Trialquin binder and method of use

Abstract

To provide linking agents with suitable properties for linking oligonucleotide-based compounds, such as RNAi agents, to targeting ligands.SOLUTION: Described are improved linking agents that are useful for facilitating the attachment of targeting groups, pharmacokinetic (PK) enhancers or modifiers, or other delivery agents to oligonucleotides. The described linking agents may exhibit improved reaction yields, stability and biological activity, particularly when used in connection with oligonucleotide-based compounds, such as RNA interference (RNAi) agents.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Application No. 62 / 631,683 filed on 17 February 2018, U.S. Provisional Application No. 62 / 646,739 filed on 22 March 2018, U.S. Provisional Application No. 62 / 663,763 filed on 27 April 2018, and U.S. Provisional Application No. 62 / 790,300 filed on 9 January 2019, all of which are incorporated herein by reference.

[0002] Field of Invention This disclosure relates to a trialukine binder suitable for use with synthetic oligonucleotides such as RNA interference (RNAi) agents. [Background technology]

[0003] background Synthetic oligonucleotides, such as antisense compounds, aptamers, ribozymes, and RNA interference (RNAi) agents or molecules, are increasingly being used in biomedical research, diagnosis, and therapy. These synthetic oligonucleotides have been used to sequence-dependently inhibit or knock down gene expression in vitro, in situ, and in vivo.

[0004] In particular, in in vivo therapeutic delivery, it is often useful to conjugate or link target ligands or other pharmacological or pharmacokinetic enhancers or modifiers to synthetic oligonucleotides. For usefulness, the conjugating chemical should be modular to allow for easy adaptation to different synthetic oligonucleotides, different target ligands, and pharmacological modifiers. Furthermore, the conjugating chemical should have simple reaction conditions, be efficient (i.e., yield high chemical yields), require no toxic or other harmful products, and produce no toxic or other harmful byproducts. The conjugating chemical should also be stable outside the target cell, such as in circulation, subcutaneous space, or extracellular space, but should be readily cleavable at the final site of action, such as inside the target cell. Moreover, particularly in oligonucleotide-based therapies, the length and flexibility of the linker are known to substantially affect the in vivo efficacy of the therapeutic compound, especially by altering cellular uptake in certain cases.

[0005] There is a need for binders with appropriate properties to bind oligonucleotide-based compounds, such as RNAi agents, to target ligands. [Overview of the project]

[0006] overview In one embodiment, the present invention relates to formula I:

[0007] [ka]

[0008] (In the above formula, L 1 , L 2 and L 3 These are linkers containing alkylenes, each independently and optionally substituted. Q is a tetravalent carbon, a tetrasubstituted phenyl, or optionally a substituted alkylene. R contains a coupling moiety or an RNAi agent, and X is NR x or a bond, and R x is H or optionally substituted C1-C6 alkyl), provides a compound having the structure or a pharmaceutically acceptable salt thereof.

[0009] In one aspect, the present invention relates to Formula II:

[0010]

Chemical formula

[0011] (wherein, L 1 , L 2 and L 3 are each independently a linker containing optionally substituted alkylene, L 4 is a linker, which linker contains optionally substituted alkylene, optionally substituted arylene, or optionally substituted cycloalkylene, each R in each case 1 is optionally substituted alkyl, R 2 is optionally substituted alkyl, and R 4 is H or optionally substituted alkyl) provides a compound having the structure or a pharmaceutically acceptable salt thereof.

[0012] Another aspect of the present invention described herein is Formula III:

[0013]

Chemical formula

[0014] (wherein, L 1 , L 2 and L 3 are each independently a linker containing optionally substituted alkylene, L 4This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, or optionally a substituted cycloalkylene. R 4 is H or optionally a substituted alkyl, X is either O or S, and RNA is a compound with a structure (containing or consisting of an RNAi agent) or a pharmaceutically acceptable salt thereof.

[0015] In another embodiment, in this specification, Formula IV:

[0016] [ka]

[0017] (In the above formula, L 1 , L 2 and L 3 These are linkers containing alkylenes, each independently and optionally substituted. L 4 This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, or optionally a substituted cycloalkylene. R 1 and R 2 Each of these is an alkyl group that is independently and optionally substituted. R 4 is H or optionally a substituted alkyl, and The TL (target ligand) is described as a compound or a pharmaceutically acceptable salt thereof, based on its structure.

[0018] Another aspect of the present invention described herein is formula V:

[0019] [ka]

[0020] (In the above formula, L 1 , L 2 and L3 These are linkers containing alkylenes, each independently and optionally substituted. L 4 This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, or optionally a substituted cycloalkylene. R 4 is H or optionally a substituted alkyl, TL is the target ligand, Y is either O or S, and RNA is a compound with a structure (containing or consisting of an RNAi agent) or a pharmaceutically acceptable salt thereof.

