,9,12-tetraoxapentadecan-15-acid salt

[0377]

[0378] Following a manner substantially similar to that used in Preparation Examples 42 and 43, the title compound was prepared using (R)-7-(4-((2-(6-(4-hydroxyphenyl)pyridin-2-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthidium-1(2H)-carboxylic acid tert-butyl ester (3.0 g, 5.1 mmol) and 1-(toluenesulfonyloxy)-3,6,9,12-tetraoxapentadecano-15-oic acid tert-butyl ester (2.7 g, 5.6 mmol), yielding the title compound as a foam (3.18 g, 81%, via 2 steps). ES / MSm / z 737.4 ([M-HCl]+H).

[0379] Preparation Example 45

[0380] (S)-1-(3-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-2-yl)phenoxy)-3,6,9,12-tetraoxapentadecan-15-acid salt

[0381]

[0382] The title compound was prepared in a manner substantially similar to that used in Preparation Examples 42 and 43, using (S)-7-(4-((2-(3-hydroxyphenyl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthidium-1(2H)-carboxylic acid tert-butyl ester (3.4 g, 6.6 mmol) and 1-(toluenesulfonyloxy)-3,6,9,12-tetraoxapentadecano-15-oic acid tert-butyl ester (3.5 g, 7.3 mmol), yielding the title compound (3.11 g, 67%, via 2 steps). ES / MS m / z 660.4 ([M-HCl]+H).

[0383] Preparation Example 46

[0384] (S)-1-((5-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-2-yl)pyridin-3-yl)oxy)-3,6,9,12-tetraoxapentadecan-15-acid salt

[0385]

[0386] Following a manner substantially similar to that used in Preparation Examples 42 and 43, the title compound was prepared using (S)-7-(4-((2-(5-hydroxypyridin-3-yl)-4-methoxy-4-oxobutyl)(methyl)amino)butyl)-3,4-dihydro-1,8-naphthyl-1(2H)-carboxylic acid tert-butyl ester (9.0 g, 18 mmol) and 1-(toluenesulfonyloxy)-3,6,9,12-tetraoxapentadecano-15-oic acid tert-butyl ester (9.2 g, 19 mmol) to give the title compound as a yellow solid (8.04 g, 66%, obtained in 2 steps). ES / MSm / z 661.4 ([M-HCl]+H).

[0387] Preparation Example 47

[0388] 6-Aminohexanoate benzyl ester hydrochloride

[0389]

[0390] 6-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 in a slow-flowing form. The mixture was stirred in an ice bath while being heated to rt. After 3 days, diethyl ether (1 L) was added to the mixture, and the mixture was stirred for 5 min and kept in a -20 °C refrigerator for 4 h. The precipitate was collected by filtration, washed with diethyl ether (2 L), and dried under N2 atmosphere to give the title compound as a off-white powder (30.9 g, 79% yield). ES / MS m / z 222.4 ([M-HCl]+H).

[0391] Preparation Example 48

[0392] (S)-12-((tert-butoxycarbonyl)amino)-8-(2-((tert-butoxycarbonyl)amino)ethyl)-2,2-dimethyl-4,9,13-trioxo-3-oxa-5,8,14-triazaeicosano-20-acid benzyl ester

[0393]

[0394] 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) was added to an RBF containing HOBt (8.23 g, 60.9 mmol) and HBTU (23.1 g, 60.9 mmol). DIPEA (36.6 mL, 212 mmol) was added, and the mixture was stirred for 5 min, followed by the addition of benzyl 6-aminohexanoate hydrochloride (15.0 g, 58.2 mmol). The mixture was stirred overnight at room temperature. The mixture was diluted with DCM (500 mL), washed with water (3 x 500 mL) and brine (3 x 500 mL), dried over MgSO4, filtered, and concentrated under vacuum. The crude residue was purified by silica gel chromatography, eluting with a hexane solution containing 0-80% ethyl acetate and then with a DCM solution containing 0-20% MeOH, to give the title compound as a milky white foam (23 g, 59% yield). ES / MS m / z 736.2 (M+H).

[0395] Preparation Example 49

[0396] (S)-6-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanoylamino)benzyl hexanoate trihydrochloride

[0397]

[0398] HCl (112 mL, 448 mmol) was added to (S)-12-((tert-butoxycarbonyl)amino)-8-(2-((tert-butoxycarbonyl)amino)ethyl)-2,2-dimethyl-4,9,13-trioxo-3-oxa-5,8,14-triazaeicosano-20-acid benzyl ester (22.0 g, 29.9 mmol) in 4 N in 1,4-dioxane, and the mixture was stirred at room temperature. After 10 minutes, the mixture became a thick suspension. Dioxane (20 mL) was added, and stirring continued. 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 N2 atmosphere to give the title compound as a creamy white solid (16.3 g, 100% yield). ES / MS m / z 436.4 ([M-3HCl]+H).

