Trialkyne linkers and methods of use

Abstract

The invention relates to trialkyne linkers and methods of use. Improved linkers are described that can be used to facilitate the attachment of targeting groups, pharmacokinetic (PK) enhancers or modifiers, or other presenting agents to oligonucleotides. The linkers can exhibit improved reaction yield, stability, and biological activity, particularly when used in combination with oligonucleotide-based compounds, such as RNA interference (RNAi) agents.

Description

This application is a divisional application. The filing date of the original application is February 15, 2019, the application number is 201980013763.0 (PCT / US2019 / 018232), and the invention title is "Trialkyne Linkers and Methods of Use".

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application Serial No. 62 / 631,683, filed February 17, 2018; U.S. Provisional Application Serial No. 62 / 646,739, filed March 22, 2018; U.S. Provisional Application Serial No. 62 / 663,763, filed April 27, 2018; and U.S. Provisional Application Serial No. 62 / 790,300, filed January 9, 2019, all of which are hereby incorporated by reference in their entireties. Field of the Invention

[0002] The present disclosure relates to trialkyne linkers suitable for use with synthetic oligonucleotides, such as RNA interference (RNAi) agents. Background Synthetic oligonucleotides, such as antisense compounds, aptamers, ribozymes, and RNA interference (RNAi) agents or molecules, are increasingly used in biomedical research, diagnosis, and therapy. These synthetic oligonucleotides have been used to inhibit or knockdown gene expression in vitro, in situ, and in vivo in a sequence - dependent manner.

[0004] It is often useful to attach or link a targeting ligand or other pharmacological or pharmacokinetic enhancer or modifier to a synthetic oligonucleotide, especially for in vivo delivery for therapeutic purposes. For it to be useful, the ligation chemistry should be modular to make it easily adaptable to different synthetic oligonucleotides as well as different targeting ligands and pharmacological modifiers. In addition, the ligation chemistry should have simple reaction conditions, be efficient (i.e., provide high chemical yields), not require toxic or other harmful products and not produce toxic or other harmful by - products. The ligation chemistry should also be stable outside the target cell, such as in circulation, the subcutaneous space, or the extracellular space, but cleave readily at the final site of action, such as within the target cell. In addition, particularly for oligonucleotide - based therapies, linker length and flexibility are known to significantly affect the in vivo potency of therapeutic compounds, especially by altering cellular uptake, in certain situations.

[0005] There is a need for linkers with suitable properties for attaching oligonucleotide - based compounds, such as RNAi agents, to targeting ligands. Summary In one aspect, the present invention provides a compound having the structure according to Formula I: or a pharmaceutically acceptable salt thereof, wherein L 1, L 2 and L 3 each independently is a linking group containing an optionally substituted alkylene group; Q is a tetravalent carbon, a tetrasubstituted phenyl or an optionally substituted alkylene group; R contains a coupling moiety or an RNAi agent; and X is NR x or a bond, and R x is H or an optionally substituted C1-C6 alkyl group.

[0007] In one aspect, the present invention provides a compound having the structure according to Formula II: or a pharmaceutically acceptable salt thereof, wherein, L 1 , L 2 and L 3 each independently is a linking group containing an optionally substituted alkylene group; L 4 is a linking group containing an optionally substituted alkylene group, an optionally substituted arylene group or an optionally substituted cycloalkylene group; each occurrence of R 1 is an optionally substituted alkyl group; R 2 is an optionally substituted alkyl group; and R 4 is H or an optionally substituted alkyl group.

[0008] Another aspect of the present invention described herein is a compound having the structure according to Formula III: or a pharmaceutically acceptable salt thereof, wherein L 1 , L 2 and L 3 each independently is a linking group containing an optionally substituted alkylene group; L 4 is a linking group containing an optionally substituted alkylene group, an optionally substituted arylene group or an optionally substituted cycloalkylene group; R 4 is H or an optionally substituted alkyl group; X is O or S; and RNA contains an RNAi agent or consists of an RNAi agent.

[0009] In another aspect, compounds having the structure according to Formula IV are described herein: or a pharmaceutically acceptable salt thereof, wherein, L1 , L 2 and L 3 are each independently a linking group comprising an optionally substituted alkylene group; L 4 is a linking group comprising an optionally substituted alkylene group, an optionally substituted arylene group or an optionally substituted cycloalkylene group; R 1 and R 2 are each independently an optionally substituted alkyl group; R 4 is H or an optionally substituted alkyl group; and TL is a targeting ligand.

[0010] Another aspect of the invention described herein is a compound of the structure according to formula V: or a pharmaceutically acceptable salt thereof, wherein, L 1 , L 2 and L 3 are each independently a linking group comprising an optionally substituted alkylene group; L 4 is a linking group comprising an optionally substituted alkylene group, an optionally substituted arylene group or an optionally substituted cycloalkylene group; R 4 is H or an optionally substituted alkyl group; TL is a targeting ligand; Y is O or S; and RNA comprises an RNAi agent or consists of an RNAi agent.

[0011] In another aspect, compounds of the structure according to formula VI are described herein: or a pharmaceutically acceptable salt thereof, wherein, L 1 , L 2 and L 3 are each independently a linking group comprising an optionally substituted alkylene group; L 4 is a linking group comprising an optionally substituted alkylene group, an optionally substituted arylene group or an optionally substituted cycloalkylene group; R 3 is H, an optionally substituted alkyl group or an optionally substituted aryl group; and R 4 is H or an optionally substituted alkyl group.

[0012] Another aspect of the invention described herein is a compound of the structure according to formula VII: or a pharmaceutically acceptable salt thereof, wherein, L 1 、L 2 and L 3 are each independently a linking group comprising an optionally substituted alkylene group; L 4 is a linking group comprising an optionally substituted alkylene group, an optionally substituted arylene group or an optionally substituted cycloalkylene group; each occurrence of R 4 is H or an optionally substituted alkyl group; X is O or S; and the RNA comprises an RNAi agent or consists of an RNAi agent.

[0013] In another aspect, the present invention describes a compound according to the structure of Formula VIII: or a pharmaceutically acceptable salt thereof, wherein, L 1 、L 2 and L 3 are each independently a linking group comprising an optionally substituted alkylene group; L 4 is a linking group comprising an optionally substituted alkylene group, an optionally substituted arylene group or an optionally substituted cycloalkylene group; R 4 is H or an optionally substituted alkyl group; and TL is a targeting ligand.

[0014] Another aspect of the present invention described herein is a compound according to the structure of Formula IX: or a pharmaceutically acceptable salt thereof, wherein, L 1 、L 2 and L 3 are each independently a linking group comprising an optionally substituted alkylene group; L 4 is a linking group comprising an optionally substituted alkylene group, an optionally substituted arylene group or an optionally substituted cycloalkylene group; R 4 is H or an optionally substituted alkyl group; TL is a targeting ligand; X is O or S; and the RNA comprises an RNAi agent or consists of an RNAi agent.

[0015] Another aspect of the present invention provides a compound of Formula II: Method for reacting with an RNAi agent to form a compound of formula III: wherein, L 1 、L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene; L 4 is a linker comprising an optionally substituted alkylene, an optionally substituted arylene, and an optionally substituted cycloalkylene; each occurrence of R 1 is an optionally substituted alkyl; R 2 is an optionally substituted alkyl; and R 4 is H or an optionally substituted alkyl X is O or S; and RNA comprises or consists of an RNAi agent.

[0016] Another aspect of the present invention provides a method for reacting a compound of formula VI with an RNAi agent comprising a free amine to form a compound of formula VII: wherein, L 1 、L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene; L 4 is a linker comprising an optionally substituted alkylene, an optionally substituted arylene, or an optionally substituted cycloalkylene; R 3 is H, an optionally substituted alkyl, or an optionally substituted aryl; and R 4 is H or an optionally substituted alkyl in each occurrence; and RNA comprises or consists of an RNAi agent.

[0017] Another aspect of the present invention provides a method for reacting a compound of formula III with a targeting ligand comprising an azide to form a compound of formula V, wherein, L 1 、L 2 and L 3Each independently is a linking group containing an optionally substituted alkylene group; L 4 is a linking group containing an optionally substituted alkylene group, an optionally substituted arylene group or an optionally substituted cycloalkylene group; R 4 is H or an optionally substituted alkyl group; TL is a targeting ligand; Y is O or S; and RNA contains an RNAi agent or consists of an RNAi agent.

[0018] Another aspect of the present invention provides a method for reacting a compound of formula VII with a targeting ligand containing an azide to form a compound of formula IX, wherein, L 1 、L 2 and L 3 Each independently is a linking group containing an optionally substituted alkylene group; L 4 is a linking group containing an optionally substituted alkylene group, an optionally substituted arylene group or an optionally substituted cycloalkylene group; R 4 in each case is H or an optionally substituted alkyl group; and RNA contains an RNAi agent or consists of an RNAi agent. Detailed description Novel compounds containing phosphoramidite triynes, their synthesis and methods of use thereof are disclosed herein. The novel compounds disclosed herein exhibit improved reaction yields, stability and bioactivity when used to conjugate synthetic oligonucleotides such as RNAi agents to targeting ligands or other pharmacokinetic (PK) enhancers or modifiers.

[0020] Triyne linkers, their synthesis and methods of use thereof are disclosed herein. The triyne linkers disclosed herein can be linked to oligonucleotides, after which the oligonucleotides can be easily linked to related compounds such as targeting ligands, lipids, cholesterol, delivery agents (such as endosomolytic polymers) or pharmacological modifiers. The triyne linkers disclosed herein can facilitate the synthesis of oligonucleotide conjugates, which have improved yields and fewer impurities compared to the use of other known linkers, while maintaining or even improving in some embodiments the potency of oligonucleotide conjugates such as RNAi agents conjugated to one or more targeting ligands and / or pharmacokinetic modifiers.

[0021] As used herein, the term "linking group" refers to an organic moiety that connects two portions of a compound. A linking group typically contains a direct bond or atoms such as oxygen or sulfur, units such as NR L (wherein R L is hydrogen, acyl, aliphatic or substituted aliphatic), C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as but not limited to substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl, heteroaryl, heterocyclo, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocycloalkyl, alkylheterocycloalkenyl, alkylheterocycloalkynyl, alkenylheterocycloalkyl, alkenylheterocycloalkenyl, alkenylheterocycloalkynyl, alkynylheterocycloalkyl, alkynylheterocycloalkenyl, alkynylheterocycloalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, wherein one or more methylenes may be replaced by O, S, S(O), SO2, N(R L )(wherein R L is hydrogen, acyl, aliphatic or substituted aliphatic), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclo; (CH2) n -, (CH2) n N-,-(CH2) n O-,-(CH2) n S-,-(CH2) n -C(O)-, -C(O)-(CH2) n -C(O)-NH-(CH2) m -C(O)-NH-(CH2) x -,-C(O)-(CH2) n -C(O)-NH-(CH2) m --C(O)-(CH2) n -C(O)-(CH2) m -,-C(O)-(CH2) n -NH-C(O)-(CH2) m -,-C(O)-(CH2) n -O-(CH2-CH2-O)m -(CH2) x -,-(O-CH2-CH2) n -,-O-(CH2-CH2-O) n -,-O-(CH2-CH2-O) n -CH2-,-CH2-(O-CH2-CH2) n -,-CH2-(O-CH2-CH2) n -O-,-CH2-(O-CH2-CH2) n -O-CH2-,-CH2-CH2-(O-CH2-CH2) n -,-(CH2-CH2-O) n -,-(CH2-CH2-O) n -CH2-, Inserted or capped.

[0022] The reactive groups are those commonly available in the art and include but are not limited to activated esters, NHS, TFP, PFP, tetrazines, norbornenes, trans-cyclooctenes, hydrazines (such as HYNIC), aminooxy reagents, and aldehydes (such as 4-formylbenzoic acid).

[0023] Targeting ligands (which may sometimes be referred to in the art as targeting groups) are used to target or improve the delivery of a compound to a target cell or tissue, or to a specific cell type. The targeting ligand enhances the association of the molecule with the target cell. Thus, the targeting ligand can enhance the pharmacokinetic or biodistribution properties of the conjugate to which it is attached to improve the cellular distribution and cellular uptake of the conjugate. Binding of the targeting group to a cell or cell receptor can initiate endocytosis. The targeting group can be monovalent, divalent, trivalent, tetravalent, or of higher valence. The targeting group can be, but is not limited to, a compound having an affinity for a cell surface molecule, a cell receptor ligand, an antibody, a monoclonal antibody, an antibody fragment, and an antibody mimetic having an affinity for a cell surface molecule, a hydrophobic group, cholesterol, cholesteryl, or a steroid. In some embodiments, the targeting group comprises a cell receptor ligand. A variety of targeting groups have been used to target drugs and genes to cells and specific cell receptors. Cell receptor ligands can include but are not limited to: carbohydrates, glycans, sugars (including but not limited to: galactose, galactose derivatives (such as N-acetylgalactosamine), mannose, and mannose derivatives), haptens, vitamins, folic acid, biotin, aptamers, and peptides (including but not limited to: RGD-containing peptides, RGD mimetics, insulin, EGF, and transferrin).

[0024] Unless otherwise specified, the term "alkyl" as used herein refers to a saturated aliphatic straight-chain or branched-chain hydrocarbon group having 1 to 10 carbon atoms. For example, "C1-C6 alkyl" includes alkyl groups having 1, 2, 3, 4, 5, or 6 carbons in a straight-chain or branched-chain arrangement. Non-limiting examples of alkyl include methyl, ethyl, isopropyl, tert-butyl, and n-hexyl. The term "aminoalkyl" as used herein refers to an alkyl group as defined above substituted at any position with one or more amino groups as permitted by normal valency. The amino group may be unsubstituted, monosubstituted, or disubstituted. Non-limiting examples of aminoalkyl include aminomethyl, dimethylaminomethyl, and 2-aminopropan-1-yl.

[0025] The term "alkylene" as used herein refers to a divalent group of an alkyl as described herein. An alkylene is a subset of an alkyl and refers to the same residue as an alkyl but having two substitution points. Examples of alkylene are methylene, -CH2-, or ethylene, -CH2CH2-, or and propylene, -CH2CH2CH2-

[0026] Unless otherwise specified, the term "cycloalkyl" as used herein refers to a saturated or unsaturated non-aromatic hydrocarbon ring group having 3 to 14 carbon atoms. Non-limiting examples of cycloalkyl include, but are not limited to, cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, and cyclohexyl. Cycloalkyl may include multiple spiro or fused rings. Cycloalkyl is optionally mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by normal valency.

[0027] The term "cycloalkylene" as used herein refers to a divalent group of a cycloalkyl as described herein. A cycloalkylene is a subset of a cycloalkyl and refers to the same residue as a cycloalkyl but having two substitution points. Examples of cycloalkylene include cyclopropylene 1,4-cyclohexylene and 1,5-cyclooctylene Cycloalkylene is optionally mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by normal valency. Cycloalkylene can be monocyclic, bicyclic, or tricyclic.

[0028] Unless otherwise specified, the term "alkenyl" as used herein refers to a non-aromatic straight-chain or branched-chain hydrocarbon group containing at least one carbon-carbon double bond and having 2 to 10 carbon atoms. Up to 5 carbon-carbon double bonds may be present in such groups. For example, "C2-C6" alkenyl is defined as an alkenyl having 2 to 6 carbon atoms. Examples of alkenyl include, but are not limited to, vinyl, propenyl, butenyl, and cyclohexenyl. The straight-chain, branched-chain, or cyclic portion of the alkenyl may contain double bonds and may optionally be mono-, di-, tri-, tetra-, or penta-substituted at any position permitted by normal valency. The term "cycloalkenyl" refers to a monocyclic hydrocarbon group having the specified number of carbon atoms and at least one carbon-carbon double bond.

[0029] Unless otherwise specified, the term "alkynyl" as used herein refers to a straight-chain or branched-chain hydrocarbon group containing 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond. Up to 5 carbon-carbon triple bonds may be present. Thus, "C2-C6 alkynyl" refers to an alkynyl having 2 to 6 carbon atoms. Examples of alkynyl include, but are not limited to, ethynyl, 2-propynyl, and 2-butynyl. The straight-chain or branched-chain portion of the alkynyl may optionally be mono-, di-, tri-, tetra-, or penta-substituted at any position permitted by normal valency.

[0030] As used herein, "alkoxyl" ("alkoxyl" or "alkoxy") refers to -O-alkyl having the indicated number of carbon atoms. For example, C1–6 alkoxyl is intended to include C1, C2, C3, C4, C5, and C6 alkoxyl. For example, C1–8 alkoxyl is intended to include C1, C2, C3, C4, C5, C6, C7, and C8 alkoxyl. Examples of alkoxyl include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentyloxy, sec-pentyloxy, n-heptyloxy, and n-octyloxy.

[0031] As used herein, "keto group" refers to any alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclic group, heteroaryl, or aryl as defined herein that is bridged by a carbonyl group. Examples of keto groups include, but are not limited to, alkanoyl (e.g., acetyl, propionyl, butyryl, valeryl, or hexanoyl), alkenoyl (e.g., acryloyl), alkynoyl (e.g., ethynoyl, propynoyl, butynoyl, pentynoyl, or hexynoyl), aroyl (e.g., benzoyl), heteroaroyl (e.g., pyrroloyl, imidazoyl, quinolinoyl, or pyridinoyl).