[0021] In another embodiment, as used herein, Formula VI:

[0022] [ka]

[0023] (In the above formula, L 1 , L 2 and L 3 These are linkers containing alkylenes, each independently and optionally substituted. L 4 This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, or optionally a substituted cycloalkylene. R 3 is H, optionally a substituted alkyl, or optionally a substituted aryl, and R 4 Compounds or pharmaceutically acceptable salts thereof, based on the structure (where is H or optionally a substituted alkyl group), are described.

[0024] Another aspect of the present invention described herein is formula VII:

[0025] [ka]

[0026] (In the above formula, L 1 , L 2 and L 3 These are linkers containing alkylenes, each independently and optionally substituted. L 4 This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, or optionally a substituted cycloalkylene. R in each case 4 is H or optionally a substituted alkyl, X is either O or S, and RNA is a compound with a structure (containing or consisting of an RNAi agent) or a pharmaceutically acceptable salt thereof.

[0027] In another embodiment, Formula VIII is used herein:

[0028] [ka]

[0029] (In the above formula, L 1 , L 2 and L 3 These are linkers containing alkylenes, each independently and optionally substituted. L 4 This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, or optionally a substituted cycloalkylene. R 4 is H or optionally a substituted alkyl, and The TL (target ligand) is described as a compound or a pharmaceutically acceptable salt thereof, based on its structure.

[0030] Another aspect described herein is Formula IX:

[0031] [ka]

[0032] (In the above formula, L 1 , L 2 and L 3 These are linkers, each independently and optionally containing substituted alkylenes. L 4 This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, or optionally a substituted cycloalkylene. R 4 is H or optionally a substituted alkyl, TL is the target ligand, X is either O or S, and RNA is a compound with a structure (containing or consisting of an RNAi agent) or a pharmaceutically acceptable salt thereof.

[0033] Another aspect of the present invention is formula II:

[0034] [ka]

[0035] Reacting it with an RNAi agent, Equation III:

[0036] [ka]

[0037] (In the above formula, L 1 , L 2 and L 3 These are linkers, each independently and optionally containing substituted alkylenes. L 4 This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, and optionally a substituted cycloalkylene. R in each case 1 This is an alkyl group which may be substituted in some cases. R 2is an alkyl which may be substituted in some cases, and R 4 is H or optionally a substituted alkyl, X is either O or S, and The present invention provides a method for producing compounds (containing or consisting of RNAi agents).

[0038] Another aspect of the present invention is formula VI:

[0039] [ka]

[0040] The compound is reacted with an RNAi agent containing a free amine to produce formula VII:

[0041] [ka]

[0042] (In the above formula, L 1 , L 2 and L 3 These are linkers, each independently and optionally containing substituted alkylenes. L 4 This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, or optionally a substituted cycloalkylene. R 3 is H, optionally a substituted alkyl, or optionally a substituted aryl, and R in each case 4 is H or optionally a substituted alkyl, and The present invention provides a method for producing compounds (containing or consisting of RNAi agents).

[0043] Another aspect of the present invention is Formula III

[0044] [ka]

[0045] The compound is reacted with a target ligand containing an azide, and formula V

[0046] [ka]

[0047] (In the above formula, L 1 , L 2 and L 3 These are linkers, each independently and optionally containing substituted alkylenes. L 4 This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, or optionally a substituted cycloalkylene. R 4 is H or optionally a substituted alkyl, TL is the target ligand, Y is either O or S, and The present invention provides a method for producing compounds (containing or consisting of RNAi agents).

[0048] Another aspect of the present invention is formula VII

[0049] [ka]

[0050] The compound is reacted with a target ligand containing an azide, and formula IX

[0051] [ka]

[0052] (In the above formula, L 1 , L 2 and L 3 These are linkers, each independently and optionally containing substituted alkylenes. L 4This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, or optionally a substituted cycloalkylene. R in each case 4 is H or optionally a substituted alkyl, and The present invention provides a method for producing compounds (containing or consisting of RNAi agents). [Modes for carrying out the invention]

[0053] Detailed explanation Novel compounds containing phosphoramidite trialquines, their synthesis, and methods of use are disclosed herein. The improved compounds disclosed herein exhibit improved reaction yield, stability, and biological activity when used to conjugate synthetic oligonucleotides, such as RNAi agents, to target ligands or other pharmacokinetic (PK) enhancers or modifiers.

[0054] Disclosed herein are trialquine binders, their synthesis, and methods of use thereof. The trialquine binders disclosed herein can bind to oligonucleotides, which can then readily bind to target ligands, lipids, cholesterol, delivery agents (such as endosomal soluble polymers), or pharmacological modifiers. The trialquine binders disclosed herein can facilitate the synthesis of oligonucleotide conjugates with improved yield and fewer impurities than can be achieved using other known binders, while maintaining or, in some embodiments, improving the efficacy of the oligonucleotide conjugates, such as RNAi agents bound to one or more target ligands and / or pharmacokinetic modifiers.