[0399] Preparation Example 50

[0400] 6-[[(2S)-5-[double[2-[3-[2-[2-[2-[2-[2-[4-[3-[(1S) )-3-methoxy-1-[[methyl-[4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl]amino]methyl]-3-oxo-propyl]phenyl]phenoxy]ethoxy]ethoxy]ethoxy]propionylamino]ethyl]amino]-2-[3-[2-[2-[2-[4-[3-[(1S )-3-methoxy-1-[[methyl-[4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl]amino]methyl]-3-oxo-propyl]phenyl]phenoxy]ethoxy]ethoxy]ethoxy]propionylamino]-5-oxo-pentanoyl]amino]benzyl hexanoate

[0401]

[0402] (S)-6-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanoylamino)benzyl hexanoate trihydrochloride (70.0 mg, 0.128 mmol) and (S)-1-((3'-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxapentadecan-15-acid salt (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 under vacuum. The crude residue was purified by reversed-phase chromatography (high pH, ​​then converted to low pH) to give the title compound as a thick, yellow oil (113 mg, 34% yield). ES / MS m / z 648.2 (M / 4+H).

[0403] Preparation Example 51

[0404] (S)-1-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-19-(1-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-15-oxo-3, 6,9,12-Tetraoxa-16-azaoctadecane-18-yl)-23-(1-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxapentadecane-15-acylamino)-15,20,24-trioxa-3,6,9,12-tetraoxa-16,19,25-triazatrionecane-31-acid

[0405]

[0406] 6-[[(2S)-5-[double[2-[3-[2-[2-[2-[2-[2-[4-[3-[(1S) )-3-methoxy-1-[[methyl-[4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl]amino]methyl]-3-oxo-propyl]phenyl]phenoxy]ethoxy]ethoxy]ethoxy]propionylamino]ethyl]amino]-2-[3-[2-[2-[2-[4-[3-[(1S Benzyl hexanoate (110 mg, 0.0425 mmol) was dissolved in MeOH (12 mL) and added to a pressure flask containing 10% Pd / C (50% wet) (31.6 mg, 0.0297 mmol). The flask was sealed, and the mixture was purged three times with N2 and then three times with H2. The mixture was stirred at 40 psi H2 for 6 hours, filtered through diatomaceous earth, 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).

[0407] Preparation Example 52

[0408] (3S 3'S )-3,3'-{[19-(N-{6-[(2,5-dioxopyrrolidone-1-yl)oxy]-6-oxohexyl}-N~2~-[15-({3'-[(2S )-4-methoxy-1-{methyl[4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl]amino}-4-oxobut-2-yl][1,1'-biphenyl]-4-yl}oxy)-4,7,10,13-tetraoxapentadecan-1-acyl]-l- -Glutamyl)-15,23-dioxo-3,6,9,12,26,29,32,35-octaoxa-16,19,22-triazaheptadecane-1,37-diyl]bis(oxy[1,1'-biphenyl]-4',3-diyl)}bis(4-{methyl[4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl]amino}butyric acid) dimethyl ester

[0409]

[0410] (S)-1-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-19-(1-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-15-oxo-3,6 9,12-Tetraoxa-16-azaoctadecane-18-yl)-23-(1-((3'-((S)-4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-2-yl)-[1,1'-biphenyl]-4-yl)oxy)-3,6,9,12-tetraoxapentadecane-15-acylamino)-15,20,24-trioxo-3,6,9,12-tetraoxa-16,19,25-triazatricarne-31-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 then directly loaded onto a 4 g silica gel column. Purification was achieved by rapid chromatography using DCM elution with 0–60% MeOH to give the title compound (33 mg, 32% yield). ES / MS m / z 650.0 (M / 4+H).

[0411] Preparation Example 53

[0412] (3R,3'R)-3,3'-{[19-(N-{6-[(2,5-dioxopyrrolidone-1-yl)oxy]-6-oxohexyl}-N~2~-[15-(4-{6-[(2R)-4-methoxy-1-{methyl[4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl]amino}-4-oxobutyl-2-yl]pyridin-2-yl}phenoxy)-4,7,10,13-tetraoxapentadecan-1-acyl]-l- -Glutamyl)-15,23-dioxo-3,6,9,12,26,29,32,35-octaoxa-16,19,22-triazaheptadecane-1,37-diyl]bis(oxy-4,1-phenylenepyridin-6,2-diyl)}bis(4-{methyl[4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl]amino}butyric acid) dimethyl ester

[0413]

[0414] The title compound was prepared in a manner substantially similar to that used in Preparation Examples 50, 51, and 52, using (R)-1-(4-(6-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-2-yl)pyridin-2-yl)phenoxy)-3,6,9,12-tetraoxapentadecan-15-acid salt (3.18 g, 4.12 mmol) and (S)-6-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanoylamino)benzyl hexanoate trihydrochloride (680 mg, 1.25 mmol) to give the title compound as a white solid (2.53 g, 57.5%, obtained in 3 steps). ES / MS m / z 650.8 (M / 4+H).