[0032] As used herein, "alkoxycarbonyl" refers to any alkoxyl as defined above that is bridged by a carbonyl group (i.e., -C(O)O-alkyl). Examples of alkoxycarbonyl include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-propoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, or n-pentyloxycarbonyl.

[0033] As used herein, "aryloxycarbonyl" refers to any aryl as defined herein linked by an oxycarbonyl bridge (i.e., -C(O)O-aryl). Examples of aryloxycarbonyl include, but are not limited to, phenoxycarbonyl and naphthyloxycarbonyl.

[0034] As used herein, "heteroaryloxycarbonyl" refers to any heteroaryl as defined herein linked by an oxycarbonyl bridge (i.e., -C(O)O-heteroaryl). Examples of heteroaryloxycarbonyl include, but are not limited to, 2-pyridyloxycarbonyl, 2-oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl, or pyrimidyloxycarbonyl.

[0035] As used herein, "aryl" or "aromatic" refers to any stable monocyclic or polycyclic carbocyclic ring having up to 6 atoms in each ring, wherein at least one ring is aromatic. Examples of aryl include, but are not limited to, phenyl, naphthyl, anthryl, tetrahydronaphthyl, indanyl, and biphenyl. Where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is through the aromatic ring. The aryl is optionally mono-, di-, tri-, tetra-, or penta-substituted at any position permitted by normal valency.

[0036] As used herein, the term "arylene" refers to a divalent group of an aryl as described herein. Arylene is a subset of aryl and refers to the same residue as aryl but having two points of substitution. Examples of arylene include phenylene, which refers to divalent phenyl. The arylene is optionally mono-, di-, tri-, tetra-, or penta-substituted at any position permitted by normal valency.

[0037] As used herein, the term "coupling moiety" refers to a chemical moiety that can be used to couple two molecules together. For example, a "coupling moiety" can refer to phosphoramidite, which reacts with an alcohol on a separate molecule to form an organophosphate / ester. Further examples of coupling agents can include, but are not limited to, esters, carbonates / esters, carboxylic acids, alkenes, alcohols, amines, aldehydes, ketones, alkynes, halogens, Grignard reagents, leaving groups, and any other moiety known in the art for coupling two molecules.

[0038] As used herein, the term "halo" refers to a halogen group. For example, "halo" can refer to a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) group.

[0039] As used herein, the term "heteroaryl" represents a stable monocyclic or polycyclic ring having up to 7 atoms in each ring, wherein at least one ring is aromatic and contains 1 to 4 heteroatoms selected from O, N, and S. Examples of heteroaryl include, but are not limited to, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furyl, thienyl, benzothienyl, benzofuryl, benzimidazolinone, benzoxazolone, quinolinyl, isoquinolinyl, dihydroisoindolinone, imidazopyridyl, isoindolinone, indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, and tetrahydroquinoline. "Heteroaryl" is also understood to include N-oxide derivatives of any nitrogen-containing heteroaryl. Where the heteroaryl substituent is bicyclic and one ring is non-aromatic or does not contain heteroatoms, it is understood that the attachment is via the aromatic ring or via the ring containing heteroatoms. Heteroaryl is optionally mono-, di-, tri-, tetra-, or penta-substituted at any position permitted by normal valency.

[0040] As used herein, the term "heteroarylene" refers to a divalent group of a heteroaryl as described herein. Heteroarylene is a subset of heteroaryl and refers to the same residue as heteroaryl, but having two attachment points. Examples of heteroarylene include pyridylene, pyrimidylene, and pyrrolylene. Heteroarylene is optionally mono-, di-, tri-, tetra-, or penta-substituted at any position permitted by normal valency.

[0041] As used herein, the terms "heterocycle", "heterocyclic", or "heterocyclic group" refer to an aromatic or non-aromatic 3- to 14-membered heterocycle containing 1 to 4 heteroatoms selected from O, N, and S, including polycyclic groups. The term "heterocyclic" as used herein is also considered synonymous with the terms "heterocycle" and "heterocyclic group" and is understood to have the same definition as set forth herein. "Heterocyclic group" includes the heteroaryls mentioned above, and their dihydro and tetrahydro analogs. Examples of heterocyclic groups include, but are not limited to, azetidinyl, benzimidazolyl, benzofuryl, benzofurazanyl, benzpyrazolyl, benzotriazolyl, benzothienyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furyl, imidazolyl, indolinyl, indolyl, indolizinyl, indazolyl, isobenzofuryl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolidinyl, oxazolyl, oxazoline, oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyridone, pyrimidinyl, pyrimidinone, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydropyranyl, tetrahydrofuryl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, tetrazolyl, tetrazolopyridinyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, 1,4-dioxanyl, hexahydroazepinyl groups such as a radical, piperazinyl, piperidinyl, pyridin-2-one group, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothiophenyl, dihydrotriazolyl, dihydroazetidinyl, sulfoxide thiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl and tetrahydrothiophenyl and their N-oxides. The attachment of the heterocyclic group substituents can be via a carbon atom or via a heteroatom. The heterocyclic group is optionally mono-, di-, tri-, tetra- or penta-substituted at any position as permitted by the normal valency.

[0042] As used herein, the term "heterocycloalkyl" refers to a 3- to 14-membered non-aromatic heterocycle containing 1 to 4 heteroatoms selected from O, N and S, including polycyclic groups. Examples of heterocyclic groups include, but are not limited to, azetidinyl, oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, oxetanyl, pyranyl, pyridinone group, pyrimidinone group, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolyl, 1,4-dioxanyl, hexahydroaza groups such as a radical, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrofuranyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothiophenyl, dihydrotriazolyl, sulfoxide thiomorpholinyl and tetrahydrothiophenyl and their N-oxides. The attachment of the heterocycloalkyl group substituents can be via a carbon atom or via a heteroatom. The heterocyclic group is optionally mono-, di-, tri-, tetra- or penta-substituted at any position as permitted by the normal valency.

[0043] As used herein, the term "subheterocycloalkyl" refers to a divalent group of a heterocycloalkyl as described herein. Subheterocycloalkyl is a subset of heterocycloalkyl and refers to the same residue as heterocycloalkyl but having two substitution points. Examples of subheterocycloalkyl include piperidinylidene, azetidinylidene and tetrahydrofuranylidene. Subheterocycloalkyl is optionally mono-, di-, tri-, tetra- or penta-substituted at any position as permitted by the normal valency.

[0044] As used herein, the terms "treatment" and the like refer to methods or steps taken to provide relief of one or more symptoms of a subject's disease or a reduction in the quantity, severity and / or frequency thereof. "Treatment" as used herein can include prevention, management, prophylactic treatment and / or suppression of the quantity, severity and / or frequency of one or more symptoms of a subject's disease.

[0045] As used herein, the phrase "introducing a cell" when referring to an RNAi agent means functionally delivering the RNAi agent into a cell. The phrase "functionally delivering" means delivering the RNAi agent into a cell in a manner that enables the RNAi agent to have the desired biological activity, such as sequence-specific inhibition of gene expression.

[0046] Unless otherwise specified, the symbols used herein mean that any one or more groups can be attached thereto, which is in line with the scope of the invention described herein. In some embodiments herein, the symbol is used multiple times in a structure to describe the attachment points of a particular variable in the compounds of Formula I. Unless otherwise specified, the indicated variables can be oriented such that any one attachment point on the variable can be attached to any one attachment point on the compounds of Formula I. For example, the variable L 1 has two attachment points to the compounds of Formula I. Although one embodiment of L 1 can be shown as this embodiment should also be understood to refer to a compound where L 1 is .

[0047] As used herein, the term "isomer" refers to compounds that have the same molecular formula but differ in the nature or order of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are called "stereoisomers". Stereoisomers that are not mirror images of each other are called "diastereomers", and stereoisomers that are non-superimposable mirror images are called "enantiomers" or sometimes "optical isomers". A carbon atom bonded to four different substituents is called a "chiral center". When the compounds described herein contain an olefinic double bond or other geometrically asymmetric center and the structure of its isomers is not explicitly specified, the compound is intended to include the E and Z geometric isomers, either independently or as a mixture. The compounds of Formula I or their pharmaceutically acceptable salts are, for example, intended to include all possible isomers, as well as their racemic and optically pure forms. Similarly, unless explicitly specified otherwise, all tautomeric forms are also intended to be included.

[0048] As used herein, a linking group is one or more atoms that connect a molecule or a part of a molecule to another molecule or a second molecule or a second part of a molecule. In the art, the terms linking group and spacers are sometimes used interchangeably. Similarly, as used in the art, the term scaffold is sometimes used interchangeably with a linking group. In some embodiments, a linking group may include a peptide-breakable linking group. In some embodiments, a linking group may include or be composed of a peptide phenylalanine-citrulline-phenylalanine-proline. In some embodiments, a linking group may include or be composed of a PEG group.

[0049] As used herein, the term "linked" when referring to a connection between two molecules refers to the two molecules being linked by a covalent bond or the two molecules being associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond). In some examples, if the term "linked" refers to the association between two molecules via a non-covalent bond, the association between the two different molecules has a molecular mass of less than 1 x 10 in a physiologically acceptable buffer (e.g., phosphate buffered saline). -4 M (e.g. less than 1x 10 -5 M, less than 1x 10 -6 M or less than 1x 10 -7 M) K D If not specified, the term "linked" as used herein may refer to a connection between a first compound and a second compound with or without the presence of any intervening atoms or groups of atoms.

[0050] Those of ordinary skill in the art will readily understand and appreciate that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending on the environment in which the compound or composition is located. Accordingly, as used herein, the structures disclosed herein contemplate that certain functional groups, such as OH, SH, or NH, may be protonated or deprotonated. As those of ordinary skill in the art will readily appreciate, the disclosure herein is intended to cover the disclosed compounds and compositions regardless of their protonation state based on the pH of the environment.

[0051] Structures may be depicted with bonds "floating" on the ring structure to indicate bonding to any carbon or heteroatom on the ring as valence permits. For example, the structure means that R can replace any hydrogen atom at any of the five available positions on the ring. A "floating" bond can also be used in a bicyclic structure to indicate bonding to any position on either ring of the bicyclic ring as valence permits. In the case of a bicyclic ring, the bond will be shown as "floating" on both rings, for example It means that R can replace any hydrogen atom at any of the seven available positions on the ring.

[0052] As used in the claims herein, the phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. When used in the claims herein, the phrase "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0053] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of at least one RNAi agent and one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance other than the active pharmaceutical ingredient (API, therapeutic product, e.g., an RNAi agent) that has been appropriately evaluated for safety and is intentionally included in a drug delivery system. An excipient does not exert, or is not intended to exert, a therapeutic effect at the intended dosage. Excipients can be used to a) assist in the processing of the drug delivery system during manufacture, b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API, c) assist in product identification, and / or d) enhance any other property of the overall safety and effectiveness of the API presentation during storage or use.

[0054] Excipients include, but are not limited to: absorption enhancers, antiadherents, antifoaming agents, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery enhancers, dextrans, dextroses, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline solutions, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, water repellents, and wetting agents. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0055] The pharmaceutical composition may contain other additional components commonly present in pharmaceutical compositions. The pharmaceutically active materials may include, but are not limited to: antipruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.). Also contemplated is that cells, tissues, or ex vivo organs expressing or containing an RNAi agent as defined herein can be used as a "pharmaceutical composition". As used herein, a "pharmacologically effective amount", "therapeutically effective amount", or simply an "effective amount" refers to the amount of an RNAi agent that produces the desired pharmacological, therapeutic, or prophylactic result.

[0056] The term polynucleotide or poly nucleic acid refers to a polymer containing at least two nucleotides. Nucleotides are the monomeric units of a polynucleotide polymer. Polynucleotides having fewer than 120 monomeric units are often referred to as oligonucleotides. Natural nucleic acids have a deoxyribose- or ribose-phosphate backbone. Unnatural or synthetic polynucleotides are polynucleotides that are polymerized in vitro or in a cell-free system and contain the same or similar bases but may contain a different type of backbone than the natural ribose or deoxyribose-phosphate backbone. Synthetic oligonucleotides can be synthesized using any known technique in the art. Polynucleotide backbones known in the art include: PNA (peptide nucleic acid), phosphorothioates / salts, phosphorodiamidates, morpholinos, and other variants of the phosphate backbone of natural nucleic acids. Bases include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs. Synthetic derivatives of purines and pyrimidines include, but are not limited to, modifications that place new reactive groups on the nucleotides, such as, but not limited to, amines, alcohols, thiols, carboxylate / salts, and alkyl halides. The term base encompasses any known base analogs of DNA and RNA. The term polynucleotide includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and combinations of DNA, RNA, and other natural and synthetic nucleotides.

[0057] The synthetic oligonucleotides of the present invention can be chemically modified. The use of chemically modified polynucleotides can improve various properties of the polynucleotides, including but not limited to: tolerance to in vivo nuclease degradation, cellular uptake, activity, and sequence-specific hybridization. Non-limiting examples of such chemical modifications include: phosphorothioate / salt internucleotide linkages, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, 2′-deoxy ribonucleotides, “universal base” nucleotides, 5-C-methyl nucleotides, 2′,3′-seco nucleotide mimics (unlocked nucleobase analogs, denoted herein as N UNA or NUNA), and inverted deoxyabasic residue incorporation. These chemical modifications, when used in various polynucleotide constructs, have shown to preserve the activity of the polynucleotides in cells while significantly enhancing the serum stability of these compounds.

[0058] In some embodiments, the synthetic oligonucleotides of the invention comprise a duplex having two strands, wherein one or both strands may be chemically modified, and wherein each strand is from about 19 to about 29 (such as about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29) nucleotides. In some embodiments, the synthetic oligonucleotides of the invention comprise one or more modified nucleotides. The synthetic oligonucleotides of the invention may comprise from about 5 to about 100% (such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) of modified nucleotides at nucleotide positions.

[0059] The synthetic oligonucleotides may comprise 5′ or 3′ end modifications. 3′ and 5′ end modifications include, but are not limited to: amine-containing groups, alkyl groups, alkylamine groups, reactive groups, TEG groups and PEG groups.

[0060] An “RNAi agent” (also referred to as an “RNAi initiator”) refers to a composition comprising an RNA or RNA-like (such as a chemically modified RNA) oligonucleotide molecule that contains an mRNA transcript of a target mRNA that can reduce or inhibit (such as reduce or inhibit under appropriate conditions) translation in a sequence-specific manner. As used herein, an RNAi agent may work through the RNA interference mechanism (i.e., by interacting with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells to induce RNA interference) or through any alternative mechanism or pathway. Although it is believed that RNAi agents, as the term is used herein, work primarily through the RNA interference mechanism, the disclosed RNAi agents are not restricted or limited to any particular pathway or mechanism of action. The RNAi agents disclosed herein consist of a sense strand and an antisense strand and include, but are not limited to: short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) and Dicer substrates. The antisense strand of the RNAi agents described herein is at least partially complementary to the targeted mRNA (i.e., HIF-2alpha mRNA). The RNAi agents may comprise one or more modified nucleotides and / or one or more non-phosphodiester bonds.

[0061] As used herein, the terms "silence", "reduce", "inhibit", "downregulate" or "knockdown", when referring to the expression of a given gene, mean that when a cell, cell population, tissue, organ or subject is treated with an RNAi agent as described herein, the gene expression, as measured by the level of RNA transcribed from the gene in the cell, cell population, tissue, organ or subject in which the gene is transcribed or the level of polypeptide, protein or protein subunit translated from the mRNA, is reduced as compared to a second cell, cell population, tissue, organ or subject that has not been so treated.

[0062] In some embodiments, the RNAi agent comprises at least two moieties, sequences that are substantially or fully complementary. In some embodiments, the two RNAi agent sequences comprise a sense strand containing a first sequence and an antisense strand containing a second sequence. In some embodiments, the two RNAi agent sequences comprise a sense strand containing a first sequence and an antisense strand containing a second sequence together, wherein the sense strand and the antisense strand together form a meroduplex. The sense strand may be linked to the antisense strand via a linking molecule such as a polynucleotide linker or a non-nucleotide linker.

[0063] The antisense strand comprises a nucleotide sequence complementary to a portion of the mRNA encoded by the target gene, and the complementary region is most preferably less than 30 nucleotides in length. The sense strand of the RNAi agent comprises a sequence having at least 85% identity with at least a portion of the target mRNA. The RNAi agent inhibits the expression of the target gene in vitro or in vivo after being delivered to a cell expressing the target gene.

[0064] In some embodiments, the RNAi agent may be composed of naturally occurring nucleotides or may be composed of at least one modified nucleotide or nucleotide mimetic. The sense and antisense strands of the RNAi agent of the present invention can be synthesized and / or modified by well-established methods in the art. The RNAi agent nucleotides or nucleobases can be linked by covalent internucleotide linkages that contain phosphate (natural) or do not contain phosphate (non-natural), i.e., the RNAi agent can have a natural or non-natural oligonucleotide backbone. In some embodiments, the RNAi agent contains non-standard (non-phosphate) linkages between the nucleobases.

[0065] In some embodiments, the RNAi agent may comprise 5′ or 3′ end modifications. 3′ and 5′ end modifications include, but are not limited to: amine-containing groups, alkyl groups, alkylamine groups, reactive groups, TEG groups and PEG groups.

[0066] In some embodiments, the RNAi agent may comprise overhangs, i.e., usually unpaired protruding nucleotides that do not directly participate in the double helix structure normally formed by the core sequences of the sense and antisense strands.