[0055] As used herein, the term “linker” refers to the organic part that joins two parts of a compound. A linker is typically a direct bond or an atom such as oxygen or sulfur, NR L (R LIt includes units or chains of atoms such as hydrogen, acyl, aliphatic or substituted aliphatic, C(O), C(O)NH, SO, SO2, SO2NH, etc., and is not limited to substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkyl Telolarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkenylheteroarylalkynyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkynyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkenylheterocyclylalkynyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkenylaryl, alkenylheteroaryl, alkenylheteroaryl, alkenylheteroaryl (one or more methylenes are O, S, S(O), SO2, N(R) L )(R L (The elements are hydrogen, acyl, aliphatic or substituted aliphatic), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycles (which can be interrupted or terminated), -(CH2) n -,-(CH2) n N-, -(CH2) n O-, -(CH2) nS-, -(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-,

Chem.

[0056] The reactive groups are generally available in the art and include, but are not limited to, activated esters, NHS, TFP, PFP, tetrazine, norbornene, trans-cyclooctene, hydrazine (e.g., hydrazinic), aminooxy reagents, and aldehydes (e.g., 4-formylbenzoic acid).

[0057] Target ligands (sometimes referred to as target groups in the art) are used to target or improve the delivery of compounds to target cells or tissues, or to specific cell types. Target ligands improve the binding of molecules to target cells. Thus, target ligands can improve the cellular distribution and uptake of conjugates by improving the pharmacokinetic or biodistribution properties of the conjugates to which they are bound. Binding of target groups to cells or cell receptors can initiate endocytosis. Target groups can be monovalent, divalent, trivalent, tetravalent, or have higher valencies. Target groups can be, but are not limited to, cell surface molecules, cell receptor ligands, antibodies, monoclonal antibodies, antibody fragments and antibody mimetic compounds with affinity for cell surface molecules, hydrophobic groups, cholesterol, cholesteryl groups, or steroids. In some embodiments, target groups include cell receptor ligands. Various target groups have been used to target drugs and genes to cells and specific cell receptors. Cell receptor ligands can be, but are not limited to, carbohydrates, glycans, sugars (including, but not limited to, galactose, galactose derivatives (such as N-acetyl-galactosamine), mannose, and mannose derivatives), haptens, vitamins, folic acid, biotin, aptamers, and peptides (including, but not limited to, RGD-containing peptides, RGD mimetic compounds, insulin, EGF, and transferrin).

[0058] As used herein, the term “alkyl” refers to a linear or branched saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms, unless otherwise specified. For example, “C1-C6 alkyl” includes alkyl groups having 1, 2, 3, 4, 5, or 6 carbon atoms in a linear or branched configuration. Non-limiting examples of alkyl groups include methyl, ethyl, isopropyl, tert-butyl, and n-hexyl. As used herein, the term “aminoalkyl” refers to the alkyl group defined above, substituted with one or more amino groups at any position as permitted by normal valence. The amino groups may be unsubstituted, monosubstituted, or disubstituted. Non-limiting examples of aminoalkyl groups include aminomethyl, dimethylaminomethyl, and 2-aminopropyl-1-yl.

[0059] As used herein, the term "alkylene" refers to the divalent group of an alkyl group as described herein. Alkylenes are a subset of alkyls, referring to the same residue as alkyls but with two substitution sites. Examples of alkylenes include methylene, -CH2- or [ka] Ethylene, -CH2CH2- or [ka] and propylene, -CH2CH2CH2- [ka] That is the case.

[0060] As used herein, the term “cycloalkyl” means a saturated or unsaturated non-aromatic hydrocarbon ring group having 3 to 14 carbon atoms, unless otherwise specified. Non-exclusive examples of cycloalkyl groups include, but are not limited to, cyclopropyl, methylcyclopropyl, 2,2-dimethylcyclobutyl, 2-ethylcyclopentyl, and cyclohexyl. Cycloalkyl groups may contain multiple spiro rings or fused rings. Cycloalkyl groups are optionally substituted with one, two, three, four, or five atoms at any position, as is permissible with normal valences.

[0061] As used herein, the term "cycloalkylene" refers to the divalent group of a cycloalkyl group as described herein. Cycloalkylenes are a subset of cycloalkyls, referring to the same residues as cycloalkyls but having two substitution sites. Examples of cycloalkylenes include cyclopropylene. [ka] 1,4-Cyclohexylene, [ka] And, 1,5-cyclooxylen, [ka] These include: The cycloalkylene group is optionally monosubstituted, disubstituted, trisubstituted, tetrasubstituted, or pensubstituted at any position, as permissible with normal valency. The cycloalkylene group can be monocyclic, bicyclic, or tricyclic.

[0062] As used herein, the term “alkenyl” means, unless otherwise specified, a linear or branched non-aromatic hydrocarbon group having 2 to 10 carbon atoms and containing at least one carbon-carbon double bond. Such a group may have up to 5 carbon-carbon double bonds. For example, a “C2-C6” alkenyl is defined as an alkenyl group having 2 to 6 carbon atoms. Examples of alkenyl groups, but not limited to, include ethenyl, propenyl, butenyl, and cyclohexenyl. The linear, branched, or cyclic portions of an alkenyl group may contain double bonds and may be optionally substituted at any position, i, di, tri, qua, or pentasubstituted, as permitted by normal valence. The term “cycloalkenyl” means a monocyclic hydrocarbon group having a specified number of carbon atoms and at least one carbon-carbon double bond.