[0415] Preparation Example 54

[0416] (3S,3'S)-3,3'-[{19-[N-{6-[(2,5-dioxopyrrolidone-1-yl)oxy]-6-oxohexyl}-N~2~-(15-{3-[(2S)-4-methoxy-1-{methyl[4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl]amino}-4-oxobut-2-yl]phenoxy}-4,7,10,13-tetraoxapentadecan-1-acyl)-l- [-glutamyl]-15,23-dioxo-3,6,9,12,26,29,32,35-octaoxa-16,19,22-triazaheptadecane-1,37-diyl}bis(oxy-3,1-phenylene)]bis(4-{methyl[4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl]amino}butyric acid) dimethyl ester

[0417]

[0418] The title compound was prepared in a manner substantially similar to that used in Preparation Examples 50, 51, and 52, using (S)-1-(3-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-2-yl)phenoxy)-3,6,9,12-tetraoxapentadecan-15-acid salt (3.08 g, 4.42 mmol) and (S)-6-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanoylamino)benzyl hexanoate trihydrochloride (730 mg, 1.34 mmol), yielding the title compound as a creamy white solid (550 mg, 17.3%, obtained via 3 steps). ES / MS m / z 593.0 (M / 4+H).

[0419] Preparation Example 55

[0420] (3S,3'S)-3,3'-{[19-(N-{6-[(2,5-dioxopyrrolidone-1-yl)oxy]-6-oxohexyl}-N~2~-[15-({5-[(2S)-4-methoxy-1-{methyl[4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl]amino}-4-oxobut-2-yl]pyridin-3-yl}oxy)-4,7,10,13-tetraoxapentadecan-1-acyl]-l- -Glutamyl)-15,23-dioxo-3,6,9,12,26,29,32,35-octaoxa-16,19,22-triazaheptadecane-1,37-diyl]bis(oxypyridine-5,3-diyl)}bis(4-{methyl[4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl]amino}butyric acid) dimethyl ester

[0421]

[0422] The title compound was prepared in a manner substantially similar to that used in Preparation Examples 50, 51, and 52, using (S)-1-((5-(4-methoxy-1-(methyl(4-(5,6,7,8-tetrahydro-1,8-naphthidin-2-yl)butyl)amino)-4-oxobut-2-yl)pyridin-3-yl)oxy)-3,6,9,12-tetraoxapentadecan-15-acid salt (7.9 g, 11 mmol) and (S)-6-(2-amino-5-(bis(2-aminoethyl)amino)-5-oxopentanoylamino)benzyl hexanoate trihydrochloride (2.0 g, 3.7 mmol), yielding the title compound as a thick, yellow oil (1.38 g, 15.9%, obtained in 3 steps). ES / MS m / z 593.6 (M / 4+H).

[0423] Example 2. Synthesis of integrin-targeting ligands and RNAi substances, and coupling of RNAi substances with integrin ligands.

[0424] The following abbreviations used for 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 cutoff value; "CPG" refers to crystalline porous glass; "LC-MS" refers to liquid chromatography-mass spectrometry; "RNA" refers to ribonucleic acid; "DNA" refers to deoxyribonucleic acid; "UV" refers to ultraviolet light; "PVDF" refers to polyvinylidene fluoride; "EDTA" refers to disodium ethylenediaminetetraacetate dihydrate; "PCR" refers to polymerase chain reaction; "SCNN1A" refers to sodium channel epithelial subunit 1. "BCP" refers to 1-bromo-3-chloropropane; "mRNA" refers to messenger RNA; "cDNA" refers to complementary DNA; "UPLC" refers to ultra-high 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.

[0425] Preparation Example 56

[0426] Synthesis of the Justice Chain

[0427] Single-stranded oligonucleotides were synthesized on a solid-phase support using a K&A H8-SE DNA / RNA synthesizer (K&A Laborgeraete GbR). Oligonucleotides were synthesized at 10, 50, or 100 µmol scales via phosphoramide chemistry. The solid-phase support was either 3'-PT-amino-modifying agent C6 CPG (Chemgenes, catalog number N-8217-05) or Universal UnyLinker CPG (Chemgenes, catalog number N-4000-05) and was commercially available. Standard reagents were used for oligosynthesis (Table 4), with a pyridine solution of 0.1 M hydroflavin used as a sulfidation agent and a 20% DEA ACN solution used as a post-synthesis auxiliary washing solution. All monomers (Table 5) were prepared in 0.1 M ACN solutions and contained in molecular sieve traps. TFA-hexylamino linker phosphoramide (CAS#: 133975-85-6) was coupled to the 5' end to generate a C6 amino-terminal linker. The positive chain of the oligonucleotide was cleaved and deprotected (C / D) for 2 hours at ambient temperature using a 28-30% ammonium hydroxide:40% methylamine (1:1) solution. CPG was filtered through a 0.45 μm PVDF needleless filter, a 0.22 μm PVDF Steriflip® vacuum filter, or a 0.22 μm PVDF Stericup® rapid release filter. The filtrate was partially concentrated using Genevac™. After concentration, the crude oligonucleotide was diluted with RNase-free water and filtered a second time, followed by anion exchange (AEX) (conditions: ES Industry Source™ 15Q column, using MPA: 20 mM 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 Source). TMPurification was performed on an AKTA™ Pure purification system using a 15Q column with MPA (20 mM NaOAc, 15% ACN, MPB (80% ACN aqueous solution)). Fractions with a mass purity greater than 85% and free of single impurities >5% were collected. The purified material was desalted using a 3K MWCO centrifuge or tangential flow filtration to obtain the title compounds in aqueous solution as shown in Tables 1 and 2. The absorbance of the solution at 260 nm was measured using a Thermo Scientific Nanodrop One C UV-Vis spectrophotometer. The concentration was then calculated using the measured absorbance and the calculated extinction coefficient. Product purity was analyzed by UPLC and LC-MS.