[0067] In some embodiments, the RNAi agent may independently contain 3′ and / or 5′ overhangs of 1-5 bases on each of the sense and antisense strands. In some embodiments, both the sense and antisense strands contain 3′ and 5′ overhangs. In some embodiments, one or more 3′ overhanging nucleotides of one strand base pair with one or more 5′ overhanging nucleotides of the other strand. In some embodiments, the one or more 3′ overhanging nucleotides of one strand are not paired with the one or more 5′ overhanging nucleotides of the other strand. The sense and antisense strands of the RNAi agent may or may not contain the same number of nucleobases. The antisense and sense strands may form a duplex wherein the 5′ end has only blunt ends, the 3′ end has only blunt ends, both the 5′ and 3′ ends are blunt ends, or neither the 5′ nor the 3′ end is a blunt end. In some embodiments, one or more of the nucleotides in the overhang contain thiophosphate / thiolate, phosphorothioate / thiolate, deoxynucleotide inverted (3′ to 3′ linkage) nucleotides, or are modified ribonucleotides or deoxynucleotides.

[0068] Lists of known mRNA sequences are available in databases maintained by various research institutions, including the database GenBank, which is maintained by a branch of the National Center for Biotechnology Information, the National Institutes of Health in the United States, as part of the International Nucleotide Sequence Database Collaboration. Known effective siRNA sequences and homologous binding sites are also well presented in the relevant literature. RNAi agent molecules are readily designed and produced by techniques known in the art. In addition, there are computational tools that increase the chances of discovering effective and specific sequence motifs (Pei et al. 2006, Reynolds et al. 2004, Khvorova et al. 2003, Schwarz et al. 2003, Ui-Tei et al. 2004, Heale et al. 2005, Chalk et al. 2004, Amarzguioui et al. 2004).

[0069] Formula I Formula I is represented by the following structure: wherein L 1 、L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group; Q is a tetravalent carbon atom, a tetrasubstituted phenyl group, or an optionally substituted alkylene group; R comprises a coupling moiety or an RNAi agent; and X is NR x or a bond, and R x is H or an optionally substituted C1-C6 alkyl group.

[0070] In some embodiments of Formula I, Q is a tetravalent carbon. In some other embodiments of Formula I, Q is wherein indicates the point of attachment. In some other embodiments, Q is wherein indicates the point of attachment.

[0071] In some embodiments of Formula I, L 1 , L 2 and L 3 are each In some embodiments of Formula I, L 1 , L 2 and L 3 are each In some embodiments of Formula I, L 1 , L 2 and L 3 are each

[0072] In some embodiments of Formula I, X is NH.

[0073] In some embodiments of Formula I, R comprises a phosphoramidite. In some embodiments of Formula I, R comprises an organophosphate / salt and an RNAi agent. In some other embodiments of Formula I, R comprises an ester. In some embodiments of Formula I, R comprises a p-nitrophenol ester. In some embodiments of Formula I, R comprises an amide and an RNAi agent. In some other embodiments of Formula I, R comprises a carbonate / salt. In some embodiments, R comprises a carbamate / salt and an RNAi agent.

[0074] In some embodiments of Formula I, R is selected from

[0075] Exemplary compounds of Formula I are shown in Table 1 below: Table 1. Compounds of Formula I

[0076] Formula II Formula II is represented by the following structure: or a pharmaceutically acceptable salt thereof, wherein, L 1 、L 2 and L 3 are each independently a linking group containing an optionally substituted alkylene group; L 4 is a linking group containing an optionally substituted alkylene group, an optionally substituted arylene group and an optionally substituted cycloalkylene group; Each occurrence of R 1 is an optionally substituted alkyl group; R 2 is an optionally substituted alkyl group; and R 4 is H or an optionally substituted alkyl group.

[0077] In some embodiments of Formula II, L 1 、L 2 and L 3 are each In some embodiments of Formula II, L 1 、L 2 and L 3 are each In some embodiments of Formula II, L 1 、L 2 and L 3 are each

[0078] In some embodiments of Formula II, each occurrence of R 1 is isopropyl.

[0079] In some embodiments of Formula II, R 2 is

[0080] In some embodiments of Formula II, L 4 is selected from: wherein indicates the point of attachment.

[0081] Formula III Formula III is represented by the following structure: or a pharmaceutically acceptable salt thereof, Wherein, L 1 、L 2 and L 3 are each independently a linking group comprising an optionally substituted alkylene group; L 4 is a linking group comprising an optionally substituted alkylene group, an optionally substituted arylene group, and an optionally substituted cycloalkylene group; R 4 is H or an optionally substituted alkyl group; X is O or S; and the RNA comprises or consists of an RNAi agent.

[0082] In some embodiments of Formula III, X is O, and the compound of Formula III is an organophosphate / salt. In some embodiments of Formula II, X is S and the compound of Formula III is a thiophosphate / salt.

[0083] In some embodiments of Formula III, L 1 、L 2 and L 3 are each In some embodiments of Formula III, L 1 、L 2 and L 3 are each In some embodiments of Formula III, L 1 、L 2 and L 3 are each

[0084] In some embodiments of Formula III, L 4 is selected from: Wherein indicates the point of attachment.

[0085] Exemplary compounds of Formula III are shown in Table 2 below: Table 2. Compounds of Formula III

[0086] Formula IV Formula IV is represented by the following structure: or a pharmaceutically acceptable salt thereof, wherein, L 1 、L 2 and L 3 are each independently a linking group comprising an optionally substituted alkylene group; L 4 is a linking group comprising an optionally substituted alkylene group, an optionally substituted arylene group and an optionally substituted cycloalkylene group; R 1 and R 2 are each independently an optionally substituted alkyl group; R 4 is H or an optionally substituted alkyl group; and TL is a targeting ligand.

[0087] In some embodiments of Formula IV, L 1 、L 2 and L 3 are each In some embodiments of Formula IV, L 1 、L 2 and L 3 are each In some embodiments of Formula IV, L 1 、L 2 and L 3 are each

[0088] In some embodiments of Formula IV, L 4 is selected from: wherein indicates the point of attachment.

[0089] In some embodiments of Formula IV, each instance of R 1 is isopropyl.

[0090] In some embodiments of Formula IV, R 2 is

[0091] Exemplary compounds of Formula IV are shown in Table 3 below.

[0092] Table 3. Exemplary Compounds of Formula IV

[0093] Formula V Formula V is represented by the following structure: or a pharmaceutically acceptable salt thereof, wherein, L 1 、L 2 and L 3 are each independently a linking group containing an optionally substituted alkylene group; L 4 is a linking group containing an optionally substituted alkylene group, an optionally substituted arylene group or an optionally substituted cycloalkylene group; R 4 is H or an optionally substituted alkyl group; TL is a targeting ligand; Y is O or S; and the RNA contains an RNAi agent or consists of an RNAi agent.

[0094] In some embodiments of Formula V, L 1 、L 2 and L 3 are each In some embodiments of Formula IV, L 1 、L 2 and L 3 are each In some embodiments of Formula V, L 1 、L 2 and L 3 are each

[0095] In some embodiments of Formula V, L 4 is selected from: wherein indicates the point of attachment.

[0096] Exemplary compounds of Formula V are shown in Table 4 below: Table 4. Exemplary Compounds of Formula V

[0097] Formula VI Formula VI is represented by the following structure or a pharmaceutically acceptable salt thereof, wherein, L 1 、L 2 and L 3 are each independently a linking group comprising an optionally substituted alkylene group; L 4 is a linking group comprising an optionally substituted alkylene group, an optionally substituted arylene group or an optionally substituted cycloalkylene group; R 3 is H, an optionally substituted alkyl group or an optionally substituted aryl group; and R 4 is H or an optionally substituted alkyl group.

[0098] In some embodiments of Formula VI, L 1 、L 2 and L 3 are each In some embodiments of Formula VI, L 1 、L 2 and L 3 are each In some embodiments of Formula VI, L 1 、L 2 and L 3 are each

[0099] In some embodiments of Formula VI, L 4 is selected from: wherein indicates the point of attachment.

[0100] In some embodiments of Formula VI, R 3 is an optionally substituted aryl group. In some embodiments of Formula VI, R 3 is p-nitrophenyl.

[0101] In some embodiments of Formula VI, R 4 is H.

[0102] Formula VII Formula VII is represented by the following structure: or a pharmaceutically acceptable salt thereof, Wherein, L 1 、L 2 and L 3 are each independently a linking group comprising an optionally substituted alkylene group; L 4 is a linking group comprising an optionally substituted alkylene group, an optionally substituted arylene group or an optionally substituted cycloalkylene group; R 4 is H or an optionally substituted alkyl group in each case; and the RNA comprises or consists of an RNAi agent.

[0103] In some embodiments of Formula VII, L 1 、L 2 and L 3 are each In some embodiments of Formula VII, L 1 、L 2 and L 3 are each In some embodiments of Formula VII, L 1 、L 2 and L 3 are each

[0104] In some embodiments of Formula VII, L 4 is selected from: wherein indicates the point of attachment.

[0105] Exemplary compounds of Formula VII are shown in Table 5 below.

[0106] Table 5. Exemplary Compounds of Formula VII

[0107] Formula VIII Formula VIII is represented by the following structure: or a pharmaceutically acceptable salt thereof, wherein, L 1 、L 2 and L 3 are each independently a linking group comprising an optionally substituted alkylene group; L 4is a linker comprising an optionally substituted alkylene, an optionally substituted arylene, or an optionally substituted cycloalkylene; R 3 is H, an optionally substituted alkyl, and an optionally substituted aryl; R 4 is H or an optionally substituted alkyl; and TL is a targeting ligand.

[0108] In some embodiments of Formula VIII, L 1 、L 2 and L 3 are each In some embodiments of Formula VIII, L 1 、L 2 and L 3 are each In some embodiments of Formula VIII, L 1 、L 2 and L 3 are each

[0109] In some embodiments of Formula VIII, L 4 is selected from: wherein indicates the point of attachment.

[0110] In some embodiments of Formula VIII, R 3 is an optionally substituted aryl. In some embodiments of Formula VIII, R 3 is p-nitrophenyl.

[0111] Exemplary compounds of Formula VIII are shown in Table 6 below.

[0112] Table 6. Exemplary Compounds of Formula VIII

[0113] Formula IX Formula IX is represented by the following structure: or a pharmaceutically acceptable salt thereof, wherein, L 1 、L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene; L 4is a linker comprising an optionally substituted alkylene, an optionally substituted arylene or an optionally substituted cycloalkylene; TL is a targeting ligand; R 4 in each occurrence is H or an optionally substituted alkyl; and the RNA comprises or consists of an RNAi agent.

[0114] In some embodiments of Formula IX, L 1 、L 2 and L 3 each is In some embodiments of Formula IX, L 1 、L 2 and L 3 each is In some embodiments of Formula IX, L 1 、L 2 and L 3 each is

[0115] In some embodiments of Formula IX, L 4 is selected from: wherein indicates the point of attachment.

[0116] Exemplary compounds of Formula IX are shown in Table 7 below.

[0117] Table 7. Exemplary Compounds of Formula IX wherein TL comprises a targeting ligand and the RNA comprises or consists of an RNAi agent.

[0118] L 1 、L 2 、L 3 In embodiments of Formulas I - IX, each occurrence of L 1 、L 2 or L 3 is a linker comprising an optionally substituted alkylene. L 1 、L 2 or L 3 can include any suitable linking moiety known in the art. In some embodiments, L 1 、L 2 or L 3 comprises a chain that is between 1 and 50 atoms in length. L 1 、L2 or L 3 The chain length of 3 indicates the number of atoms directly between the alkyne and the quaternary carbon, but additional atoms may branch off from the atoms in the chain. In some embodiments, L 1 , L 2 or L 3 may have a length of from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 atoms.

[0119] In some embodiments, each of L 1 , L 2 and L 3 is the same. In other embodiments, L 1 , L 2 and L 3 are each different moieties.

[0120] In some embodiments, L 1 , L 2 , L 3 or L 4 may contain an amide.

[0121] In some embodiments, L 1 , L 2 , L 3 or L 4 may contain a polyethylene glycol (PEG) chain.

[0122] In some embodiments, L 1 , L 2 , L 3 or L 4The optional substituted alkylene may be inserted with amide, ether, ester, thioether, thioketone, ketone, amine, sulfone, sulfonamide or an atomic chain, such as but not limited to substituted or unsubstituted alkenyl, arylalkyl, arylalkenyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl, heteroaryl, heterocyclic, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocycloalkylalkyl, alkylheterocycloalkenylalkyl, alkylheterocycloalkynylalkyl, alkenylheterocycloalkylalkyl, alkenylheterocycloalkenylalkyl, alkenylheterocycloalkynylalkyl, alkynylheterocycloalkylalkyl, alkynylheterocycloalkenylalkyl, alkynylheterocycloalkynylalkyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl or alkynylheteroaryl.

[0123] In some embodiments, L 1 , L 2 and L 3 may each independently be selected from:

[0124] L 4 In embodiments of Formulas I-IX, L 4 is a linker comprising an optionally substituted alkylene. L 4 may include any suitable linking moiety known in the art. In some embodiments, L 4 comprises a chain having a length between 1 and 50 atoms. The chain length of L 2 indicates the number of atoms directly between the alkyne and the quaternary carbon, but additional atoms may branch from the atoms in the chain. In some embodiments, L 2The length can be from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 atoms.

[0125] In some embodiments, L 4 is selected from: wherein indicates a point of attachment.

[0126] R In embodiments of Formulas I-IX, R comprises a conjugate moiety or an RNAi agent. In some embodiments, R comprises a conjugate moiety and the conjugate moiety is a phosphoramidite. In other embodiments, R comprises a conjugate moiety and the conjugate moiety is an ester. In other embodiments, R comprises a conjugate moiety and the conjugate moiety is a carbonate / salt.

[0127] In some embodiments, R comprises an RNAi agent. When R comprises an RNAi agent, R may comprise additional atoms that are not part of the RNAi sequence. For example, in some embodiments, R can be wherein RNA refers to the RNAi agent, and indicates a point of attachment. In some embodiments, the RNAi agent is bonded to the compound of Formulas I-IX at the 5' end of the sense strand.

[0128] In some embodiments, R is selected from

[0129] Pharmaceutical composition In some embodiments, the present disclosure provides a pharmaceutical composition comprising a therapeutic compound comprising one or more of the triyne linkers disclosed herein.

[0130] As used herein, "pharmaceutical composition" comprises a pharmacologically effective amount of an active pharmaceutical ingredient (API) and optionally one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the active pharmaceutical ingredient (API, therapeutic product) that are intentionally included in a drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients can be used to a) assist in the processing of the drug delivery system during manufacturing, b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API, c) assist in product identification, and / or d) enhance any other property of the overall safety and effectiveness of the API presentation during storage or use. Pharmaceutically acceptable excipients can be or not be inert substances.

[0131] Excipients include, but are not limited to: absorption enhancers, anti-adhesives, anti-foaming agents, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery enhancers, delivery polymers, dextrans, dextroses, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline solutions, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, tonicity agents, vehicles, water-repellent agents, and wetting agents.

[0132] The pharmaceutical compositions described herein may contain other additional components commonly present in pharmaceutical compositions. In some embodiments, the additional component is a pharmaceutically active material. Pharmaceutically active materials include, but are not limited to: antipruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.), small molecule drugs, antibodies, antibody fragments, aptamers, and / or vaccines.

[0133] The pharmaceutical composition may also contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts for altering osmotic pressure, buffers, coating agents, or antioxidants. They may also contain other agents with known therapeutic benefits.

[0134] The pharmaceutical composition can be administered in many ways depending on whether local or systemic treatment is required and the area to be treated. Administration can be effected by any of the methods well-known in the art, for example but not limited to locally (e.g., by transdermal patch), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, intranasal), epidermal, transdermal, oral or parenteral. Parenteral administration includes but is not limited to intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal (e.g., by means of an implanted device), intracranial, intrenchymal, intrathecal and intraventricular administration. In some embodiments, the pharmaceutical composition described herein is administered by subcutaneous injection. The pharmaceutical composition can be administered orally, for example in the form of tablets, coated tablets, lozenges, hard or soft gelatin capsules, solutions, emulsions or suspensions. It can also be administered rectally (e.g., using suppositories); locally or transdermally (e.g., using ointments, creams, gels or solutions); or parenterally (e.g., using injection solutions).

[0135] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where soluble in water) or dispersions and sterile powders for the extemporaneous preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol) and suitable mixtures thereof. The fluidity can be maintained, for example, by the use of coating agents such as lecithin, in the case of dispersions, by maintaining the required particle size and by the use of surfactants. In many cases, it is preferred to include in the composition isotonic agents, for example sugars, polyols such as mannitol, sorbitol and sodium chloride. Prolonged absorption of the injectable compositions can be achieved by including in the composition agents that delay absorption, for example aluminum monostearate and gelatin.

[0136] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound, as required, into a suitable solvent with one or a combination of the above-listed ingredients, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients selected from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and freeze-drying which yield a powder of the active ingredient + any additional required ingredients from a previously filter-sterilized solution.

[0137] Formulations suitable for intra-articular administration may be in the form of a sterile aqueous preparation of any of the ligands described herein, and the ligands may be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems may also be used to deliver any of the ligands described herein for intra-articular and intra-ocular administration.