[0063] As used herein, the term “alkynyl” refers, unless otherwise specified, to a linear or branched hydrocarbon group containing 2 to 10 carbon atoms and at least one carbon-carbon triple bond. Up to five carbon-carbon triple bonds may be present. Thus, “C2-C6 alkynyl” means an alkynyl group having 2 to 6 carbon atoms. Examples of alkynyl groups, but not limited to, include ethynyl, 2-propynyl, and 2-butynyl. The linear or branched portion of the alkynyl group may be optionally substituted at any position, as permitted by normal valence, and may be mono-, di-, tri-, tetra-, or penta-substituted.

[0064] As used herein, "alkoxyl" or "alkoxy" refers to an -O-alkyl group having the indicated number of carbon atoms. For example, C 1-6 Alkoxy groups are intended to include C1, C2, C3, C4, C5, and C6 alkoxy groups. For example, C 1-8Alkoxy groups are intended to include C1, C2, C3, C4, C5, C6, C7, and C8 alkoxy groups. Examples of alkoxy groups, though not limited to them, include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, s-pentoxy, n-heptoxy, and n-octoxy.

[0065] As used herein, “keto” means any alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, or aryl group linked via carbonyl crosslinking, as defined herein. Examples of keto groups include, but are not limited to, alkanoyl (e.g., acetyl, propionyl, butanoyl, pentanoyl, or hexanoyl), alkenoyl (e.g., acryloyl), alkinoyl (e.g., ethinoyl, propinoyl, butinoyl, pentinoyl, or hexinoyl), arylloyl (e.g., benzoyl), and heteroarylloyl (e.g., pyrroloyl, imidazoloyl, quinolinoyl, or pyridinoyl).

[0066] As used herein, “alkoxycarbonyl” refers to any alkoxy group (i.e., -C(O)O-alkyl) as defined above, linked via a carbonyl crosslink. Examples of alkoxycarbonyl groups, but not limited to, include methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-propoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, or n-pentoxycarbonyl.

[0067] As used herein, “aryloxycarbonyl” refers to any aryl group (i.e., -C(O)O-aryl) linked via an oxycarbonyl crosslink. Examples of aryloxycarbonyl groups include, but are not limited to, phenoxycarbonyl and naphthyloxycarbonyl.

[0068] As used herein, “heteroaryloxycarbonyl” refers to any heteroaryl group (i.e., -C(O)O-heteroaryl) linked via an oxycarbonyl crosslink, as defined herein. Examples of heteroaryloxycarbonyl groups include, but are not limited to, 2-pyridyloxycarbonyl, 2-oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl, or pyrimidinyloxycarbonyl.

[0069] As used herein, “aryl” or “aromatic” means any stable monocyclic or polycyclic carbocyclic ring having up to six atoms in each ring, with at least one ring being aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, tetrahydronaphthyl, indanyl, and biphenyl. When the aryl substituent is bicyclic and one ring is non-aromatic, the bond is understood to be via an aromatic ring. The aryl group may be substituted at any position, optionally i, di, tri, tetra, or pentasubstituted, as is permissible by normal valency.

[0070] As used herein, the term “arirene” refers to a divalent aryl group as described herein. Arirenes are a subset of aryls, referring to the same residue as aryls but with two substitution sites. An example of an arirene is phenylene, which refers to a divalent phenyl group. Arirene groups are optionally substituted with one, two, three, four, or five at any position, as is permissible with normal valences.

[0071] As used herein, the term “coupling moiety” refers to a chemical part that can be used to bond two molecules together. For example, a “coupling moiety” may refer to a phosphoramidite that reacts with an alcohol on another molecule to form an organophosphate. Further examples of coupling agents include, but are not limited to, esters, carbonates, carboxylic acids, olefins, alcohols, amines, aldehydes, ketones, alkynes, halogens, Grignard reagents, leaving groups, and other parts known in the art that are used to bond two molecules together.

[0072] As used herein, the term "halo" refers to a halogen group. For example, "halo" may refer to a fluoro(F), chloro(Cl), bromo(Br), or iodo(I) group.

[0073] As used herein, the term “heteroaryl” refers to a stable monocyclic or polycyclic ring with up to seven atoms in each ring, where at least one ring is aromatic and contains one to four heteroatoms selected from the group consisting of O, N, and S. Examples of heteroaryl groups, but not limited to, include acridinyl, carbazolyl, synnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, benzimidazolonyl, benzoxazolonyl, quinolinyl, isoquinolinyl, dihydroisoindonyl, imidazopyridinyl, isoindonyl, indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl, pyrazinyl, pyridadinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinoline. “Heteroaryl” is also understood to include N-oxide derivatives of any nitrogen-containing heteroaryl. When a heteroaryl substituent is bicyclic and one ring is non-aromatic or does not contain a heteroatom, the bond is understood to be mediated either through the aromatic ring or the heteroatom-containing ring. Heteroaryl groups are optionally substituted at any position, possibly i, di, tri, tetra, or pentasubstituted, as is permissible by their normal valence.