[0428] Table 2 – SCNN1A Justice Chain

[0429]

[0430] Abbreviations – “m” represents 2'-OMe; “f” represents 2'-fluorine; " indicates a thiophosphate linkage; "iAb" indicates a reverse non-base nucleoside; "C6Am" indicates a 3' or 5' C6 amino group linked via a phosphate ester.

[0431] Preparation Example 57

[0432] Synthesis of antisense chains

[0433] Following the protocol described in Preparation Example 56, antisense chains were prepared using standard CPG (mC: Chemgenes catalog number: N-7905-05 or mG: Chemgenes catalog number: N-7912-05). The title compounds, which are aqueous solutions, were obtained as shown in Table 3.

[0434] Table 3 – SCNN1A Antisense Chain

[0435]

[0436] Abbreviations – “m” represents 2'-OMe; “f” represents 2'-fluorine; " " indicates a thiophosphate linkage; "VP" indicates a 5'-vinylphosphonate.

[0437] Table 4 – Reagents for Oligonucleotide Synthesis

[0438]

[0439] Table 5 – Phosphite

[0440]

[0441] Preparation Example 58

[0442] Coupling of amine-functionalized sense chains

[0443] The aqueous solution of the amine-functionalized positive chain from Preparation Example 56 was buffered with phosphate buffer (0.5 M, pH 7.5, 10% v / v positive chain). An NHS-ester solution (50 mM, in ACN, 5.0 eq / C6Am) was added. The mixture was shaken at 1000 rpm for 4–16 hours at ambient temperature. For Preparation Examples 58-4, 58-5, 58-6, 58-7, 58-8, 58-9, 58-10, and 58-11, an aqueous solution of LiOH (200 eq, 1N) was added, followed by shaking for 1–2 hours. The conjugates were purified by anion exchange or reversed-phase chromatography. The purified material was desalted using a 3 K MWCO centrifuge to obtain the title compounds as aqueous solutions, as shown in Table 6.

[0444] Table 6 – Coupling of Amine-Functionalized Sensitive Chains

[0445]

[0446]

[0447]

[0448] Preparation Example 59

[0449] Annealing of the justice chain and the antithesis chain

[0450] Equimolar amounts of the sense and complementary antisense strands were mixed in solution in RNase-free water and vortexed for 20 seconds (Table 7).

[0451] Uncoupled double strands: The solution was shaken at 90°C for 2 min, allowed to stand, and cooled to 25°C for 45 min. The solution was lyophilized and reconstituted with 1X PBS.

[0452] Targeting ligands and alkyne-functionalized duplexes: lyophilized solutions or concentrated using a Genevac evaporator and reconstituted in RNase-free water to a concentration ≥50 mg / mL.

[0453] The concentration of the double-stranded compound was calculated based on the measured absorbance (260 nm) and extinction coefficient of the solution, corrected for by a factor of 0.85. Purity was analyzed by UPLC and LC-MS.

[0454] For in vivo samples, endotoxin levels (upper limit 2 EU / mg) were measured using the Charles River Endosafe® device.

[0455] Table 7 – Annealing of Justice Chain and Antithesis Chain

[0456]

[0457] Preparation Example 60

[0458] Coupling of targeted ligands with alkyne-functionalized duplexes

[0459] The alkyne-functionalized duplexes (Table 7, Preparation Examples 59-2; 59-3 and 59-4) were coupled with the targeting ligands (Table 8) via a copper-catalyzed click reaction (CuAAC) as follows: Stock solutions of copper sulfate pentahydrate (II) (0.5 M), THPTA (0.5 M), and sodium ascorbate (2 M) were prepared in RNase-free water. A 0.1 M solution of the targeting ligand in DMSO and a 50 mg / mL solution of the duplexes in RNase-free water were prepared. DMSO (0.1 M, ~8 eq / alkyne) was added to a Falcon tube containing the alkyne-functionalized duplexes. The solution of the targeting ligand in DMSO (2 eq / alkyne) was added and vortexed. Borate buffer (50 mM, pH 8.5, 2 eq / alkyne) was added and vortexed. The solution was placed in a shaker cooled to 10°C. Add a 4:1 v / v mixture of THPTA and copper(II) sulfate pentahydrate (2 eq / acetylene) and vortex. Immediately add sodium ascorbate (16 eq / acetylene) and shake the reaction mixture at 10°C until complete (30–100 min). After completion, add EDTA (0.05 M, 3.5 eq / acetylene), as determined by IP-RP LCMS, and purify by non-denaturing anion exchange chromatography or reversed-phase chromatography. Desalt the purified material using a 3K MWCO centrifuge or tangential flow filtration to obtain the title duplex RNAi material as an aqueous solution. Lyophilize the duplexes, reconstitute them in 1X PBS, and ultrafilter using a 100K MWCO filter unit to obtain the duplex RNAi material as shown in Table 9. For in vivo samples, endotoxin levels (upper limit 2 EU / mg) were measured using a Charles River Endosafe® device.