[0138] The active compound may be prepared with a carrier that prevents the rapid elimination of the compound from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. The methods of preparing such formulations are obvious to those skilled in the art. Liposomal suspensions may also be used as pharmaceutically acceptable carriers. These may be prepared by methods known to those skilled in the art, as described, for example, in U.S. Patent No. 4,522,811.

[0139] The pharmaceutical composition may contain other additional components commonly present in pharmaceutical compositions. Such additional components include, but are not limited to: antipruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.). As used herein, "pharmacologically effective amount," "therapeutically effective amount," or simply "effective amount" refers to the amount of a pharmaceutically active agent that produces a pharmacological, therapeutic, or prophylactic result.

[0140] Agents containing a triyne linker are also an object of the present invention, and the method of manufacturing such agents is also an object of the present invention, the method comprising making a pharmaceutically acceptable form of one or more compounds containing a triyne linker and, if desired, one or more other substances having known therapeutic benefits.

[0141] The triyne linker and pharmaceutical compositions containing the triyne linker disclosed herein may be packaged or included in a kit, container, pack, or dispenser. The triyne linker and pharmaceutical compositions containing the triyne linker may be packaged in pre-filled syringes or vials.

[0142] Targeting ligands, pharmacokinetic (PK) modulators, and delivery vehicles In some embodiments, the triyne linker is conjugated to one or more non-nucleotide groups, including but not limited to targeting ligands, pharmacokinetic (PK) modulators, delivery polymers, or delivery vehicles. The non-nucleotide groups may enhance the targeting, delivery, or ligation of the cargo molecule. The non-nucleotide groups may be covalently linked to the RNAi agent at the 3' or 5' end of the sense strand. In some embodiments, the non-nucleotide groups are linked to the 5′ end of the sense strand of the RNAi agent. In some embodiments, the triyne linker of formula I is linked to the RNAi agent via an unstable, labile, or reversible bond or linker.

[0143] In some embodiments, the non-nucleotide enhances the pharmacokinetic or biodistribution properties of the RNAi agent or conjugate to which it is attached to improve the cellular or tissue-specific distribution and cellular-specific uptake of the conjugate. In some embodiments, the non-nucleotide enhances the endocytosis of the RNAi agent.

[0144] The targeting ligand or targeting moiety enhances the pharmacokinetic or biodistribution properties of the cargo molecule to which it is attached to improve the cellular-specific (including organ-specific in some cases) distribution and cellular-specific (or organ-specific) uptake of the RNAi agent. In some embodiments, the targeting ligand comprises a targeting compound and a PK enhancer or modulator. In some embodiments, the targeting ligand is directed to a cell receptor.

[0145] Conjugated to a targeting ligand In some embodiments, the triyne linker of Formula I can be conjugated to an RNAi agent via a coupling agent. An exemplary protocol for conjugating the triyne linker of Formula I to an RNAi molecule is shown in the following reaction scheme: wherein L 1 、L 2 、L 3 、Q and X are all as described in Formula I, R contains a coupling moiety, RG is a reactive group and L 4 is a linker comprising an optionally substituted alkylene, an optionally substituted arylene or an optionally substituted cycloalkylene.

[0146] In some embodiments, the targeting ligand (TL) can be conjugated to the triyne moiety first and then to the RNAi molecule. An example of such a reaction is shown in the following scheme: wherein L 1 、L 2 、L 3 、Q and X are all as described in Formula I, R contains a coupling moiety and L 4 is a linker comprising an optionally substituted alkylene, an optionally substituted arylene or an optionally substituted cycloalkylene.

[0147] RNAi molecules having reactive groups, such as amino groups (also referred to herein as amines), can be synthesized. In some embodiments, the reactive group can be attached at the 5'-end and / or 3'-end of the RNAi agent. In some embodiments, the RNAi agent can be double-stranded. In embodiments where the RNAi agent is double-stranded, the reactive group can be on the sense strand or the antisense strand of the RNAi agent.

[0148] For example, in some embodiments, the synthesis results in an RNAi agent having an NH2-C6H 12 (hexanediamine) group at the 5′-end of the sense strand of the RNAi agent. The terminal amino group can then react to form a conjugate with a coupling moiety of a compound such as Formula I. In some embodiments, the coupling moiety is an ester, and the reactive group on the RNAi agent is a primary amine, forming an amide bond between the RNAi agent and the triyne linker. An example of such a reaction using a compound of Formula VI is shown in the following scheme: where L 1 、L 2 、L 3 、L 4 、R 3 、R 4 and RNA are all defined as in Formulas VI and VII.

[0149] In other embodiments, an RNAi agent having a terminal -CH2OH group is synthesized. In some embodiments, the coupling agent in R of Formula I comprises a phosphoramidite. As shown in the following reaction scheme, the RNAi agent comprising a terminal alcohol can be reacted with a triyne of Formula II to form a phosphate ester / salt: In some embodiments, the targeting ligand (TL) can be conjugated to the triyne linker as described herein after the triyne linker has been conjugated to the RNAi agent. An example of such a reaction is shown in the following scheme: Examples

[0150] Example 1. Synthesis of Compound 1 (2-cyanoethyl ((1r,4r)-4-((1,7-dioxo-4-(3-oxo-3-(prop-2-yn-1-ylamino)propyl)-1,7-bis(prop-2-yn-1-ylamino)hept-4-yl)carbamoyl)cyclohexyl)diisopropylphosphoramidite). To a solution of 1 (12.00 g, 25.6 mmol) and DIPEA (12.22 g, 16.47 mL, 94.6 mmol) in DMF (50 mL) at 0 °C was added TBTU (28.72 g, 89.5 mmol). The internal temperature was raised from 0 °C to 16 °C. Propargylamine (4.93 g, 5.73 mL, 89.5 mmol) was added dropwise while maintaining an internal temperature of less than 20 °C. The cooling bath was removed and the reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with DCM (100 mL) and washed with 1 N HCl (2 x 100 mL) and saturated aqueous NaHCO3 (2 x 100 mL). The organic layer became turbid and was stirred at room temperature. After 1.5 h, the precipitate was collected by filtration, rinsed with DCM (100 mL) and dried. Yield of 2: 10.4 g (70%). C 34 H 36 Calculated [M+H] for C N4O5: 581.70, found: 581.79.

[0151] To a solution of 2 (12.17 g, 21.0 mmol) in DMF (60 mL) at room temperature was added triethylamine (10.6 g, 14.7 mL, 105 mmol). The reaction mixture was stirred overnight. The reaction mixture was then concentrated and purified by using silica gel as the stationary phase and eluting with a gradient of MeOH / DCM containing 1% triethylamine (0 - 13%). Yield of 3: 6.08 g (81%). C 19 H 26 Calculated [M+H] for C N4O3: 359.45, found: 359.35. 4 (2.55 g, 17.69 mmol) was treated with acetic anhydride (12.8 mL, 135 mmol) in pyridine (26 mL) and stirred at room temperature for 4 h. After completion, all volatiles were removed and 5 was isolated as follows: separated by silica gel, eluting with a gradient of ethyl acetate / hexane containing 1% acetic acid. Yield: 2.56 g (78%). 1 H NMR (400 MHz, DMSO-d6): δ 12.11 (s, br, 1H) 4.56 (m, 1H), 2.21 (m, 1H), 1.97 (s, 3H), 1.90 (m, 4H), 1.38 (m, 4H). To a solution of 5 (400 mg, 2.15 mmol) in DCM (5 mL) at 0 °C was added DMF (16 mg, 17 μL, 0.215 mmol) and oxalyl chloride (1.36 g, 922 μL, 10.74 mmol). After 30 m, the cooling bath was removed and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated and the product was used in the next step without further purification.

[0152] To a solution of 3 (1000 mg, 2.79 mmol) in DCM (10 mL) was added pyridine (1.88 g, 1.92 mL, 23.7 mmol). The reaction mixture was cooled to 0 °C and a solution of 6 (398 mg, 1.95 mmol) in DCM (5 mL) was added dropwise. The cooling bath was removed and the mixture was stirred overnight at room temperature. Water (10 mL) was added to quench the reaction. The mixture was diluted with DCM (30 mL) and washed with saturated aqueous NH4Cl (20 mL) and brine (10 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by using silica gel as the stationary phase and eluting with a MeOH / DCM (0 - 7%) gradient. Yield of 7: 630 mg (43%). C 28 H 38 Calculated [M+H] for C H N4O6: 527.64, found: 527.69. To a solution of 7 (288 mg, 0.55 mmol) in THF (1.75 mL) at room temperature was added 1 M NaOH solution (2.73 mL, 2.73 mmol). The reaction mixture was stirred at room temperature for 1.5 h and then heated to 35 °C for an additional 30 m. After the starting material was consumed, the reaction mixture was acidified to pH = 5 with 2 M HCl and concentrated. The residue was co-evaporated with ACN (20 mL). After drying, the residue was purified by using silica gel as the stationary phase and eluting with a MeOH / DCM (0 - 10%) gradient. Yield of 8: 216 mg (81%). C 26 H 36 Calculated [M+H] for C H N4O5: 485.61, found: 485.56. 8 (213 mg, 0.44 mmol) was azeotropically dried from anhydrous ACN (2 x 10 mL) and then dissolved in ACN (4 mL). The reaction mixture was cooled to 0 °C. 4,5-Dicyanoimidazole (26 mg, 0.22 mmol) was added, followed by 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (199 mg, 0.66 mmol). The reaction mixture was stirred at 0 °C for 30 m. After the starting materials were exhausted, triethylamine (44 mg, 61 μL, 0.44 mmol) was added and the reaction mixture was concentrated to an oil. The oil was purified by using silica gel as the stationary phase and eluted with a gradient of EtOAc / DCM containing 1% triethylamine (50 - 100%). Yield of 9 (Compound 1): 174 mg (58%). C 35 H 53 Calculated [M+H] for C Example 2. Synthesis of Compound 2 (2-cyanoethyl ((1s,4s)-4-((1,7-dioxo-4-(3-oxo-3-(prop-2-yn-1-ylamino)propyl)-1,7-bis(prop-2-yn-1-ylamino)hept-4-yl)carbamoyl)cyclohexyl)diisopropylphosphoramidite) To a solution of 10 (cis-4-hydroxycyclohexanecarboxylic acid, 2.00 g, 13.87 mmol) in pyridine (19.75 g, 20.20 mL, 250 mmol) at 0 °C was added acetic anhydride (10.83 g, 10.03 mL, 106 mmol). The cooling bath was removed and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated and the residue was purified by using silica gel as the stationary phase and eluted with a gradient of EtOAc / hexane (0 - 30%). Yield of 11: 1.75 g (68%). Calculated [M-H] for C9H 14 O4: 185.20, found: 185.35. To a solution of 11 (420 mg, 2.26 mmol) in DCM (5 mL) at 0 °C was added DMF (16.5 mg, 17.4 μL, 0.226 mmol) and oxalyl chloride (1.43 g, 0.97 mL, 11.3 mmol). After 30 m, the cooling bath was removed and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated, co-evaporated with toluene, and the product 12 was used in the next step without further purification.