[0074] As used herein, the term “heteroarylene” refers to the divalent group of a heteroaryl group as described herein. Heteroarylenes are a subset of heteroaryls, referring to the same residues as heteroaryls but having two substitution sites. Examples of heteroaryls include pyridinylene, pyrimidinylene, and pyrrolylene. Heteroarylene groups are optionally mono-, dya-, tri-, qua-, or penta-substituted at any position, as is permissible with normal valences.

[0075] As used herein, the terms “heterocyclic,” “heterocyclic formula,” or “heterocyclyl” mean a 3- to 14-membered aromatic or non-aromatic heterocyclic ring containing one to four heteroatoms selected from the group consisting of O, N, and S, including a polycyclic group. As used herein, the term “heterocyclic formula” is also considered to be synonymous with the terms “heterocyclic” and “heterocyclyl,” and is understood to have the same definitions as set forth herein. “Heterocyclyl” includes the heteroaryl compounds and their dihydro and tetrahydro analogs.Examples of heterocyclyl groups, though not limited to them, include azetidinyl, benzimidazolyl, benzofuranil, benzoflazanil, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbonyl, cinnolinyl, furanil, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranil, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthopyridinyl, and oxadia. Zolyl, oxoxazolidinil, oxazolyl, oxazoline, oxopiperazinil, oxopyrrolidinil, oxomorpholinil, isoxazoline, oxetanil, pyranil, pyrazinil, pyrazolyl, pyridadinil, pyridopyridinil, pyridadinil, pyridyl, pyridinonil, pyrimidyl, pyrimidinoyl, pyrrolyl, quinazolinil, quinolyl, quinoxalinil, tetrahydropyranil, tetrahydrofuranil, tetrahydrothiopyranil, tetrahydroisoquinolinil, tetra Lazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, 1,4-dioxanil, hexahydroazepinyl, piperazinil, piperidinil, pyridine-2-onyl, pyrrolidinil, morpholinil, thiomorpholinil, dihydrobenzimidaryl, dihydrobenzofuranil, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranil, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl Examples include dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazolyl, dihydropyrizolyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dioxidethiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl, and tetrahydrothienyl and their N-oxides. Bonding of heterocyclyl substituents can occur via carbon atoms or heteroatoms. Heterocyclyl groups are optionally substituted in one, two, three, four, or five positions at any position, as permitted by their normal valence.

[0076] As used herein, the term "heterocycloalkyl" means a 3- to 14-membered non-aromatic heterocycle containing 1 to 4 heteroatoms selected from the group consisting of O, N, and S, including a polycyclic group. Examples of heterocyclyl groups, but not limited to, include azetidinyl, oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, oxetanyl, pyranyl, pyridinonyl, pyrimidinonyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrofuranyl, and di Examples include hydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydroxazolyl, dihydropyrazinel, dihydropyrazinel, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dioxidethiomorpholinyl, and tetrahydrothienyl and their N-oxides. The bonding of heterocycloalkyl substituents may occur via carbon atoms or via heteroatoms. Heterocyclyl groups are optionally substituted with one, two, three, four, or five atoms at any position, as permitted by their normal valence.

[0077] As used herein, the term “heterocycloalkylene” refers to the divalent group of a heterocycloalkyl group as described herein. Heterocycloalkylenes are a subset of heterocycloalkyls, referring to the same residues as heterocycloalkyls but having two substitution sites. Examples of heterocycloalkylenes include piperidinylene, azetidinylene, and tetrahydrofuranylene. Heterocycloalkylene groups are optionally substituted at any position, possibly i, d, 3, 4, or 5, as permitted by their normal valence.

[0078] As used herein, terms such as “to treat” and “treatment” mean methods or processes taken to reduce or alleviate the number, severity and / or frequency of one or more symptoms of a disease in a subject. As used herein, “to treat” and “treatment” may include prevention, control, prophylactic treatment and / or inhibition of the number, severity and / or frequency of one or more symptoms of a disease in a subject.

[0079] As used herein, the phrase "introduce into cells" means functionally delivering an RNAi agent to cells when referring to an RNAi agent. The phrase "functional delivery" means delivering an RNAi agent to cells in a manner that enables the RNAi agent to have its expected biological activity, such as sequence-specific inhibition of gene expression.

[0080] Unless otherwise specified, when used herein, the symbol [ka] The use of means that any one or more groups may be bonded to it in accordance with the scope of the invention as described herein. In some embodiments herein, symbols are used in the structure to describe the bonding sites of specific variants in the compound of formula I. [ka] This is used multiple times. Unless otherwise specified, the shown mutants may be oriented such that any of the binding sites on the mutant can bind to any of the binding sites on the compound of formula I. For example, mutant L 1 It has two bonding sites to the compound of formula I. 1 One embodiment of this is, [ka] Although it is shown as such, the embodiment also includes L 1 but [ka] It should be understood to refer to a compound that is.