[0460] Table 8 – Coupling of Target Ligands with Alkyne-Functionalized Duplexes

[0461]

[0462] Note – “a” refers to a compound containing three ligands; “b” refers to a compound containing two ligands; and “c” refers to a compound containing one targeting ligand.

[0463] Table 9 – Double-stranded RNAi substances

[0464]

[0465] NA – without ligand

[0466] Intranasal administration of SCNN1A RNAi substance in mice

[0467] The efficacy of RNAi substances was investigated in wild-type male C57BL / 6 mice. Four mice were administered the drug to each group. Animals were randomly assigned to groups based on body weight prior to the study. Mice were anesthetized with isoflurane and administered 100 µL + 100 µL (total 200 µL, with 100 µL doses administered at 5–10 min intervals) intranasally (0.75 mg / kg). Animals were held upright for 1 minute after administration. They were then returned to their cages and their recovery was monitored. Clinical observations and body weight were monitored on day 1 and on the day of necropsy. Animals were euthanized on the day of necropsy (day 8 or day 15), and lung tissue (right / left lung weighed separately and rapidly frozen) was collected.

[0468] RNA extraction and TaqMan analysis

[0469] The day before tissue processing, fill each lysis matrix A tube (MP BioMedicals, 116920500) with 1 mL of TRIzol (Invitrogen, 15596026) and chill overnight at 4°C. On the day of tissue processing, remove the tissue sample cassette from -80°C and pack it in dry ice in an ice bucket to keep it frozen during processing. For processing, quickly cut 3-4 slices of the upper right lung (each ~50-100 mg) and place them in lysis matrix A tubes and cap them. Once all samples have been added to their respective tubes, homogenize the tissues at 1800 RPM for 1 minute using FastPrep-96 (MP BioMedicals, 116010500). After homogenization, add 300 µL of BCP (Sigma-Aldrich, B9673) to each tube. Cap the tubes and shake vigorously to mix the contents. Centrifuge the tubes at 15000xg for 15 minutes at 4°C. After centrifugation, remove 300µL of aqueous solution from each tube and transfer it to a 1.2 mL deep-well plate. Add an equal volume (300µL) of 100% ethanol to each well and mix thoroughly. After mixing, transfer the entire 600µL solution to a silica gel plate equipped with the Quick RNA 96 Isolation Kit (Zymo Research, R1053). Isolate total RNA according to the kit instructions, using 50µL of RNase-free water for final elution. Measure the concentration of isolated RNA using a Nanodrop 8000. Dilute the isolated RNA to 100 ng / µL with RNase-free water and aliquot 10 µL from the dilution plate for cDNA synthesis. Perform cDNA synthesis using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (4368814) according to the instructions. After synthesis, dilute the cDNA to 20 ng / µL with RNase-free water. Using 5 µL of cDNA diluted (total 100 ng), and following the instructions, a total volume of 20 µL was used for TaqMan assays, with the SCNN1A probe (Mm00803386_m1) and HPRT probe (Mm03024075) from ThermoFisher Scientic as endogenous controls. The assay plate was run on an Applied Biosystems QuantStudio 6. The Ct method is used for data analysis, and GraphPad Prism is used to draw charts.

[0470] Example 3. In vivo intranasal administration of uncoupled and ligand-coupled SCNN1A RNAi substances in mice.

[0471] Mice were given 0.75 mg / kg of RNAi substance on day 1 and sacrificed on day 8 (Table 10).

[0472] Table 10 – Mean relative SCNN1A mRNA knockdown (%KD) at sacrifice (day 8)

[0473]

[0474] As shown in Table 10, each of the SCNN1A RNAi substances showed reduced mRNA expression in mice compared to the control. Overall, integrin ligand conjugates showed better potency compared to the unconjugated RNAi substances. For example, tridentate integrin ligand conjugates 60-1a, 60-2a, and 60-3a showed 56%, 56%, and 53% knockdown, respectively, compared to the unconjugated RNAi substance 59-1, which showed a 41% knockdown. Significantly, bidentate integrin ligand conjugates also showed improved potency relative to the unconjugated RNAi. For example, bidentate conjugates 60-1b, 60-3b, and 60-4b showed 52%, 52%, and 58% knockdown, respectively. Furthermore, monodentate integrin ligand conjugates also showed improved potency compared to the unconjugated substances. For example, RNAi substances 60-1c and 60-4c showed 54% and 51% knockdown, respectively, compared to the uncoupled substance 59-1 (41% knockdown). The data in Table 10 demonstrate the benefit of using integrin ligands in facilitating RNAi substance uptake, as they showed up to 17% improvement in target mRNA knockdown compared to the uncoupled substances.