[0153] To a solution of 3 (400 mg, 1.12 mmol) in DMF (2 mL) was added pyridine (750 mg, 767 μL, 9.50 mmol). The reaction mixture was cooled to 0 °C and a solution of 12 (457 mg, 2.23 mmol) in DCM (2 mL) was added dropwise. The cooling bath was removed and the mixture was stirred at room temperature for 1.5 h. The mixture was diluted with DCM (20 mL) and quenched with saturated aqueous NH4Cl (10 mL). The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by using silica gel as the stationary phase and eluted with a MeOH / DCM (0 - 10%) gradient. Yield of 13: 365 mg (62%). 1 1H NMR (400 MHz, DMSO-d6): δ 8.21 (t, 3H), 7.07 (s, 1H), 4.84 (m, 1H), 3.81 (dd, 6H), 3.07 (t, 3H), 2.18 (m, 1H), 1.99 (m, 9H), 1.80 - 1.72 (m, 8H), 1.64 - 1.42 (m, 6H). To a solution of 13 (360 mg, 0.68 mmol) in THF (2.2 mL) at room temperature was added 1 M NaOH solution (3.42 mL, 3.42 mmol). The reaction mixture was stirred at room temperature for 2 h and then heated to 35 °C for an additional 1.5 h. After the starting material was consumed, the reaction mixture was acidified to pH = 5 with 2 M HCl and concentrated. The residue was co-evaporated with ACN (20 mL). After drying, the residue was purified by using silica gel as the stationary phase and eluted with a MeOH / DCM (0 - 12%) gradient. Yield of 8: 250 mg (75%). 1 1H NMR (400 MHz, DMSO-d6): δ 8.22 (t, 3H), 6.96 (s, 1H), 4.24 (d, 1H), 3.81 (dd, 6H), 3.75 (s, br, 1H), 3.07 (t, 3H), 2.10 (m, 1H), 1.99 (m, 6H), 1.82 - 1.58 (m, 10H), 1.36 (m, 4H). 14 (245 mg, 0.51 mmol) was co-distilled dry from anhydrous ACN (2 x 10 mL) and then dissolved in ACN (4 mL). The reaction mixture was cooled to 0 °C. 4,5-Dicyanoimidazole (30 mg, 0.25 mmol) was added, followed by 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (229 mg, 0.76 mmol). The reaction mixture was stirred at 0 °C for 30 min and then at room temperature for 1.5 h. The reaction mixture was concentrated to an oil and then dissolved in DCM (15 mL). The mixture was washed with saturated aqueous NaHCO3 (2 x 5 mL) and brine (5 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by using silica gel as the stationary phase and eluted with a gradient of EtOAc / DCM containing 1% triethylamine (50 - 100%). Yield of 15 (Compound 2): 204 mg (59%). C 35 H 53 Calculated [M+H] for C Example 3. Synthesis of Compound 3 (2-Cyanoethyl (5-((1,7-dioxo-4-(3-oxo-3-(prop-2-yn-1-ylamino)propyl)-1,7-bis(prop-2-yn-1-ylamino)hept-4-yl)amino)-5-oxopentyl)diisopropylphosphoramidite) To a solution of 3 (475 mg, 1.33 mmol) in DMF (5 mL) at room temperature was added triethylamine (402 mg, 555 μL, 3.98 mmol) and glutaric anhydride (190 mg, 1.65 mmol). The reaction mixture was stirred for 1 h and then DMAP (8.1 mg, 0.066 mmol), MeOH (424 mg, 536 μL, 13.25 mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (508 mg, 2.65 mmol) were added at room temperature. The mixture was stirred overnight at room temperature. The reaction mixture was diluted with DCM (20 mL) and washed with saturated aqueous NaHCO3 (10 mL). The aqueous layer was back-extracted with DCM (2 x 5 mL). The combined organic phases were dried over Na2SO4, filtered and concentrated. The residue was purified by using silica gel as the stationary phase and eluted with a gradient of MeOH / DCM (0 - 7.5%). Yield of 16: 273 mg (42%). C 25 H 34 Calculated [M+H] for C Sodium borohydride (54 mg, 1.42 mmol) was added to a solution of 16 (173 mg, 0.36 mmol) in MeOH (0.87 mL) and iPrOH (1.74 mL) at 0 °C. After 30 m, the cooling bath was removed and lithium chloride (10 mg) was added. The reaction mixture was stirred overnight at room temperature. The next day, another portion of sodium borohydride (27 mg, 0.71 mmol) was added and the reaction was continued for 1 h. The reaction mixture was concentrated and purified by using silica gel as the stationary phase and eluting with a MeOH / DCM (0 - 12%) gradient. Yield of 17: 93 mg. C 24 H 34 Calculated [M+H] for C H N4O5: 459.57, found: 459.63. Compound 17 (175 mg, 0.38 mmol) was azeotropically dried from anhydrous ACN (2 x 5 mL) and then dissolved in ACN (3 mL). The reaction mixture was cooled to 0 °C. 4,5 - Dicyanoimidazole (22.5 mg, 0.19 mmol) was added, followed by 2 - cyanoethyl N,N,N′,N′ - tetraisopropylphosphorodiamidite (173 mg, 0.57 mmol). The reaction mixture was stirred at 0 °C for 30 m and then at room temperature for 30 m. The reaction mixture was concentrated to an oil and then dissolved in DCM (15 mL). The mixture was washed with saturated aqueous NaHCO3 (5 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by using silica gel as the stationary phase and eluting with an EtOAc / DCM containing 1% triethylamine (50 - 100%) gradient. Yield of 18 (Compound 3): 132 mg (53%). C 33 H 51 Calculated [M+H] for C H N6O6P: 659.79, found: 659.93. Example 4. Synthesis of Compound 4 (2 - cyanoethyl ((1r,4r) - 4 - ((11,17 - dioxo - 14 - (3 - oxo - 3 - ((2 - (2 - (prop - 2 - ynyloxy)ethoxy)ethyl)amino)propyl) - 4,7,21,24 - tetraoxa - 10,18 - diazaheptacosa - 1,26 - diyn - 14 - yl)carbamoyl)cyclohexyl)diisopropylphosphoramidite) Triethylamine (3.63 g, 5.00 mL, 35.9 mmol) was added to a solution of 19 (4.42 g, 5.23 mmol) in DMF (25 mL) at room temperature. The reaction mixture was stirred overnight. Then the reaction mixture was concentrated and purified by Silica gel was used as the stationary phase for purification and eluted with a MeOH / DCM (0 - 20%) gradient. Yield of 20: 3.08 g (95%). 1 HNMR (400 MHz, DMSO-d6): δ 7.82 (t, 3H), 4.14 (d, 6H), 3.58 - 3.49 (m, 12H), 3.42 - 3.36 (m, 9H), 3.17 (q, 6H), 2.05 (m, 6H), 1.41 (m, 6H). To a solution of 20 (900 mg, 1.45 mmol) and compound 5 (404 mg, 2.17 mmol) in DMF (7 mL) at 0 °C was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 550 mg, 2.17 mmol), followed by DIEA (374 mg, 503 μL, 2.90 mmol). The cooling bath was removed and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated to an orange oil, which was dissolved in DCM (25 mL). The mixture was washed with 1 M HCl (2 x 10 mL) and saturated aqueous NaHCO3 (10 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by using silica gel as the stationary phase and eluted with a MeOH / DCM (0 - 8%) gradient. Yield of compound 27: 880 mg (77%). C 40 H 62 N4O 12 Calculated [M+H] for: 791.96, found: 792.08. To a solution of compound 27 (925 mg, 1.17 mmol) in THF (6 mL) at room temperature was added 1 M NaOH (5.85 mL, 5.85 mmol). The mixture was heated to 35 °C for 2 h. The reaction mixture was neutralized to pH = 6 using 2 M HCl. Sodium chloride (ca. 3 g) was added to the aqueous phase and the mixture was extracted with DCM (3 x 40 mL). The combined organic phases were dried over Na2SO4, filtered and concentrated. The residue was purified by using silica gel as the stationary phase and eluted with a MeOH / DCM (0 - 12%) gradient. Yield of compound 28: 580 mg (66%). 11H NMR (400 MHz, DMSO-d6): δ 7.82 (t, 3H), 7.04 (s, 1H), 4.51 (d, 1H), 4.14 (d, 6H), 3.58 - 3.49 (m, 12H), 3.42 - 3.36 (m, 9H), 3.18 (q, 6H), 2.06 - 1.92 (m, 7H), 1.88 - 1.62 (m, 10H), 1.35 (m, 2H), 1.10 (m, 2H). Compound 28 (577 mg, 0.77 mmol) was co - dried from anhydrous ACN (2 x 20 mL) by azeotropic distillation and then dissolved in ACN (10 mL). The reaction mixture was cooled to 0 °C. 4,5 - Dicyanoimidazole (45.5 mg, 0.39 mmol) was added, followed by 2 - cyanoethyl N,N,N′,N′ - tetraisopropylphosphorodiamidite (348 mg, 1.12 mmol). The cooling bath was removed and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated to an oil and then dissolved in DCM (30 mL). The mixture was washed with saturated aqueous NaHCO3 (2 x 10 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by using silica gel as the stationary phase and eluted with a gradient of MeOH / DCM (0 - 2%) containing 1% triethylamine. Yield of 29 (Compound 4): 610 mg (83%). 1 1H NMR (400 MHz, DMSO-d6): δ 7.82 (t, 3H), 7.07 (s, 1H), 4.14 (d, 6H), 3.76 - 3.60 (m, 2H), 3.58 - 3.48 (m, 14H), 3.42 - 3.36 (m, 9H), 3.18 (q, 6H), 2.74 (t, 2H), 2.12 - 2.04 (m, 1H), 2.02 - 1.89 (m, 8H), 1.83 - 1.67 (m, 8H), 1.45 - 1.31 (m, 2H), 1.30 - 1.21 (m, 2H), 1.13 (dd, 12H). 31 31P NMR (400 MHz, DMSO-d6): δ 144.6. Example 5. Synthesis of Compound 5 (2 - cyanoethyl ((1s,4s)-4 - ((11,17 - dioxo - 14 - (3 - oxo - 3 - ((2-(2-(prop - 2 - ynyloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24 - tetraoxa - 10,18 - diazapentacos - 1,26 - diyn - 14 - yl)carbamoyl)cyclohexyl)diisopropylphosphoramidite) Pyridine (1.22 g, 1.25 mL, 15.5 mmol) was added to a solution of 20 (1070 mg, 1.72 mmol) in DCM (7 mL). The reaction mixture was cooled to 0 °C and a solution of 12 (1.06 g, 5.15 mmol) in DCM (3.5 mL) was added dropwise. The cooling bath was removed and the mixture was stirred at room temperature for 2 h. The mixture was diluted with DCM (20 mL) and quenched with saturated aqueous NH4Cl (10 mL). The layers were separated and the organic phase was washed with saturated aqueous NaHCO3 (10 mL) and brine (10 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by using silica gel as the stationary phase and eluted with a MeOH / DCM (0 - 7%) gradient. Yield of compound 24: 295 mg (22%). C 40 H 62 N4O 12 Calculated [M + H] for: 791.96, found: 792.08. 1 M NaOH (1.83 mL, 1.83 mmol) was added to a solution of compound 24 (290 mg, 0.37 mmol) in THF (2 mL) at room temperature. The mixture was heated to 35 °C for 3 h. The reaction mixture was quenched with saturated aqueous NH4Cl (8 mL) and further acidified to pH = 6 with 2 M HCl. The mixture was extracted with DCM (3 x 15 mL). The combined organic phases were dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash using silica gel as the stationary phase and eluted with a MeOH / DCM (0 - 12%) gradient. Yield of compound 25: 183 mg (67%). 1 H NMR (400 MHz, DMSO-d6): δ 7.83 (t, 3H), 6.97 (s, 1H), 4.24 (d, 1H), 4.14 (d, 6H), 3.75 (s, br, 1H), 3.58 - 3.49 (m, 12H), 3.42 - 3.36 (m, 9H), 3.18 (q, 6H), 2.10 (m, 1H), 1.97 (m, 6H), 1.82 - 1.60 (m, 10H), 1.38 (m, 4H). Compound 25 (180 mg, 0.24 mmol) was azeotropically dried from anhydrous ACN (2 x 5 mL) and then dissolved in ACN (2 mL). The reaction mixture was cooled to 0 °C. 4,5-Dicyanoimidazole (14.2 mg, 0.12 mmol) was added, followed by 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (109 mg, 0.36 mmol). The reaction mixture was stirred at 0 °C for 30 m and then at room temperature for 1.5 h. Another portion of 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (36 mg, 0.12 mmol) was added and the reaction mixture was stirred for an additional 3 h. The reaction mixture was concentrated to an oil and then dissolved in DCM (15 mL). The mixture was washed with a mixture of saturated aqueous NaHCO3 (2.5 mL) and water (2.5 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by using silica gel as the stationary phase and eluted with a gradient of MeOH / DCM (0 - 2%) containing 1% triethylamine. Yield of 26 (Compound 5): 116 mg (51%). C 47 H 77 N6O 12 Calculated [M+H] for: 950.15, found: 950.18. Example 6. Synthesis of Compound 6 (2-Cyanoethyl (11,16-dioxo-14,14-bis(3-oxo-3-((2-(2-(prop-2-ynyloxy)ethoxy)ethyl)amino)propyl)-4,7-dioxa-10,15-diazaeicos-1-yn-20-yl)diisopropylphosphoramidite) To a solution of 1 (3.00 g, 6.39 mmol) and DIPEA (2.89 g, 3.89 mL 16.47 mL, 22.4 mmol) in DMF (50 mL) at 0 °C was added TBTU (6.77 g, 21.1 mmol). Then a solution of propargyl-PEG2-amine (3.02 g, 21.1 mmol) in DMF (5 mL) was added dropwise. The cooling bath was removed and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with DCM (30 mL) and washed with 1N HCl (2 x 30 mL) and saturated aqueous NaHCO3 (2 x 30 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by using silica gel as the stationary phase and eluted with a gradient of MeOH / DCM (0 - 10%). Yield of 19: 4.42 g (82%). To a solution of 20 (960 mg, 1.54 mmol) in DCM (8 mL) was added triethylamine (468 mg, 645 μL, 4.62 mmol) and glutaric anhydride (220 mg, 1.93 mmol). The reaction mixture was stirred overnight at room temperature. The next day, DMAP (9.4 mg, 0.077 mmol), MeOH (494 mg, 624 μL, 15.42 mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (591 mg, 3.08 mmol) were added at room temperature. The reaction mixture was stirred for 5 h. The reaction mixture was concentrated to an oil, which was dissolved in DCM (45 mL) and then washed with saturated aqueous NaHCO3 (10 mL) and saturated aqueous NH4Cl (10 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by using silica gel as the stationary phase and eluted with a MeOH / DCM (0 - 7.5%) gradient. Yield of 21: 880 mg (76%). C 37 H 58 N4O 12 Calculated [M + H] for: 751.90, found: 751.90. To a solution of 21 (877 mg, 1.17 mmol) in THF (4 mL) and MeOH (1.75 mL) was added a solution of lithium chloride (25 mg, 0.58 mmol) in water (1.75 mL). The mixture was cooled to 0 °C and sodium borohydride (265 mg, 7.01 mmol) was added in one portion. The cooling bath was removed and the reaction mixture was stirred overnight at room temperature. The reaction mixture was quenched by the addition of saturated aqueous NH4Cl (5 mL). After stirring for 10 m, the mixture was concentrated to remove THF and MeOH. The remaining aqueous phase was diluted with water (5 mL) and extracted with DCM (3 x 20 mL). The combined organic phases were dried over Na2SO4, filtered and concentrated. The residue was purified by using silica gel as the stationary phase and eluted with a MeOH / DCM (0 - 12%) gradient. Yield of 22: 562 mg (66%). C 36 H 58 N4O 11 Calculated [M + H] for: 723.19, found: 723.81. 22 (560 mg, 0.77 mmol) was azeotropically dried from anhydrous ACN (2 x 10 mL) and then dissolved in ACN (5 mL). The reaction mixture was cooled to 0 °C. 4,5-Dicyanoimidazole (45.7 mg, 0.39 mmol) was added, followed by 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (350 mg, 1.16 mmol). The reaction mixture was stirred at 0 °C for 30 min and then at room temperature for 30 min. The reaction mixture was concentrated to an oil and then dissolved in DCM (30 mL). The mixture was washed with saturated aqueous NaHCO3 (2 x 10 mL) and brine (10 mL). The organic phase was dried over Na2SO4, filtered, and concentrated. The residue was purified by using silica gel as the stationary phase and eluted with a gradient of MeOH / DCM (0 - 2%) containing 1% triethylamine. Yield of compound 6: 434 mg (61%). 1 1H NMR (400 MHz, DMSO-d6): δ 7.82 (t, 3H), 7.13 (s, 1H), 4.14 (d, 6H), 3.72 - 3.65 (m, 2H), 3.58 - 3.48 (m, 16H), 3.42 - 3.36 (m, 9H), 3.17 (q, 6H), 2.75 (t, 2H), 2.09 - 1.92 (m, 8H), 1.83 - 1.72 (m, 6H), 1.52 (m, 4H), 1.13 (dd, 12H). 31 31P NMR (400 MHz, DMSO-d6): δ 146.3. Example 7. Synthesis of compound 7 (2-cyanoethyl (4-((11,17-dioxo-14-(3-oxo-3-((2-(2-(prop-2-ynyloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacos-1,26-diyne-14-yl)carbamoyl)phenyl)diisopropylphosphoramidite) Step 1. To a solution of 1 (200 mg, 0.32 mmol), 4-acetoxybenzoic acid (86.4 mg, 0.48 mmol), and N,N-diisopropylethylamine (123.8 mg, 0.17 mL, d = 0.742 g / mL, 0.96 mmol) in DMF (2 mL) was added HATU (243.2 mg, 0.64 mmol). The reaction mixture was stirred at room temperature. After confirming the exhaustion of all starting materials by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL × 3). The combined organic layers were successively washed with HCl(aq) and brine. The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA: DCM, MPB: 20% MeOH / DCM, 0 - 50% ramp in 30 min) to afford the product. Yield: 133 mg. Step 2. The amide product from Step 1 was dissolved in 2 mL of MeOH and 100 mg of K2CO3 was added to the reaction. After stirring overnight at room temperature, the reaction mixture was filtered through a short silica gel pad. The filtrate was collected and concentrated under reduced pressure. Yield: 115 mg, 48% for the two steps. C 38 H 53 N4O 11 MS (ESI) m / z calculated for [M-H] 741.37, found: 741.67. To a solution of 2 (100 mg, 0.1346 mmol)), diisopropylammonium tetrazolide (11.5 mg, 0.0673 mmol), and molecular sieve (20 mg) in DCM (2 mL) was added 2-cyanoethyl Ν,Ν,Ν’,Ν’-tetraisopropylphosphorodiamidite (60.9 mg, 0.064 mL, 0.2019 mmol, 1.5 eq). The reaction mixture was stirred at room temperature. After confirming the exhaustion of all starting materials by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL × 3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA: 1% TEA / DCM, MPB: 1% TEA and 4% MeOH in DCM, 0 - 50% ramp in 30 min) to afford Compound 7. Yield: 105 mg (83%). C 47 H 70 N6O 12 MS (ESI) m / z calculated for [M-H] P 941.48, found 941.88. Synthesis of Example 8. Compound 8 (2-Cyanoethyl (3-((11,17-dioxo-14-(3-oxo-3-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacos-1,26-diyne-14-yl)carbamoyl)phenyl)diisopropylphosphoramidite) Step 1. To a solution of 1 (200 mg, 0.32 mmol), 3-acetoxybenzoic acid (86.7 mg, 0.48 mmol), and N,N-diisopropylethylamine (123.8 mg, 0.17 mL, d = 0.742 g / mL, 0.96 mmol) in DMF (2 mL) was added HATU (243.2 mg, 0.64 mmol). The reaction mixture was stirred at room temperature. After confirming the exhaustion of all raw materials by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL × 3). The combined organic layers were successively washed with HCl(aq) and brine. The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica gel column and purified (MPA: DCM, MPB: 10% MeOH / DCM, 0 - 40% ramp in 30 min) to afford the product. Yield: 148 mg. Step 2. The amide product from Step 1 was dissolved in 2 mL of MeOH and 100 mg of K2CO3 was added to the reaction. After stirring overnight at room temperature, the reaction mixture was filtered through a short silica gel pad. The filtrate was collected and concentrated under reduced pressure. Yield: 126 mg, 53% for two steps. C 38 H 55 N4O 11 MS (ESI) m / z calculated for [M+H] 743.39, found: 743.65. To 3 (125 mg, 0.1683 mmol)), diisopropylammonium tetrazolate (14.4 mg, 0.0841 mmol) and To a solution of molecular sieve (20 mg) in DCM (2 mL) was added 2-cyanoethyl Ν,Ν,Ν’,Ν’-tetraisopropylphosphorodiamidite (76.1 mg, 0.08 mL, 0.2524 mmol, 1.5 eq). The reaction mixture was stirred at room temperature. After confirmation by LC-MS that all starting materials were exhausted, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL × 3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica gel column and purified (MPA: 1% TEA / DCM, MPB: 1% TEA and 4% MeOH in DCM, 0 - 50% ramp in 30 min) to afford compound 8. Yield: 130 mg (82%). C 47 H 70 N6O 12 MS (ESI) m / z calcd for [M-H] 941.48, found 941.79.

[0154] Example 9. Synthesis of Compound 9 (2-Cyanoethyl (2-((11,17-dioxo-14-(3-oxo-3-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacosa-1,26-diyne-14-yl)carbamoyl)phenyl)diisopropylphosphoramidite) Step 1. To a solution of 1 (200 mg, 0.32 mmol), triethylamine (97.3 mg, 0.134 mL, d = 0.726 g / mL, 0.96 mmol) in DCM (2 mL) was added o-acetylsalicyl chloride (127.6 mg, 0.6423 mmol, 1.2 eq, CAS Registry No.: 5538-51-2). The reaction mixture was stirred at room temperature. After confirmation by LC-MS that all starting materials were exhausted, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed successively with HCl(aq) and brine. The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica gel column and purified (MPA: DCM, MPB: 10% MeOH / DCM, 0 - 50% ramp in 30 min) to afford the product. Yield: 177.8 mg.