[0081] As used herein, the term "isomer" refers to compounds that have the same molecular formula but differ in the nature or order of the bonds of their atoms or in the arrangement of those atoms in space. Isomers that differ in the spatial arrangement of their atoms 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 for which the isomeric structure is not specifically defined, it is intended that the compounds can contain both the E and Z geometric isomers individually or as a mixture. The compounds of formula I or their pharmaceutically acceptable salts are intended to include, for example, all possible isomers, as well as their racemic and optically pure forms. Similarly, all tautomers are intended to be included unless otherwise explicitly stated.

[0082] As used herein, a linking group is one or more atoms that link one molecule or part of a molecule to another second molecule or second part of a molecule. In the art, the terms linking group and spacer are sometimes used interchangeably. Similarly, as used in the art, the term scaffold is sometimes used interchangeably with linking group. In some embodiments, the linking group can include a peptide-cleavable linking group. In some embodiments, the linking group can include or consist of peptidylphenylalanine-citrulline-phenylalanine-proline. In some embodiments, the linking group can include or consist of a PEG group.

[0083] As used herein, the term "bonded," when referring to a bond between two molecules, means that the two molecules are bonded by a covalent bond or that the two molecules are bonded via a non-covalent bond (e.g., a hydrogen bond or an ionic bond). In some examples where the term "bonded" refers to a bond between two molecules via a non-covalent bond, the bond between the two different molecules is in a physiologically acceptable buffer (e.g., phosphate buffered saline) at 1×10 -4 M or less (e.g., 1×10 -5 M or less, 1×10 -6 M or less or 1×10 -7 M or less) of K D . Unless otherwise specified, as used herein, the term "bonded" can refer to a bond between a first compound and a second compound, with or without intervening atoms or groups of atoms.

[0084] One of ordinary skill in the art will readily understand and recognize that the compounds and compositions disclosed herein can 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 placed. Thus, as used herein, the structures disclosed herein are envisioned such that certain functional groups such as OH, SH or NH can be protonated or deprotonated. The disclosure herein is intended to encompass the disclosed compounds and compositions regardless of the state of protonation based on the pH of the environment, as will be readily understood by one of ordinary skill in the art.

[0085] Structures can be depicted as having "floating" bonds on the ring structure to show bonds to carbon or heteroatoms on the ring as permitted by valence. For example, the structure

Chemical Formula

[0086] When used in the claims of this specification, the phrase "consists of" excludes elements, processes, or components not specified in the claims. When used in the claims of this specification, the phrase "essentially consists of" limits the scope of the claims to those that do not substantially affect the specific materials or processes and the fundamental and novel characteristics of the claimed invention.

[0087] When used herein, “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 pharmaceutically active ingredient (a therapeutic product such as an API or RNAi agent) that has been appropriately evaluated for safety and is intentionally included in a drug delivery system. Excipients do not exert, or are not intended to exert, a therapeutic effect at the intended dose. Excipients 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 the overall safety, efficacy or other attributes of delivery of the API during storage or use.

[0088] Excipients include, but are not limited to, absorption enhancers, antifungals, defoamers, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, lubricants, humectants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, isotonic agents, vehicles, water repellents, and wetting agents. Pharmaceutically acceptable excipients may or may not be inert substances.

[0089] A pharmaceutical composition may include other additional components commonly found in pharmaceutical compositions. Examples of pharmaceutically active materials include, but are not limited to, antipruritic agents, astringents, topical anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramines, etc.). It is also conceivable that cells, tissues, or isolated organs expressing or containing RNAi agents as defined herein may be used as a “pharmaceutical composition.” Where used herein, “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” refers to the amount of RNAi agent that produces the intended pharmacological, therapeutic, or prophylactic effect.

[0090] The term polynucleotide or polynucleic acid refers to a polymer containing at least two nucleotides. A nucleotide is the monomer unit of a polynucleotide polymer. Polynucleotides with fewer than 120 monomer units are often called oligonucleotides. Natural nucleic acids have a deoxyribose or ribose phosphate backbone. Non-natural or synthetic polynucleotides are polynucleotides that are polymerized in vitro or in a cell-free system and contain the same or similar bases, but can contain types of backbones other than the natural ribose or deoxyribose-phosphate backbone. Synthetic oligonucleotides can be synthesized using any technique known in the art. Polynucleotide backbones known in the art include PNA (peptide nucleic acid), phosphorothioates, phosphorodiamidates, morpholino, and other variants of the phosphate backbone of natural nucleic acids. Bases include purines and pyrimidines, as well as 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 novel reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides on the nucleotide. The term "base" encompasses any known base analogues of DNA and RNA. The term "polynucleotide" includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as combinations of DNA, RNA, and other natural and synthetic nucleotides.