[0475] Example 4. In vivo intranasal administration of uncoupled and ligand-coupled SCNN1A RNAi substances in mice.

[0476] Mice were given 0.75 mg / kg of RNAi substance on day 1 and sacrificed on day 15 (Table 11).

[0477] Table 11 – Mean relative SCNN1A mRNA knockdown at sacrifice (%KD) (Day 15)

[0478]

[0479] As shown in Table 11, each of the SCNN1A RNAi substances showed reduced mRNA expression in mice compared to the control. All integrin ligand conjugates except bidentate conjugate 60-2b and monodentate conjugate 6-2c showed improved knockdown 2 weeks after intranasal administration of RNAi. For example, tridentate integrin ligand conjugates 60-1a and 60-2a showed knockdown rates of 58% and 55%, respectively, compared to the unconjugated substance 59-1 (47% knockdown). Bidentate integrin ligand conjugate 60-1b showed an even higher 65% mRNA knockdown, an 18% improvement compared to 59-1 (47%). The data in Table 11 demonstrate the benefits of using integrin ligands in facilitating RNAi substance uptake compared to the unconjugated RNAi substance.

[0480] Example 5. In vivo intranasal administration of uncoupled and ligand-coupled SCNN1A RNAi substances in mice.

[0481] Mice were given 0.75 mg / kg of RNAi substance on day 1 and sacrificed on day 15 (Table 12).

[0482] Table 12 – Mean relative SCNN1A mRNA knockdown at sacrifice (%KD) (Day 8)

[0483]

[0484] Note: %KD is the average of the two studies.

[0485] As shown in Table 12, each of the SCNN1A RNAi substances showed reduced mRNA expression in mice compared to the control. Generally, integrin ligand conjugates exhibited better potency than unconjugated RNAi substances. For example, tridentate integrin ligand conjugates 59-9, 59-10, 59-11, and 59-12 achieved 46%, 44%, 49%, and 52% knockdown, respectively, compared to the unconjugated RNAi substance 59-1, which showed a 25% knockdown.

[0486] Example 6. In vivo intranasal administration of uncoupled and ligand-coupled SCNN1A RNAi substances in mice

[0487] Mice were given RNAi substance at doses of 0.75 mg / kg, 0.5 mg / kg, 0.25 mg / kg or 0.01 mg / kg on day 1 and were sacrificed on day 8 or day 15 (Table 13).

[0488] Table 13 – Mean relative SCNN1A mRNA knockdown at sacrifice (%KD)

[0489]

[0490] As shown in Table 13, two of the uncoupled RNAi substances (59-5 and 59-8) exhibited extremely good reductions in target mRNA expression at all tested doses, with no dose-response observed. These knockdowns are comparable to their integrin ligand-coupled analogs (59-9 and 59-12). Ligand-coupling benefits were observed only at very low doses (0.001 mg / kg) (59-5 and 59-9), although the percentage reduction in mRNA was very low. The dose-titer analysis of RNAi substance 59-5 showed a very steep curve (data not shown), which could explain why the uncoupled and coupled substances have comparable potency.

[0491] Example 7. Binding activity of integrin-targeting ligands

[0492] The experimentally measured potency and selectivity of integrin-targeting ligands against various integrins are shown in Table 14:

[0493] αvβ6 and αvβ8 binding assay – 96-well plates were coated overnight at 4°C with LAP (TGF-β) (0.4 µg / mL) dissolved in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) (Acros Biosystems). The plates were washed with washing buffer (PBS, 0.1% Tween 20) and then blocked for 1 hour at ambient temperature with blocking buffer (Superblock TBS, Thermofisher Scientific). Serially diluted ligands in blocking buffer were added to the plates along with purified αvβ6 (0.4 µg / mL, R&D) or αvβ8 (0.4 µg / mL, R&D) and incubated for 1 hour at ambient temperature. The plate was washed with washing buffer and then incubated at room temperature for 1 hour with the primary antibody (anti-αv mouse anti-human, Millipore), followed by incubation at room temperature for 1 hour with the secondary antibody (anti-mouse IgG (H+L), HRP conjugate, Promega). The plate was developed using Pierce Step 1 Ultra TMB substrate solution (ThermoFisher Scientific), and then imaged using a Biotek Neos2 microplate reader after terminating the reaction with 2M H2SO4. The IC50 value was calculated by plotting the test compound concentration versus binding percentage using a 3-parameter logistic curve fitting equation.