[0155] Step 2. The amide product from Step 1 was dissolved in 2 mL of MeOH and 100 mg of K2CO3 was added to the reaction. After stirring overnight at room temperature, the reaction mixture was filtered through a short silica gel pad. The filtrate was collected and concentrated under reduced pressure. Yield: 126 mg, 53% for two steps. C 38 H53 N4O 11 The calculated MS(ESI) m / z of [M-H] is 741.39, found: 741.67. To a solution of 4 (105 mg, 0.1457 mmol), diisopropylammonium tetrazolide (12.5 mg, 0.0728 mmol), and molecular sieve (20 mg) in DCM (2 mL) was added 2-cyanoethyl Ν,Ν,Ν’,Ν’-tetraisopropylphosphorodiamidite (66 mg, 0.069 mL, 0.2185 mmol, 1.5 eq). The reaction mixture was stirred at room temperature. After confirming the depletion of all starting materials by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL × 3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica gel column and purified (MPA: 1% TEA / DCM, MPB: 1% TEA and 4% MeOH in DCM, 0 - 50% gradient over 30 min) to afford Compound 9. Yield: 181 mg (83%). C 47 H 70 N6O 12 The calculated MS(ESI) m / z of P[M-H] is 941.48, found 941.79. Example 10. Synthesis of Compound 10 (2-cyanoethyl (4'-((11,17-dioxo-14-(3-oxo-3-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacosa-1,26-diyne-14-yl)carbamoyl)-[1,1'-biphenyl]-4-yl)diisopropylphosphoramidite) To a solution of 1 (200 mg, 0.3212 mmol), 4′-hydroxy-4-biphenylcarboxylic acid (103.2 mg, 0.4817 mmol) and N,N-diisopropylethylamine (124.5 mg, 0.17 mL, d = 0.742 g / mL, 0.96 mmol) in DMF (2 mL) was added HATU (244.2 mg, 0.64 mmol). The reaction mixture was stirred at room temperature. After confirming the depletion of all starting materials by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed successively with HCl(aq) and brine. The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica gel column and purified (MPA: DCM, MPB: 10% MeOH / DCM, 0 - 50% gradient in 30 min) to afford the product. Yield: 138 mg, 52%. C 44 H 59 N4O 11 MS (ESI) m / z calcd for [M+H] 819.42, found: 819.90. To a solution of 5 (138 mg, 0.1685 mmol), diisopropylammonium tetrazolide (14.4 mg, 0.0843 mmol) and molecular sieve (20 mg) in DCM (2 mL) was added 2-cyanoethyl Ν,Ν,Ν’,Ν’-tetraisopropylphosphorodiamidite (76.2 mg, 0.08 mL, 0.2528 mmol, 1.5 eq). The reaction mixture was stirred at room temperature. After confirming the depletion of all starting materials by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL × 3). Dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica gel column and purified (MPA: 1% TEA / DCM, MPB: 1% TEA and 4% MeOH in DCM, 0 - 50% gradient in 30 min) to afford compound 10. Yield: 171 mg (99%). C 53 H 74 N6O 12 MS (ESI) m / z calcd for [M-H] 1017.51, found 1017.99. Example 11. Synthesis of Compound 11 (2-cyanoethyl ((1r,3r)-3-((11,17-dioxo-14-(3-oxo-3-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacos-1,26-diyne-14-yl)carbamoyl)cyclobutyl)diisopropylphosphoramidite) To a solution of 1 (300 mg, 0.4817 mmol), trans-3-hydroxycyclobutanecarboxylic acid (83.9 mg, 0.7226 mmol, CAS No.: 1268521-85-2), and N,N-diisopropylethylamine (186.8 mg, 0.252 mL, d = 0.742 g / mL, 1.4452 mmol) in DMF (3 mL) was added HATU (366.3 mg, 0.9635 mmol). The reaction mixture was stirred at room temperature. After confirming the depletion of all starting materials by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed successively with HCl(aq) and brine. The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica gel column and purified (MPA: DCM, MPB: 10% MeOH / DCM, 0 - 100% ramp in 30 min) to give the product. Yield: 333.2 mg, 88%. C 36 H 57 N4O 11 MS (ESI) m / z calculated for [M+H] 721.40, found 721.96. To a solution of 6 (166.5 mg, 0.2310 mmol)), diisopropylammonium tetrazolide (19.8 mg, 0.1155 mmol), and molecular sieve (20 mg) in DCM (2 mL) was added 2-cyanoethyl Ν,Ν,Ν’,Ν’-tetraisopropylphosphorodiamidite (104.4 mg, 0.11 mL, 0.3465 mmol, 1.5 eq). The reaction mixture was stirred at room temperature. After confirming the depletion of all starting materials by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL × 3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica gel column and purified (MPA: 1% TEA / DCM, MPB: 1% TEA and 4% MeOH in DCM, 0 - 50% ramp in 30 min) to give Compound 11. Yield: 200 mg (94%). C 45 H72 N6O 12 MS (ESI) m / z calculated value of P[MH]: 919.50, found value: 919.73. Example 12. Synthesis of Compound 12 (2-cyanoethyl (4-((11,17-dioxo-14-(3-oxo-3-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptadecanoyl-1,26-diyn-14-yl)carbamoyl)bicyclo[2.2.2]oct-1-yl)diisopropylphosphoramidite) HATU (732.7 mg, 1.9269 mmol) was added to a solution of 1 (600 mg, 0.9635 mmol), 4-hydroxybicyclo[2.2.2]octane-1-carboxylic acid (245.1 mg, 0.1561 mmol, CAS No.: 1127-13-5) and N,N-diisopropylethylamine (373.6 mg, 0.503 mL, d = 0.742 g / mL, 2.8904 mmol) in DMF (5 mL). The reaction mixture was stirred at room temperature. After all the starting materials were exhausted by LC-MS, the reaction mixture was quenched with 2 mL of saturated NaHCO3 aqueous solution and extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed successively with HCl (aq) and brine. The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA:DCM, MPB: 10% MeOH / DCM, 0-100% ramp in 30 min) to afford the product. Yield: 398 mg, 54%. 40 H 61 N4O 11 MS (ESI) m / z calculated value for [MH]: 773.45, found value: 773.80. 7 (200 mg, 0.2581 mmol), diisopropylammonium tetrazolium salt (22.1 mg, 0.1290 mmol) and To a solution of molecular sieve (20 mg) in DCM (2 mL) was added 2-cyanoethyl Ν,Ν,Ν’,Ν’-tetraisopropylphosphorodiamidite (116.7 mg, 0.123 mL, 0.3871 mmol, 1.5 eq). The reaction mixture was stirred at room temperature. After confirming the depletion of all starting materials by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL × 3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica gel column and purified (MPA: 1% TEA / DCM, MPB: 1% TEA and 4% MeOH in DCM, 0 - 50% ramp in 30 min) to afford Compound 12. Yield: 40 mg (16%). C 49 H 78 N6O 12 MS (ESI) m / z calculated for [M-H] 973.54, found 973.75. Example 13. Synthesis of Compound 13 (2-cyanoethyl (3-((11,17-dioxo-14-(3-oxo-3-((2-(2-(prop-2-ynyloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacos-1,26-diyne-14-yl)carbamoyl)bicyclo[1.1.1]pentan-1-yl)diisopropylphosphoramidite) To a solution of 1 (400 mg, 0.6423 mmol), 3-hydroxybicyclo[1.1.1]pentane-1-carboxylic acid (98.7 mg, 0.7708 mmol, CAS No.: 83249-08-5) and N,N-diisopropylethylamine (249.1 mg, 0.336 mL, d = 0.742 g / mL, 1.9269 mmol) in DMF / DCM (10 mL, 1:1 v / v) was added HATU (488.4 mg, 1.2846 mmol). The reaction mixture was stirred at room temperature. After confirming the depletion of all starting materials by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed successively with HCl (aq) and brine. The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica gel column and purified (MPA: DCM, MPB: 10% MeOH / DCM, 0 - 100% ramp in 30 min) to afford 8. Yield: 387.6 mg, 82%. C 37 H 57 N4O 11 MS (ESI) m / z calculated for [M+H] 733.40, found: 733.66. To a solution of 8 (387.6 mg, 0.5289 mmol), diisopropylammonium tetrazolide (45.3 mg, 0.2644 mmol) and molecular sieve (20 mg) in DCM (2 mL) was added 2-cyanoethyl Ν,Ν,Ν’,Ν’-tetraisopropylphosphorodiamidite (239.1 mg, 0.252 mL, 0.7933 mmol, 1.5 eq). The reaction mixture was stirred at room temperature. After confirming the depletion of all starting materials by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL × 3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica gel column and purified (MPA: 1% TEA / DCM, MPB: 1% TEA and 4% MeOH in DCM, 0 - 50% ramp in 30 min) to afford the pure phosphoramidite product. Yield: 206.7 mg (42%). C 46 H 72 N6O 12 MS (ESI) m / z calcd for [M-H] 931.50, found 931.71. Example 14. Synthesis of Compound 14 (2-cyanoethyl (11,16,20-trioxo-14,14-bis(3-oxo-3-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7-dioxa-10,15,21-triazatricos-1-yn-27-yl)diisopropylphosphoramidite) and Compound 22 (4-nitrophenyl 11,16-dioxo-14,14-bis(3-oxo-3-((2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7-dioxa-10,15-diazatricos-1-yn-20-oate) 500 mL of DCM and 4 (75.0 g, 0.16 mol) were added to a 3 L jacketed reactor. The internal temperature of the reaction was cooled to 0 °C and TBTU (170.0 g, 0.53 mol) was added. The suspension was then treated dropwise with amine 5 (75.5 g, 0.53 mol) while maintaining the internal temperature below 5 °C. The reaction was then treated slowly with DIPEA (72.3 g, 0.56 mol) while maintaining the internal temperature below 5 °C. After the addition was complete, the reaction was warmed to 23 °C over 1 hour and allowed to stir for 3 hours. A 10% supplemental feed (kicker charge) of all three reagents was added and allowed to stir for an additional 3 hours. When less than 1% of 4 remained, the reaction was considered complete. The reaction mixture was washed with saturated ammonium chloride solution (2 x 500 mL) and once with saturated sodium bicarbonate solution (500 mL). The organic layer was then dried over sodium sulfate and concentrated to an oil. The mass of the crude oil was 188 g and it was determined by QNMR to contain 72% of 6. The crude oil was sent to the next step. C 46 H 60 N4O 11 Calculated mass = 845.0 m / z. Observed [M+H] = 846.0. 121.2 g of the crude oil containing 72 wt% of compound 6 (86.0 g, 0.10 mol) was dissolved in DMF (344 mL) and treated with TEA (86 mL, 20 v / v%) while maintaining the internal temperature below 23 °C. The consumption relative to Fmoc-amine 6, the formation of dibenzofulvene (DBF), were monitored by HPLC method 1 and the reaction was complete within 10 hours. Glutaric anhydride (12.8 g, 0.11 mol) was added to the solution and the intermediate amine 7 was converted to compound 8 within 2 hours. After completion, DMF and TEA were removed under reduced pressure at 30 °C to yield 100 g of crude oil. Due to the high solubility of compound 7 in water, aqueous workup may not be possible and chromatography is the only way to remove DBF, TMU, and glutaric anhydride. The crude oil (75 g) was purified in three portions on a Teledyne ISCO purification system. The crude oil (25 g) was loaded onto a 330 g silica gel column and eluted with 0–20% methanol / DCM over 30 minutes to yield 42 g of compound 8 (54% yield over 3 steps). C 36 H 55 N4O 12 Calculated mass = 736.4 m / z. Observed [M+H] = 737.0. Compound 8 (42.0 g, 0.057 mol) was co-stripped with 10 volumes of acetonitrile prior to use to remove any residual methanol from the chromatography solvent. The oil was redissolved in DMF (210 mL) and cooled to 0 °C. The solution was treated with 4-nitrophenol (8.7 g, 0.063 moL), followed by EDC hydrochloride (12.0 g, 0.063 mol) and found to be complete within 10 hours. The solution was cooled to 0 °C and 10 volumes of ethyl acetate were added, followed by 10 volumes of saturated ammonium chloride solution, keeping the internal temperature below 15 °C. The layers were allowed to separate and the ethyl acetate layer was washed with brine. The combined aqueous layers were extracted twice with 5 volumes of ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated to an oil. The crude oil (55 g) was purified in three portions on a Teledyne ISCO purification system. The crude oil (25 g) was loaded onto a 330 g silica gel column and eluted from 0–10% methanol / DCM over 30 minutes to yield 22 g of pure 9 (Compound 22) (50% yield). C 42 H 59 N5O 14 Calculated mass for = 857.4 m / z. Found [M+H] = 858.0. A solution of ester 9 (49.0 g, 57.1 mmol) and 6-amino-1-hexanol (7.36 g, 6.28 mmol) in dichloromethane (3 volumes) was treated dropwise with triethylamine (11.56 g, 111.4 mmol). The reaction was monitored by observing the disappearance of compound 9 on HPLC method 1 and found to be complete within 10 minutes. The crude reaction mixture was diluted with 5 volumes of dichloromethane and washed with saturated ammonium chloride (5 volumes) and brine (5 volumes). The organic layer was dried over sodium sulfate and concentrated to an oil. The crude oil was purified on a Teledyne ISCO purification system using a 330 g silica gel column. 4-Nitrophenol was eluted with 100% ethyl acetate and the column was rinsed with 20% methanol / DCM for 10 to yield a colorless oil (39 g, 81% yield). C 42 H 69 N5O 12 Calculated mass for = 836.0 m / z. Found [M+H] = 837.0. The alcohol 10 was co - stripped twice with 10 volumes of acetonitrile to remove any residual methanol from the chromatographic solvent and then co - stripped once with anhydrous dichloromethane (KF < 60 ppm) to remove trace water. The alcohol 10 (2.30 g, 2.8 mmol) was dissolved in 5 volumes of anhydrous dichloromethane (KF < 50 ppm) and treated with diisopropylammonium tetrazolide (188 mg, 1.1 mmol). The solution was cooled to 0 °C and treated dropwise with 2 - cyanoethyl N,N,N’,N’ - tetraisopropylphosphoramidite (1.00 g, 3.3 mmol). The solution was removed from the ice bath and stirred at 20 °C. The reaction was found to be complete within 3 - 6 hours. The reaction mixture was cooled to 0 °C and treated with 10 volumes of a 1:1 saturated ammonium bicarbonate / brine solution, then warmed to ambient temperature over 1 minute and allowed to stir at 20 °C for an additional 3 minutes. The two - phase mixture was transferred to a separatory funnel and 10 volumes of dichloromethane were added. The organic layer was separated and washed with 10 volumes of saturated sodium bicarbonate solution to hydrolyze the unreacted bis - phosphorus reagent. The organic layer was dried over sodium sulfate and concentrated to an oil to afford 3.08 g of 94 wt% of compound 14.C 51 H 86 N7O 13 Calculated mass for P = 1035.6 m / z. Found [M + H]=1036. Example 15. Synthesis of compound 15 (4 - nitrophenyl 5 - ((1,3 - bis(prop - 2 - ynyloxy)-2 - ((prop - 2 - ynyloxy)methyl)prop - 2 - yl)amino)-5 - oxopentanoate) Step 1. A solution of di - tert - butyl dicarbonate (2.35 g, 10.7 mmol) in t BuOH (10 mL) was added to a suspension of tris(hydroxymethyl)aminomethane (1.00 g, 8.20 mmol, CAS number: 77 - 86 - 1) in a 1:1 MeOH / tBuOH mixture (15 mL) and the mixture was stirred at room temperature for 18 h. The solvent was removed under reduced pressure to afford a residue, which was purified by precipitation with cold EtOAc. Vacuum filtration gave the pure compound as a white solid (1.4449, 80% yield). C9H 20 MS (ESI) m / z calculated for C9HNO5[M + H] 222.13, found 222.24. Step 2. A solution of triol-NHBoc 10 (500 mg, 2.26 mmol) in anhydrous DMF (6 mL) was stirred at 0 °C with propargyl bromide (80 wt% in toluene, 1.46 mL, 13.6 mmol). Several portions of finely ground KOH (951 mg, 13.6 mmol) were added over a period of 15 min. The mixture was then heated to 35 °C and stirred under a nitrogen atmosphere for 24 h. The reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (20 mL × 3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA: hexane, MPB: EA, 0 - 10% ramp in 30 min) to afford the pure product 11. Yield: 483 mg (64%). Steps 3 and 4. TFA (2.3 mL) was added dropwise to a solution of trialk-NHBoc 11 (483 mg, 1.44 mmol) in anhydrous DCM (5.6 mL) at 0 °C. The brown mixture was then stirred at room temperature for 2 h. Without further purification, it was concentrated under high vacuum to afford a solid. The crude was dissolved in DMF / TEA (6 mL, 5 / 1 v / v) at room temperature. Glutaric anhydride (328 mg, 2.877 mmol) was added to the mixture. After overnight, the solvent was removed under reduced pressure. Purification using silica gel as the stationary phase afforded 0.9357 g of product 14. (MPA: DCM, MPB: 20% MeOH / DCM, 0 - 50% ramp in 30 min). C H 18 H 22 MS (ESI) m / z calcd for C H NO6 [M-H] 348.15, found 348.28. EDC HCl salt (1.28 g, 6.7 mmol, 5 eq) was added to a solution of 14 (470 mg, 1.3 mmol) and p-nitrophenol (936 mg, 6.7 mmol, 5 eq) in DCM (10 mL) at 0 °C. The reaction mixture was then stirred at room temperature. After confirming by LC-MS that all starting materials were consumed, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL × 3). It was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA: hexane, MPB: EA, 0 - 60% ramp in 30 min) to afford the pure product as a yellow oil. Yield: 471 mg (77%). C 24 H 27 MS (ESI) m / z calcd for C H N2O8 [M+H] 471.18, found 471.33. Synthesis of Example 16. Compound 16 (4-Nitrophenyl 5-(((S)-1-(((R)-1,5-dioxo-1,5-bis(prop-2-yn-1-ylamino)pentan-2-yl)amino)-1,5-dioxo-5-(prop-2-yn-1-ylamino)pentan-2-yl)amino)-5-oxopentanoate) Step 1. To a solution of methyl 3,4,5-trihydroxybenzoate 15 (4.6 g, 25 mmol, CAS No. 99-24-1) and propargyl bromide (11.9 g, 11.1 mL, d = 1.57 g / mL, 100 mmol, 4 eq) in DMF (50 mL) was added K2CO3 (13.8 g, 100 mmol, 4 eq). The reaction mixture was then stirred overnight at room temperature. After confirming the depletion of the starting materials by TLC, the reaction mixture was filtered and concentrated under reduced pressure.