[0091] The synthetic oligonucleotides of the present invention can be chemically modified. Chemical modification of polynucleotides can improve various properties of the polynucleotides, including, but not limited to, resistance to nuclease degradation in vivo, intracellular uptake, activity, and sequence-specific hybrid formation. Non-limiting examples of such chemical modifications include phosphorothioate nucleotide interlinking, 2'-O-methylribonucleotide, 2'-deoxy-2'-fluororibonucleotide, 2'-deoxyribonucleotide, "universal base" nucleotide, 5-C-methylnucleotide, and 2',3'-seconucleotide mimetic (unlocked nucleic acid base analogue, referred to herein as N). UNA Examples include the incorporation of (or represented as NUNA) and reverse deoxydebase residues. These chemical modifications have been shown to dramatically improve the serum stability of these compounds while maintaining intracellular polynucleotide activity when used in various polynucleotide structures.

[0092] In some embodiments, the synthetic oligonucleotides of the present invention comprise a double helix having two chains, one or both of which may be chemically modified, with each chain having about 19 to about 29 nucleotides (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29). In some embodiments, the synthetic oligonucleotides of the present invention comprise one or more modified nucleotides. The synthetic oligonucleotides of the present invention may contain modified nucleotides at about 5 to about 100% of the nucleotide positions (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%).

[0093] Synthetic oligonucleotides may contain 5' or 3' terminal modifications. Examples of 3' and 5' terminal modifications, though not limited to them, include amine-containing groups, alkyl groups, alkylamine groups, reactive groups, TEG groups, and PEG groups.

[0094] An "RNAi agent" (also called an "RNAi trigger") means a composition comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of reducing or inhibiting (e.g., reducing or inhibiting under appropriate conditions) the sequence-specific translation of a messenger RNA (mRNA) transcript of a target mRNA. When used herein, an RNAi agent may act by an RNA interference mechanism (i.e., by inducing RNA interference through interaction with the RNA interference pathway mechanism (RNA-induced silencing complex or RISC) in mammalian cells) or by any alternative mechanism or pathway. While an RNAi agent is considered to act primarily by an RNA interference mechanism when the term is used herein, the disclosed RNAi agents are not constrained or limited to a specific pathway or mechanism of action. The RNAi agents disclosed herein include, but are not limited to, sense and antisense strands, 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 agent described herein is at least partially complementary to the targeted mRNA (i.e., HIF-2 alpha mRNA). The RNAi agent may contain one or more modified nucleotides and / or one or more non-phosphodiester bonds.

[0095] When used herein, the terms “silence,” “reduction,” “inhibition,” “downregulate,” or “knockdown” refer to the expression of a given gene, and mean that the expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptides, proteins, or protein subunits translated from mRNA, is reduced in the cells, cell populations, tissues, organs, or subjects on which the gene is transcribed, compared to a second set of cells, cell populations, tissues, organs, or subjects that have not been treated in the same manner, when the cells, cell populations, tissues, organs, or subjects have been treated with the RNAi agents described herein.

[0096] In some embodiments, the RNAi agent comprises at least two sequences that are partially, substantially, or completely complementary to each other. 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 each comprise two sense strands containing a first sequence and an antisense strand containing a second sequence, and the sense and antisense strands together form a meloduplex. The sense strands may be bound to the antisense strand via binding molecules such as polynucleotide linkers or non-nucleotide linkers.

[0097] The antisense strand contains a nucleotide sequence complementary to a portion of the mRNA encoded by the target gene, the complementary region most preferably being less than 30 nucleotides in length. The RNAi agent sense strand contains a sequence having at least 85% identity with at least a portion of the target mRNA. When delivered to cells expressing the target gene, the RNAi agent inhibits the expression of the target gene in vitro or in vivo.

[0098] In some embodiments, the RNAi agent may contain naturally occurring nucleotides or may contain at least one modified nucleotide or nucleotide mimetic. The sense and antisense strands of the RNAi agent of the present invention may be synthesized and / or modified by methods well established in the art. The nucleosides or nucleotide bases of the RNAi agent may be linked by phosphate-containing (natural) or non-phosphate-containing (non-natural) covalent nucleoside bonds; that is, the RNAi agent may have a natural or non-natural oligonucleotide backbone. In some embodiments, the RNAi agent includes non-standard (non-phosphate) bonds between nucleotide bases.

[0099] In some embodiments, the RNAi agent may include 5' or 3' terminal modifications. Examples of 3' and 5' terminal modifications include, but are not limited to, amine-containing groups, alkyl groups, alkylamine groups, reactive groups, TEG groups, and PEG groups.

[0100] In some embodiments, the RNAi agent may include overhangs, i.e., typically unpaired overhang nucleotides that do not directly participate in the double helix structure normally formed by the core sequences of the sense and antisense strands.

[0101] In some embodiments, the RNAi agent may independently contain 1 to 5 nucleotide 3' and / or 5' overhangs in both 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' overhang nucleotides of one strand are paired with one or more 5' overhang nucleotides of the other strand. In some embodiments, one or more 3' overhang nucleotides of one strand are not paired with one or more 5' overhang nucleotides of the other strand. The sense and antisense strands of the RNAi agent may or may not contain the same number of nucleotide bases. The antisense and sense strands may form a double helix with only the 5' end blunt, only the 3' end blunt, both the 5' and 3' ends blunt, or neither the 5' nor the 3' ends blunt. In some embodiments, one or more nucleotides within the overhang include a thiophosphate, a phosphorothioate, a deoxynucleotide inverted (3'-3' bond) nucleotide, or a modified ribonucleotide or deoxynucleotide.