[0494] αvβ3, αvβ5, and αvβ1 binding assay – 96-well plates were coated overnight at 4°C with fibronectin (2 µg / mL, R&D) or hylocinin (2 µg / mL, R&D) dissolved in coating buffer (15 mmol / L Na2CO3, 35 mmol / L NaHCO3, 7.7 mmol / L NaN3, pH 9.6, containing 1 mmol / L CaCl2 and 1 mmol / L MgCl2). The plates were washed with washing buffer (0.05% Tween-20 in TBS solution, pH 7.4, containing 1 mmol / L MnCl2) and then blocked at 37°C for 1.5 hours with blocking buffer (2% BSA, in washing buffer, pH 7.4). Serially diluted ligands in sample dilution buffer (0.5% BSA, in wash buffer, pH 7.4) were added to the plate along with purified αvβ3 (1.2 µg / mL, Acros Biosystems), αvβ5 (0.8 µg / mL, Acros Biosystems), or αvβ1 protein (4 µg / mL, Acros Biosystems), and incubated at 37°C for 1 hour. The plate was washed with wash buffer and then incubated at 37°C for 1 hour with primary antibody (2 µg / mL human integrin β3 biotinylated antibody, R&D; 2 ng / mL human integrin β1 biotinylated antibody, R&D). The plate was washed with wash buffer and then incubated at 37°C for 1 hour with HRP-conjugated anti-His labeled antibody (AcrosBiosystems; for αvβ5) or HRP-conjugated streptavidin (ThermoFisher Scientific; for αvβ1 and αvβ3). The plate was developed using Pierce Step 1 Ultra TMB substrate solution (Thermo Fisher Scientific), and then imaged using a Biotek Neos2 microplate reader after terminating the reaction with 2M H2SO4. IC50 values ​​were calculated by plotting the test compound concentration versus binding % using a 3-parameter logic curve fitting equation.

[0495] Table 14 – Integrins are firmly bound together

[0496]

[0497] As shown in Table 14, the compounds from Preparation Examples 19, 20, 21, and 22 exhibited strong binding to integrins αvβ6, αvβ8, αvβ5, and αvβ3, with all compounds showing a particular preference for binding to αvβ6 and αvβ5. For example, the compound from Preparation Example 19 showed strong binding to integrins αvβ6, αvβ8, and αvβ5 (IC50 = 5.0 nM, 12.9 nM, and 8.9 nM, respectively), compared to weakened binding to αvβ3 (284 nM) and αvβ1 (114.5 nM). Compared to the compound from Preparation Example 19, the compounds from Preparation Examples 20, 21, and 22 all showed a stronger preference for binding to integrin αvβ5. However, the compound from Preparation Example 19 showed better binding to integrins αvβ6 and αvβ8 compared to the compounds from Preparation Examples 20, 21, and 22.

[0498] Example 8. In vitro activity of SCNN1A RNAi substance in mouse lung epithelial cells

[0499] MLE-12 cells were seeded in 96-well plates (10,000 MLE12 cells / well; Falcon, 353072) and incubated overnight at 37°C. Cells were transfected with serially diluted 1:5 siRNA at a starting concentration of 100 nM in Opti-Mem medium (Gibco, 31985-070) and delivered to cells using lipofectamine (Invitrogen, 13778-150) according to Invitrogen's RNAiMax transfection protocol. Cells were incubated at 37°C for 24 hours. Medium was aspirated from the 96-well plates, and cells were lysed in each well with 150 µL of lysis buffer (Zymo, R1053). The 96-well plates were frozen overnight at -80°C. RNA was purified using a total RNA purification kit (Zymo, R1053). Purified RNA was reverse transcribed into cDNA according to the manufacturer's instructions for the High-Capacity cDNA Reverse Transcription Kit (AppliedBiosystems, 4368813). The cDNA was diluted 2.5x with RNase-free water, placed in 384-well plates, and run on a QuantStudio7 Flex (AppliedBiosystems) according to the manufacturer's instructions. TaqMan PCR assays were run using the SCNN1A (Mm00803386_m1) TaqMan probe and GAPDH as an endogenous control (Mm99999915_g1) (ThermoFisher Scientific). GraphPad was used for computation. CT scan - CT and magnification changes were plotted and graphed (Tables 15, 16, and 17).

[0500] Table 15 – In vitro activity of RNAi substances in mouse lung epithelial cells

[0501]

[0502] %KD refers to the percentage reduction of the SCNN1A gene in MLE12 cells relative to the control.

[0503] Table 16 – In vitro activity of RNAi substances in mouse lung epithelial cells

[0504]

[0505] %KD refers to the percentage reduction of the SCNN1A gene in MLE12 cells relative to the control.

[0506] Table 17 – In vitro activity of IRNAi substances in mouse lung epithelial cells

[0507]

[0508] %KD refers to the percentage reduction of the SCNN1A gene in MLE12 cells relative to the control.

[0509] Example 9. In vivo intranasal administration of uncoupled and ligand-coupled Muc5B RNAi substances in mice.

[0510] Mice were given 3 mg / kg or 10 mg / kg of RNAi substance on day 1 and sacrificed on day 15 (Table 18).

[0511] Table 18 – In vitro and in vivo activities of Muc5B RNAi substances

[0512]

[0513] %KD refers to the percentage reduction in the Muc5B gene. MLE12 cells were used for in vitro assays.