[0156] Step 2. The above crude product was dissolved in EtOH / H2O (200 mL, 1:1 v / v), and then 90 mL of 4 M aqueous NaOH was added to the reaction. After confirming the depletion of all starting materials by TLC, the reaction mixture was concentrated under reduced pressure to remove EtOH and filtered to afford a white solid 17 (6.18 g). This solid was used in the next step without further purification.

[0157] Step 3. To a solution of 17 (73 mg, 0.35 mmol) and PNP (139 mg, 1 mmol, 3 eq) in DCM (5 mL) at 0 °C was added EDC HCl salt (191 mg, 1 mmol, 3 eq). The reaction mixture was then stirred at room temperature. After confirming the depletion of all starting materials by TLC, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL × 3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica gel column and purified (MPA: hexane, MPB: EA, 0 - 40% gradient in 30 min) to afford Compound 16. C 22 H 14 MS (ESI) m / z calculated for C25H22N7O7 [M - H] 404.08, found: 404.48. Synthesis of Example 17. Compound 17 (4-Nitrophenyl 5-(((S)-1-(((R)-1,5-dioxo-1,5-bis(prop-2-yn-1-ylamino)pentan-2-yl)amino)-1,5-dioxo-5-(prop-2-yn-1-ylamino)pentan-2-yl)amino)-5-oxopentanoate) Step 1. To a solution of acid 18 (4.225 g, 10 mmol), amine 19 (2.959 g, 10 mmol), and N,N - diisopropylethylamine (3.87 g, 0.52 mL, d = 0.742 g / mL, 30 mmol) in DMF (20 mL) at 0 °C was added HBTU (5.685 g, 15 mmol). The reaction mixture was stirred at room temperature. After confirming the exhaustion of all raw materials by LC - MS, the reaction was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (20 mL×3). The combined organic layers were washed with brine. The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica gel column and purified (MPA: hexane, MPB: EA, 0 - 33% ramp in 30 min) to afford product 20, which was used for the next step. C 37 H 51 MS (ESI) of N2O9[M + H] m / z calculated 667.36, found: 667.49. Step 2. The product from Step 1 was dissolved in TFA / DCM (20 mL, 1:1 v / v). The reaction was stirred at room temperature for 3 h. After confirming the exhaustion of all raw materials by LC - MS, the mixture was concentrated under reduced pressure overnight. Yield 3.4 g. C 25 H 25 MS (ESI) of N2O9[M - H] m / z calculated 497.16, found: 497.35. Step 3. To a flame - dried round - bottom flask were added tri - acid 21 (1.000 g, 2.008 mmol), DMF (14 mL), propargylamine (0.3645 g, 0.42 mL, d = 0.86 g / mL, 6.6265 mmol), and DIEA (0.9066 g, 1.222 mL, d = 0.742 g / mL, 7.0281 mmol). The mixture was cooled to 0 °C and TBTU (2.256 g, 7.0281 mmol, 3.5 eq) was added. After confirming the exhaustion of all raw materials by LC - MS, the reaction mixture was concentrated under reduced pressure. The product was obtained by filtration and washed with DCM (5 mL) and H2O (5 mL). It was freeze - dried overnight to afford 0.8818 g of white solid 22. C 34 H 36 MS (ESI) of N5O6[M + H] m / z calculated 610.27, found: 610.41. Steps 4, 5, and 6. 22 (100 mg, 0.1642 mmol) in DMF (1 mL) was added to triethylamine (0.1658 g, 0.228 mL, d = 0.726 g / mL, 1.6420 mmol) at room temperature. The reaction mixture was stirred overnight. After confirming the depletion of all starting materials by LC-MS, glutaric anhydride (28.1 mg, 0.2463 mmol) and DMAP (2.0 mg, 0.0164 mmol) were added. The reaction mixture was stirred overnight. PNP (114.1 mg, 0.821 mmol) and EDC-HCl (156.8 mg, 0.8210 mmol) were added. After the consumption of starting materials, the reaction mixture was concentrated and purified by using silica gel as the stationary phase and eluted with a MeOH / DCM (0 - 20%) gradient. Yield: 34 mg, 34%. C 30 H 35 MS (ESI) m / z calculated for C H N6O9 [M+H] 623.25, found: 623.38. Synthesis of Compound 18 of Example 18 (4-Nitrophenyl 5-((1,7-dioxo-4-(3-oxo-3-(prop-2-yn-1-ylamino)propyl)-1,7-bis(prop-2-yn-1-ylamino)hept-4-yl)amino)-5-oxopentanoate) To a solution of 3 (1.00 g, 2.79 mmol) in DMF (5 mL) at room temperature was added triethylamine (0.847 g, 1.17 mL, 8.37 mmol) and glutaric anhydride (493 mg, 4.32 mmol). The reaction mixture was stirred overnight. The next day, 4-nitrophenol (896 mg, 6.44 mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1.23 g, 6.44 mmol) were added at room temperature and the reaction mixture was stirred overnight. The reaction mixture was concentrated. The residue was purified by using silica gel as the stationary phase and eluted with a MeOH / DCM (0 - 6%) gradient. Yield of 31 (Compound 18): 1.13 g (74%). C 30 H 35 Calculated [M+H] for C H N5O8: 594.65, found: 594.39. Example 19. Synthesis of Compound 19 (2,3,5,6-Tetrafluorophenyl 3-((1,7-dioxo-4-(3-oxo-3-(prop-2-yn-1-ylamino)propyl)-1,7-bis(prop-2-yn-1-ylamino)hept-4-yl)carbamoyl)bicyclo[1.1.1]pentane-1-carboxylate) Step 1. To a solution of acid 26 (52.2 mg, 0.3073 mmol, 1.1 eq), TBTU (134.5 mg, 0.4190 mmol, 1.5 eq) and DIEA (108.1 mg, 0.1457 mL, d = 0.742 g / mL, 0.8380 mmol) in DMF (0.5 mL) was added amine 25 (100 mg, 0.2793 mmol). The reaction was stirred at room temperature. After confirming the depletion of all starting materials by LC-MS, the reaction mixture was concentrated under reduced pressure. Purification on (MPA: DCM, MPB: 20% MeOH / DCM, 0 - 50% ramp in 30 min) afforded the pure product 27. Yield: 114 mg, 80%. C 27 H 35 MS (ESI) m / z calculated for C H N4O6 [M+H] 511.26, found: 511.75. Step 2. The above product was dissolved in THF / H2O (0.6 mL, 2:1 v / v), and then LiOH (16 mg, 0.66 mmol, 3 eq) was added to the reaction. After confirming the depletion of all starting materials by LC-MS, the reaction mixture was neutralized by adding 0.66 mmol HCl(aq). The mixture was concentrated under reduced pressure and lyophilized over the weekend. The crude product was used in the next step without further purification.

[0158] Step 3. At 0 °C, EDC HCl salt (210.1 mg, 1.1 mmol, 5 eq) was added to a solution of 28, TFP (182.6 mg, 1.1 mmol, 5 eq) and DIEA (179.6 mg, 0.242 mL, d = 0.742 g / mL, 1.39 mmol) in DCM (5 mL). The reaction mixture was then stirred at room temperature. After confirming the depletion of all starting materials by TLC, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL × 3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica gel column and purified (MPA: hexane, MPB: EA, 0 - 50% ramp in 30 min) to afford compound 19. Yield: 89 mg (63%). C 32 H 33 MS (ESI) m / z calculated for C H F4N4O6 [M+H] 645.23, found: 645.79. Synthesis of Example 20. Compound 20 (2,3,5,6-Tetrafluorophenyl 4'-((1,7-dioxo-4-(3-oxo-3-(prop-2-yn-1-ylamino)propyl)-1,7-bis(prop-2-yn-1-ylamino)hept-4-yl)carbamoyl)-[1,1'-biphenyl]-4-carboxylate) Step 1. To a solution of acid 29 (78.7 mg, 0.3073 mmol, 1.1 eq), TBTU (134.5 mg, 0.4190 mmol, 1.5 eq) and DIEA (108.1 mg, 0.1457 mL, d = 0.742 g / mL, 0.8380 mmol) in DMF (0.5 mL) was added 25 (100 mg, 0.2793 mmol). The reaction was stirred at room temperature. After confirming by LC-MS that all starting materials were exhausted, the reaction mixture was concentrated under reduced pressure. Purification on (MPA: DCM, MPB: 20% MeOH / DCM, 0 - 50% ramp in 30 min) afforded the pure product 30. Yield: 165 mg, 99%. C 34 H 37 MS (ESI) m / z calculated for C27H24N4O6 [M+H] 597.27, found: 597.81. Step 2. The product from Step 1 was dissolved in THF / H2O (0.6 mL, 2:1 v / v), then LiOH (20 mg, 0.83 mmol, 3 eq) was added. After confirming by LC-MS that all starting materials were exhausted, the reaction mixture was neutralized by adding 0.83 mmol HCl(aq). The mixture was concentrated under reduced pressure and lyophilized over the weekend. The crude product was used in the next step without further purification.

[0159] Step 3. At 0 °C, EDC HCl salt (266 mg, 1.39 mmol, 5 eq) was added to a solution of 31, TFP (231 mg, 1.39 mmol, 5 eq) and DIEA (179.6 mg, 0.242 mL, d = 0.742 g / mL, 1.39 mmol) in DCM (5 mL). The reaction mixture was then stirred at room temperature. After confirming by TLC that all starting materials were exhausted, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL × 3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica gel column and purified (MPA: hexane, MPB: EA, 0 - 50% ramp in 30 min) to afford the pure product Compound 20. Yield: 87 mg (42%). C 39 H 35 F4N4O6 [M+H]+ Calculated MS(ESI) m / z value: 731.25, found: 731.85. Example 21. Synthesis of Compound 21 ((4-Nitrophenyl) Carbonate (1r,4r)-4-((1,7-Dioxo-4-(3-Oxo-3-(Prop-2-YnylAmino)Propyl)-1,7-Bis(Prop-2-YnylAmino)Hept-4-Yl) Carbamoyl) Cyclohexyl Ester) To a solution of Compound 8 (see Example 1) (0.048 g, 0.10 mmol) and DIEA (0.18 mL, 1.0 mmol) in THF (0.5 mL) was added 4-Nitrophenyl Chloroformate (0.044 g, 0.22 mmol) and the reaction was stirred at 50 °C. After completion, all volatiles were removed and Compound 21 was isolated by silica gel column chromatography eluting with a MeOH / DCM gradient. Yield: 0.035 g (54%). Example 22. Synthesis of Tridentate Ligands and Conjugation of Targeting Ligands to RNAi Agents Targeting ligands can be conjugated to one or more RNAi agents, which can be used to inhibit the expression of one or more target genes. Targeting ligands facilitate the delivery of RNAi agents to target cells and / or tissues. Targeting ligands can contain certain moieties that interact with cell surface receptors, thereby introducing the RNAi agent into the cell. A general procedure for synthesizing certain targeting ligand-RNAi agent conjugates using the triyne linkers described herein, exemplified in the non-limiting examples given herein, is described below.

[0160] A. Synthesis of RNAi Agents RNAi agents can be synthesized using methods well known in the art. To synthesize the RNAi agents exemplified in the examples given herein, the sense and antisense strands of the RNAi agents were synthesized on a solid support for oligonucleotide synthesis according to the phosphoramidite technique. Depending on the scale, use (Bioautomation), (Bioautomation) or OPPilot 100 (GE Healthcare). The synthesis was carried out on a solid support made of controlled pore glass (CPG, or obtained from PrimeSynthesis, Aston, PA, USA). All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the following 2'-O-methyl phosphoramidites were used: (5'-O-Dimethoxytrityl-N 6-(Benzoyl)-2′-O-methyl-adenosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, 5′-O-dimethoxy-trityl-N 4 -(Acetyl)-2′-O-methyl-cytidine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, (5′-O-dimethoxytrityl-N 2 -(Isobutyryl)-2′-O-methyl-guanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite and 5′O-dimethoxytrityl-2′-O-methyluridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite. 2′-Deoxy-2′-fluoro-phosphoramidites carry the same protecting groups as the 2′-O-methyl RNA amidites. 5′-Dimethoxytrityl-2′-O-methyl-inosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was purchased from Glen Research (Virginia). Reverse abasic (3′-O-dimethoxytrityl-2′-deoxyribose-5′-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was purchased from ChemGenes (Wilmington, MA, USA). The following UNA phosphoramidites were used: 5′-(4,4'-dimethoxytrityl)-N6-(benzoyl)-2′,3′-seco-adenosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′(4,4'dimethoxytrityl)-N-acetyl-2′,3′-seco-cytidine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′-(4,4'-dimethoxytrityl)-N-isobutyryl-2′,3′-seco-guanosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and 5′-(4,4'-dimethoxy-trityl)-2′,3′-seco-uridine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. TFA aminolink phosphoramidites are also available (ThermoFisher).

[0161] Alternatively, the triyne moiety is introduced after solid support synthesis (see Section F below). For this route, the sense strand is functionalized with 5′- and / or 3′-terminal nucleotides containing a primary amine. The TFA aminolink phosphoramidite is dissolved in anhydrous acetonitrile (50 mM) and molecular sieves are added 5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used as the activator solution. The coupling times were 10 minutes (RNA), 90 seconds (2′O-Me), and 60 seconds (2′F). To introduce phosphorothioate / phosphonate linkages, a 100 mM solution of 3-phenyl-1,2,4-dithiazol-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was used.

[0162] In some embodiments, the compound of Formula II is reacted to synthesize the compound of Formula III by adding it to the terminus of an RNAi agent. In some embodiments, the triyne linker of Formula II is added to the 5′-end of the sense strand of a double-stranded RNAi agent. In some embodiments, the triyne linker of Formula II is added to the 3′-end of the sense strand of a double-stranded RNAi agent. In some embodiments, the compound of Formula II is added to the 5′-end of the antisense strand of a double-stranded RNAi agent. In some embodiments, the compound of Formula II is added to the 3′-end of the antisense strand of a double-stranded RNAi agent. An exemplary reaction of this type is shown in the following scheme: When used in combination with the RNAi agents given in certain examples herein, the triyne-containing phosphoramidite is dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other amidites are dissolved in anhydrous acetonitrile (50 mM), and molecular sieves are added. 5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used as the activator solution. The coupling times were 10 minutes (RNA), 90 seconds (2′O-Me), and 60 seconds (2′F). To introduce phosphorothioate / phosphonate linkages, a 100 mM solution of 3-phenyl-1,2,4-dithiazol-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was used.

[0163] B. Cleavage and deprotection of the support bound oligomer. After completion of the solid-phase synthesis, the dried solid support was treated at 30 °C with a 1:1 volume solution of 40 wt% methylamine in water and 28% to 31% ammonium hydroxide solution (Aldrich) for 1.5 hours. The solution was evaporated and the solid residue was reconstituted in water (see below).

[0164] C. Purification. The crude oligomers were purified by anion-exchange HPLC using a TSKgel SuperQ-5PW 13 μm column and a Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0 and contained 20% acetonitrile, and buffer B was the same as buffer A with the addition of 1.5 M sodium chloride. The UV trace was recorded at 260 nm. Appropriate fractions were pooled and then run on size-exclusion HPLC using a GE Healthcare XK 16 / 40 column packed with Sephadex G25 fine with a running buffer of 100 mM ammonium bicarbonate, pH 6.7 and 20% acetonitrile or filtered water.

[0165] D. Annealing. Complementary strands were mixed to form the RNAi agent by combining equimolar RNA solutions (sense and antisense) in 1× PBS (phosphate-buffered saline, 1×, Corning, Cellgro). Some of the RNAi agents were lyophilized and stored at -15 to -25 °C. The duplex concentration was determined by measuring the solution absorbance in 1× PBS on a UV-Vis spectrophotometer. The solution absorbance at 260 nm was then multiplied by the conversion factor and the dilution factor to determine the duplex concentration. The conversion factor used was 0.037 mg / (mL·cm), or for some experiments, the conversion factor was calculated from the extinction coefficient determined experimentally.

[0166] E. Conjugation of Targeting Ligands Compounds of Formulas IV, V, VIII, and IX can be synthesized by conjugating a targeting ligand to the triyne compounds described herein. An exemplary reaction is shown in the following scheme: where each variable is as described in Formula I, and TL is a targeting ligand.

[0167] In some embodiments, the following procedure can be used for the conjugation of targeting ligands. The following procedure describes the conjugation of a targeting ligand to a compound of Formula I in which R contains an RNAi agent, but the conjugation of a targeting ligand can also be carried out on a compound of Formula I in which R does not contain an RNAi agent.

[0168] Before or after annealing, a 5′ or 3′ trialkynyl-functionalized sense strand is conjugated to a targeting ligand. The following examples describe the conjugation of a targeting ligand to an annealed duplex: Prepare stock solutions of 0.5 M tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 0.5 M copper(II) sulfate pentahydrate (Cu(II)SO4·5H2O), and 2 M sodium ascorbate in deionized water. Make a 75 mg / mL solution of the targeting ligand in DMSO. In a 1.5 mL centrifuge tube containing the trialkyne-functionalized duplex (3 mg, 75 μL, 40 mg / mL in deionized water, ∼15,000 g / mol), add 25 μL of 1 M Hepes pH 8.5 buffer. After vortexing, add 35 μL of DMSO and vortex the solution. Add the targeting ligand to the reaction (6 eq / duplex, 2 eq / alkyne, ∼15 μL) and vortex the solution. Using pH paper, check the pH and confirm it is pH ∼8. In a separate 1.5 mL centrifuge tube, mix 50 μL of 0.5 M THPTA with 10 μL of 0.5 M Cu(II)SO4·5H2O, vortex, and incubate at room temperature for 5 min. After 5 min, add the THPTA / Cu solution (7.2 μL, 6 eq 5:1 THPTA:Cu) to the reaction vial and vortex. Immediately thereafter, add 2 M ascorbate (5 μL, 50 eq / duplex, 16.7 / alkyne) to the reaction vial and vortex. Once the reaction is complete (usually complete within 0.5 - 1 h), immediately purify the reaction by non-denaturing anion exchange chromatography.