[0514] As shown in Table 18, compared with a moderate but significant 32% knockdown achieved using integrin ligands, uncoupled RNAi material (59-17) did not show knockdown of the target gene at a dose of 3 mpk, thus demonstrating the benefit of using integrin ligands for RNAi material uptake.

[0515] Example 10. In vivo intranasal administration of uncoupled and ligand-coupled RAGE RNAi substances in mice.

[0516] Mice were given 0.5 mg / kg of RNAi substance on day 1 and sacrificed on day 15 (Table 19).

[0517] Table 19 – Mean relative RAGE mRNA knockdown at sacrifice (%KD)

[0518]

[0519] As shown in Table 19, each RAGE RNAi substance showed reduced mRNA expression in mice compared to the control. Integrin ligand conjugates (59-20) showed better efficacy compared to unconjugated RNAi substances (5-19), thus demonstrating the benefit of using integrin ligands for RNAi substance uptake.

[0520]

[0521] .

Claims

1. The following compound: ， in: R1 is H or C 1- C6 alkyl, For (C5-C) 14 aryl)-O- or (C5-C)- having 1-5 heteroatoms selected from N, O and S 14 (heteroaryl)-O-, L may include a connector or not. Z contains oligonucleotides, and q is an integer between 1 and 3. Or its pharmaceutically acceptable salt.

2. The compound of claim 1, wherein the compound has the following formula: 。 3. The compound of claim 1, wherein the compound has the following formula: 。 4. The compound according to any one of claims 1-3, wherein Selected from: 。 5. The compound according to any one of claims 1-4, wherein It has the following formula: 。 6. The compound according to any one of claims 1-4, wherein It has the following formula: 。 7. The compound according to any one of claims 1-4, wherein It has the following formula: 。 8. The compound according to any one of claims 1-4, wherein It has the following formula: 。 9. The compound of any one of claims 1-8, wherein L comprises a connector of the following formula: , , , or , Each side of L can be with Or Z-connected, and n and m are independently selected from integers from 1 to 10.

10. The compound of any one of claims 1-9, wherein L comprises a connector of the following formula: ， Each side of L can be with Or Z-connected, and n and m are independently selected from integers from 1 to 10.

11. The compound of any one of claims 1, 2, or 4-10, wherein the compound has the following formula: Or, or a pharmaceutically acceptable salt thereof.

12. The compound of any one of claims 1, 2, or 4-10, wherein the compound has the following formula: Or, or a pharmaceutically acceptable salt thereof.

13. The compound of any one of claims 1, 2, or 4-10, wherein the compound has the following formula: Or, or a pharmaceutically acceptable salt thereof.

14. The compound of any one of claims 1, 2, or 4-10, wherein the compound has the following formula: ， Or its pharmaceutically acceptable salt.

15. The compound of any one of claims 1, 2, or 4-10, wherein the compound has the following formula: Or, or a pharmaceutically acceptable salt thereof.

16. The compound of any one of claims 1, 2, or 4-10, wherein the compound has the following formula: Or, or a pharmaceutically acceptable salt thereof.

17. The compound of any one of claims 1, 2, or 4-10, wherein the compound has the following formula: Or, or a pharmaceutically acceptable salt thereof.

18. The compound of any one of claims 1, 2, or 4-10, wherein the compound has the following formula: Or, or a pharmaceutically acceptable salt thereof.

19. The compound of any one of claims 1-9, wherein L comprises a connector of the following formula: ， Each side of L can be with Alternatively, Z can be connected, where m is an 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 has the following formula: ， Or its pharmaceutically acceptable salt.

22. The compound of claim 19 or 20, wherein the compound has the following formula: ， Or its pharmaceutically acceptable salt.

23. The compound of claim 19 or 20, wherein the compound has the following formula: ， Or its pharmaceutically acceptable salt.

24. The compound of claim 19 or 20, wherein the compound has the following formula: ， Or its pharmaceutically acceptable salt.

25. The compound of any one of claims 1-24, wherein the oligonucleotide comprises the 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 ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22 or SEQ ID NO.

23.

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

27. The compound of claim 26, wherein the compound is used to treat lung disease.

28. Use of the compound of any one of claims 1-25 or a pharmaceutically acceptable salt thereof in the preparation of a medicament.

29. Use of any compound of claims 1-25 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating lung diseases.

30. A method of treating lung disease, comprising administering to a patient in need of treatment a composition comprising any one of the compounds of claims 1-25.

31. The following compounds: ， Where R1 is H or C 1- C6 alkyl, and For (C5-C) 14 aryl)-OH or (C5-C)-OH having 1-5 heteroatoms selected from N, O and S 14 (heteroaryl)-OH.

32. The compound of claim 31, wherein Selected from: 。 33. The compound of claim 31, wherein Selected from: 。 34. The compound of claim 31, wherein the compound has the following formula: 。 35. The compound of claim 31, wherein the compound has the following formula: 。 36. The compound of claim 31, wherein the compound has the following formula: 。 37. The compound of claim 31, wherein the compound has the following formula: 。

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