[0169] F. Addition of a Trialkyne Linker after Solid Support Synthesis RNAi molecules with reactive groups, such as amino groups (also referred to as amines herein), can be synthesized. In some embodiments, the reactive group can be attached to the 5′-end and / or 3′-end of the RNAi agent. In some embodiments, the RNAi agent can be double-stranded. In embodiments where the RNAi agent is double-stranded, the reactive group can be on the sense strand or the antisense strand of the RNAi agent.

[0170] For example, in some embodiments, an RNAi agent is synthesized that has an NH2-C6H 12 (hexanediamine) group at the 5′-end of the sense strand of the RNAi agent. The terminal amino group can then react to form a conjugate with a coupling moiety of a compound such as Formula I. In some embodiments, the coupling moiety is an ester, and the reactive group on the RNAi agent is a primary amine, forming an amide bond between the RNAi agent and the trialkyne linker. An example of such a reaction using a compound of Formula VI is shown in the following scheme: where L 1 、L 2 、L3 , L 4 , R 3 , R 4 and RNA are both defined as in Formulas VI and VII.

[0171] When the RNAi molecule has been cleaved from the solid support, the addition of the triyne linker described herein can be carried out as follows. The sense strand is functionalized with 5′- and / or 3′-terminal nucleotides containing a primary amine. The amine-functionalized duplex is dissolved in 90% DMSO / 10% H2O at ~50 - 70 mg / mL. 40 equivalents of triethylamine are added, followed by 3 equivalents of the triyne ester of Formula VI. Once complete, the conjugate is precipitated twice in a solvent system of 1x phosphate buffered saline / acetonitrile (1:14 ratio) and dried.

[0172] In Vivo Examples The linkers described herein can be used in combination with various RNAi agents. The following examples demonstrate the use of the linkers described herein with RNAi agents directed against the Alpha-ENaC and HIF2α mRNA sequences and are intended to provide examples of the application of the linkers and not to limit the scope of the invention to any particular RNAi agent. The RNAi agents used in the following examples are shown in Table 8 below. The compounds in Table 8 are shown as the structures cleaved from the solid support. In some cases, further modification of the compounds is made prior to in vivo administration. For AD5614 - 5617, AD5620, AD5858, AD5860, and AD5919, as part of the synthesis on the solid support, the triyne linker is added as a phosphoramidite of Formula II to the sense strand. In the case of AD04546, AD5347, and AD5453, the sense strand is cleaved from the support in the structure shown in Table 8. The respective triyne linkers are added as compounds of Formula VI in an amide coupling reaction. The targeting ligands are added after cleavage from the resin, and thus for AD5614 - 5617, AD5620, AD5858, AD5860, and AD5919, the triyne linker is represented as a compound of Formula III. In Table 8 below, a, c, g, and u represent 2′-O-methyladenosine, cytidine, guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent 2′-fluoroadenosine, cytidine, guanosine, or uridine, respectively; and s represents a phosphorothioate / phosphate bond, and cPrpu represents 5′-cyclopropyl phosphonate-2′-O-methyl uridine:

[0173] Example 23. Renal tumor-bearing mouse model (orthotopic xenograft). Generation of clear cell renal cell carcinoma (ccRCC) A498 cells expressing SEAP A pCR3.1 expression vector expressing the reporter gene secreted alkaline phosphatase (SEAP) under the CMV promoter was prepared by directional cloning of the SEAP coding sequence by PCR amplified from Clontech’s pSEAP2-basic vector. Convenient restriction sites were added to the primers used to amplify the SEAP coding sequence for cloning into the pCR3.1 vector (Invitrogen). The resulting construct pCR3-SEAP was used to generate A498 ccRCC cell lines expressing SEAP. Briefly, the pCR3-SEAP plasmid was transfected into A498 ccRCC cells by electroporation according to the manufacturer's recommendations. Stable transfectants were selected by G418 resistance. SEAP expression and integration stability of the selected A498-SEAP clone lines were evaluated.

[0174] Implantation of clear cell renal cell carcinoma (ccRCC) A498 cells expressing SEAP Female athymic nude mice were anesthetized with ~3% isoflurane and placed in the right lateral decubitus position. A small 0.5-1 cm longitudinal abdominal incision was made in the left flank. Using a moistened cotton swab, the left kidney was removed from the peritoneum and gently stabilized. Immediately prior to injection, a 1.0 ml syringe was filled with the cell / Matrigel mixture and a 27-gauge needle cannula was attached to the syringe tip. The filled syringe was then attached to a syringe pump (Harvard Apparatus, model PHD2000) and primed to remove air. The tip of the 27-gauge needle cannula attached to the syringe was inserted just beneath the renal capsule near the caudal pole, and then the needle tip was carefully advanced cranially along the capsule 3-4 mm. A 10 μl aliquot of the 2:1 (vol:vol) cell / Matrigel mixture containing approximately 300,000 cells was slowly injected into the renal parenchyma using the syringe pump. The needle was left in the kidney for 15-20 seconds to ensure complete injection. The needle was then removed from the kidney and a cotton swab was pressed against the injection site for 30 seconds to prevent cell leakage or bleeding. The kidney was then carefully replaced in the abdomen and the abdominal wall was closed. Serum was collected every 7-14 days after implantation to monitor tumor growth using a commercial SEAP detection kit. For most studies, tumor-bearing mice were used 5-6 weeks after implantation, at which time tumor measurements were typically approximately 4-8 mm.

[0175] Determination of HIF2 mRNA expression For the studies reported in the examples herein, mice were euthanized at the designated days after injection and total RNA was isolated from renal tumors using Trizol reagent according to the manufacturer's recommendations. Relative HiF2α mRNA levels were determined by RT-qPCR as described below and compared to mice treated with only delivery buffer (isotonic glucose).

[0176] In preparation for quantitative PCR, total RNA was isolated from tissue samples homogenized in TriReagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's procedure.

[0177] Approximately 500 ng of RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Life Technologies). For human (tumor) Hif2α (EPAS1) expression, TaqMan Gene Expression Master Mix (Life Technologies) or VeriQuest Probe Master Mix (Affymetrix) was used in duplicate in a biplex reaction with pre-made TaqMan gene expression assays for human Hif2α (Catalog# 4331182) and CycA (PPIA) Catalog#: 4326316E). Quantitative PCR was performed using the 7500 Fast or StepOnePlus Real-Time PCR System (Life Technologies). Relative gene expression was calculated using the ΔΔC T method.

[0178] Example 24. In Vivo Administration of an Integrin-Targeting Ligand Conjugated to an RNAi Agent Targeting HIF-2alpha (EPAS1) in Renal Tumor-Bearing Mice. The RNAi agent comprising the sense and antisense strand sequences given in Table 8 was synthesized on a solid phase according to the phosphoramidite technique according to general procedures known and commonly used in the art for oligonucleotide synthesis. (See Example 22 herein). The RNAi agent comprises an antisense strand having a nucleobase sequence that is at least partially complementary to the HIF-2alpha (Hif2α or EPAS1) gene. EPAS1 is a member of the HIF (hypoxia-inducible factor) gene family and encodes a subunit of a transcription factor that is involved in oxygen-regulated gene induction and is induced when oxygen levels decline (a condition known as hypoxia). Hif2α is known to be frequently overexpressed in clear cell renal carcinoma (ccRCC) cells. The Hif2α RNAi agent was designed to reduce or inhibit the translation of messenger RNA (mRNA) transcripts of Hif2α in a sequence-specific manner, thereby inhibiting the expression of the EPAS1 gene.

[0179] On Study Day 1, the renal tumor-bearing mice (see Example 23) were dosed via tail vein injection according to a dosing regimen comprising the following groups: Table 9. Dosing Groups of Renal Tumor-Bearing Mice in Example 5

[0180] The RNAi agent of Example 24 was synthesized with a functionalized amine-reactive group (NH2-C6) at the 5′ end of the sense strand to facilitate conjugation to the respective triyne linker compounds shown. In the case of Groups 4-8 and 10, the triyne linker was added to the RNAi agent using phosphoramidite compounds 1, 2, 3, 4, and 6, respectively. The respective integrin-targeting ligands were synthesized, which have azide-reactive groups (see, for example, Example 22), which were subsequently conjugated to the triyne moiety of the linker. A 40 kilodalton (kDa) PEG moiety was linked to act as a pharmacokinetic (PK) modulator to increase the circulation time of the drug product-conjugate. The structures of the targeting ligands αvβ3 integrin ligands 4.1 and 4.5 are shown below:

[0181] Three (3) tumor-bearing mice were dosed in each group (n = 3). The mice were sacrificed on Study Day 8 after injection, and total RNA was isolated from the renal tumors according to the procedure set forth in Example 4. Then, as explained in Example 23, the relative human HIF2α mRNA expression was quantified by probe-based quantitative PCR (RT-qPCR), normalized to cyclophilin A (PPIA) expression, and expressed as a fraction (geometric mean, + / - 95% confidence interval) of the vehicle control group (isotonic glucose).

[0182] Table 10. Mean Relative huHif2α mRNA Expression at Sacrifice in Example 24

[0183] Example 25. In Vivo Administration of an Integrin-Targeting Ligand Conjugated to an RNAi Agent Targeting HIF-2alpha (EPAS1) in Mice Bearing Renal Tumors The RNAi agent comprising the sense and antisense strand sequences given in Table 8 was synthesized on a solid phase according to the phosphoramidite technique according to general procedures known in the art and commonly used for oligonucleotide synthesis. (See Example 22 herein). The RNAi agent comprises an antisense strand having a nucleobase sequence that is at least partially complementary to the (Hif2α)(EPAS1) gene.

[0184] On Study Day 1, the mice bearing renal tumors (see Example 23) were administered by tail vein injection according to the following dosing groups: Table 11. Dosing Groups for Mice Bearing Renal Tumors in Example 5

[0185] The RNAi agent of Example 25 was synthesized, which has a nucleotide sequence designed to target the human Hif2α gene and, in the case of Groups 3-6, comprises a functionalized amine-reactive group (NH2-C6) at the 5′ end of the sense strand to facilitate conjugation to the triyne linker compounds 15-18. In the case of Groups 3 and 7-9, the triyne linker was added to the RNAi agent using phosphoramidite compounds 14, 10, 12, and 13, respectively. The respective integrin-targeting ligands were synthesized, which have an azide-reactive group (see, for example, Example 22), which was subsequently conjugated to the triyne moiety of the linker. A 40 kDa PEG moiety and a C-18 diacid moiety were linked to act as a pharmacokinetic (PK) modulator by increasing the circulation time of the drug product-conjugate. The structure of the C-18 diacid moiety is shown below: The C-18 diacid moiety was linked to the 3′ end of the sense strand via an amide bond. The structure of the targeting ligand αvβ3 integrin ligand 2 is shown below: where indicates the point of attachment to the linker.

[0186] Three (3) tumor-bearing mice were dosed in each group (n = 3). The mice were sacrificed on Study Day 8 after injection, and total RNA was isolated from the renal tumors according to the procedure described in Example 4. Then, as explained in Example 23, the relative human HIF2α mRNA expression was quantified by probe-based quantitative PCR (RT-qPCR), normalized to human cyclophilin A (PPIA) expression, and expressed as a fraction (geometric mean, + / - 95% confidence interval) of the vehicle control group (isotonic glucose).

[0187] Table 12. Mean relative huHif2α mRNA expression at sacrifice in Example 25

[0188] Example 26. In Vivo Oropharyngeal Aspiration Administration of Alpha-ENaC RNAi Agent Conjugated to an Epithelial Cell-Targeting Ligand in Rats The triyne linker can be used with a variety of RNAi constructs. RNAi constructs containing the linker of the present invention can be administered in a variety of different administration methods as described in this example. The triyne linker can also be used with a variety of targeting ligands. In this example, the targeting ligand conjugated to the triyne linker is the αvβ6 targeting ligand.

[0189] In this example, the triyne linker of Compound 22 was added to the sense strand after solid support synthesis in the method as described in Example 22.

[0190] On Study Day 1, male Sprague Dawley rats were administered by 200 μL oropharyngeal aspiration (OP) using a pipette according to the following dosing groups: Table 13. Dosing Groups of Rats in Example 8

[0191] In each group, Compound 22 was reacted with the amine bond at the 5'-end of the sense strand. The structure of the αvβ6 targeting ligand is shown below: where indicates the point of attachment to the linker.

[0192] Four (4) rats were dosed in each group (n = 4). The rats were euthanized on Study Day 9 after injection, and total RNA was isolated from both lungs after collection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantified by probe-based quantitative PCR, normalized to GAPDH expression and expressed as a fraction of the vehicle control group (geometric mean, + / - 95% confidence interval).

[0193] Table 14. Mean relative rENaC mRNA Expression at Sacrifice (Day 9) in Example 8

[0194] As shown in Table 14 above, various different targeting ligand structures linked to their respective RNAi agents using the triyne linking compounds disclosed herein exhibited inhibition of gene expression compared to the controls.

[0195] Other embodiments It is to be understood that although the invention has been described in detail in connection therewith, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A compound of formula I: or a pharmaceutically acceptable salt thereof, wherein L 1 , L 2 and L 3 are each independently a linking group comprising an optionally substituted alkylene group; Q is a tetravalent carbon, a tetrasubstituted phenyl or an optionally substituted alkylene group; R comprises a coupling moiety or an RNAi agent; and X is NR x or a bond, and R x is H or an optionally substituted C1-C6 alkyl group.

2. A compound selected from: or a pharmaceutically acceptable salt thereof.

3. A compound of formula II, or a pharmaceutically acceptable salt thereof, wherein, L 1 , L 2 , and L 3 each independently is a linking group containing an optionally substituted alkylene group; L 4 is a linking group comprising an optionally substituted alkylene, an optionally substituted arylene and an optionally substituted cycloalkylene; each occurrence of R 1 is an optionally substituted alkyl; R 2 is an optionally substituted alkyl; and R 4 is H or an optionally substituted alkyl group.

4. A compound of formula V, or a pharmaceutically acceptable salt thereof, wherein, L 1 、L 2 and L 3 each independently is a linking group containing an optionally substituted alkylene group; L 4 is a linking group containing an optionally substituted alkylene, an optionally substituted arylene or an optionally substituted cycloalkylene; R 4 is H or an optionally substituted alkyl; TL is a targeting ligand; Y is O or S; and RNA comprises an RNAi agent or consists of an RNAi agent.

5. A compound selected from: or a pharmaceutically acceptable salt thereof, wherein TL is a targeting ligand and RNA comprises an RNAi agent or consists of an RNAi agent.

6. A compound of formula IX or a pharmaceutically acceptable salt thereof, wherein, L 1 、L 2 and L 3 each independently is a linking group containing an optionally substituted alkylene group; L 4 is a linking group comprising an optionally substituted alkylene, an optionally substituted arylene or an optionally substituted cycloalkylene; TL is a targeting ligand; R 4 each occurrence is H or an optionally substituted alkyl; and RNA comprises an RNAi agent or consists of an RNAi agent.

7. A method for reacting a compound of formula II with an RNAi agent to form a compound of formula III: wherein, L 1 、L 2 and L 3 each independently is a linking group containing an optionally substituted alkylene group; L 4 is a linking group comprising an optionally substituted alkylene, an optionally substituted arylene and an optionally substituted cycloalkylene; each R 1 is, in each case, an optionally substituted alkyl group; R 2 is an optionally substituted alkyl; and R 4 is H or an optionally substituted alkyl X is O or S; and RNA comprises an RNAi agent or consists of an RNAi agent.

8. A method for reacting a compound of formula VI with an RNAi agent comprising a free amine to form a compound of formula VII: wherein, L 1 、L 2 and L 3 each independently is a linking group containing an optionally substituted alkylene group; L 4 is a linking group comprising an optionally substituted alkylene, an optionally substituted arylene or an optionally substituted cycloalkylene; R 3 is H, an optionally substituted alkyl or an optionally substituted aryl; and R 4 in each case is H or an optionally substituted alkyl; and RNA comprises an RNAi agent or consists of an RNAi agent.

9. A method for reacting a compound of formula III with a targeting ligand comprising an azide to form a compound of formula V wherein, L 1 , L 2 and L 3 each independently is a linking group containing an optionally substituted alkylene group; L 4 is a linking group containing an optionally substituted alkylene, an optionally substituted arylene or an optionally substituted cycloalkylene; R 4 is H or an optionally substituted alkyl; TL is a targeting ligand; Y is O or S; and RNA comprises an RNAi agent or consists of an RNAi agent.

10. A method for reacting a compound of formula VII with a targeting ligand comprising an azide to form a compound of formula IX, wherein, L 1 、L 2 and L 3 each independently is a linking group containing an optionally substituted alkylene group; L 4 is a linking group comprising an optionally substituted alkylene, an optionally substituted arylene or an optionally substituted cycloalkylene; R 4 each instance of which is H or an optionally substituted alkyl; and RNA comprises an RNAi agent or consists of an RNAi agent.

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