[0102] Lists of known mRNA sequences can be found in databases maintained by various research institutions, including the database GenBank, which is managed by the National Center for Biotechnology Information, a branch of the National Institutes of Health, as part of the International Nucleotide Sequence Database Collaboration. Known effective siRNA sequences and homologous binding sites are also well represented in the relevant literature. RNAi agent molecules can be readily designed and produced by techniques known in the art. Furthermore, there are computational tools (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) that can increase the chances of finding effective and specific sequence motifs.

[0103] Formula I Formula I has the structure:

[0104] [Chemical formula]

[0105] (In the above formula, L 1 , L 2 and L 3 are each independently a linker containing optionally substituted alkylene, Q is a tetravalent carbon atom, tetrasubstituted phenyl or optionally substituted alkylene, R contains a coupling moiety or an RNAi agent, and, X is NR x or a bond, and R x is H or optionally substituted C1-C6 alkyl).

[0106] In some embodiments of Formula I, Q is a tetravalent carbon. In other embodiments of Formula I, Q is [Chemical formula] And, [ka] indicates a bonding point. In other embodiments, Q is [ka] And, [ka] The symbol indicates a connection point.

[0107] In some embodiments of formula I, L 1 , L 2 and L 3 Each [ka] In some embodiments of formula I, L 1 , L 2 and L 3 Each [ka] In some embodiments of formula I, L 1 L 2 and L 3 Each [ka] That is the case.

[0108] In some embodiments of formula I, X is NH.

[0109] In some embodiments of Formula I, R comprises a phosphoramidite. In some embodiments of Formula I, R comprises an organophosphate and an RNAi agent. In other embodiments of Formula I, R comprises an ester. In some embodiments of Formula I, R comprises a para-nitrophenol ester. In some embodiments of Formula I, R comprises an amide and an RNAi agent. In other embodiments of Formula I, R comprises a carbonate. In some embodiments, R comprises a carbamate and an RNAi agent.

[0110] In some embodiments of formula I, R is selected from the group consisting of the following: [ka]

[0111] Examples of compounds representing Formula I are shown in Table 1 below.

[0112] [Table 1-1]

[0113] [Table 1-2]

[0114] [Table 1-3]

[0115] [Table 1-4]

[0116] [Table 1-5]

[0117] [Table 1-6]

[0118] Formula II Equation II is the structure:

[0119] [ka]

[0120] (In the above formula, L 1 , L 2 and L 3 These are linkers, each independently and optionally containing substituted alkylenes. L 4 This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, and optionally a substituted cycloalkylene. R in each case 1 This is an alkyl group which may be substituted in some cases. R 2 is an alkyl which may be substituted in some cases, and R 4 A is represented by (where is H or optionally a substituted alkyl group), or a pharmaceutically acceptable salt thereof.

[0121] In some embodiments of formula II, L 1 , L 2 and L 3 Each [ka] In some embodiments of formula II, L 1 , L 2 and L 3 Each [ka] In some embodiments of formula II, L 1 , L 2 and L 3 Each [ka] That is the case.

[0122] In some embodiments of Equation II, R in each case 1 It is isopropyl.

[0123] In some embodiments of formula II, R 2 teeth [ka] That is the case.

[0124] In some embodiments of formula II, L 4 teeth: [ka] Selected from the group consisting of, [ka] The symbol indicates a connection point.

[0125] Formula III Equation III is the structure:

[0126] [ka]

[0127] (In the above formula, L 1 , L 2 and L 3 These are linkers, each independently and optionally containing substituted alkylenes. L 4 This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, and optionally a substituted cycloalkylene. R 4 is H or optionally a substituted alkyl, X is either O or S, and RNA is represented by (containing or consisting of) an RNAi agent, or a pharmaceutically acceptable salt thereof.

[0128] In some embodiments of formula III, X is O, and the compound of formula III is an organophosphate. In some embodiments of formula II, X is S, and the compound of formula III is a phosphorothioate.

[0129] In some embodiments of formula III, L 1 , L 2 and L 3 Each [ka] In some embodiments of Equation III, L 1 , L 2 and L 3 Each [ka] In some embodiments of Equation III, L 1 , L 2 and L 3 Each [ka] That is the case.

[0130] In some embodiments of formula III, L 4 teeth: [ka] Selected by a group consisting of, [ka] The symbol indicates a connection point.

[0131] Examples of compounds representing Equation III are shown in Table 2 below.

[0132] [Table 2-1]

[0133] [Table 2-2]

[0134] [Table 2-3]

[0135] [Table 2-4]

[0136] [Table 2-5]

[0137] [Table 2-6]

[0138] [Table 2-7]

[0139] Formula IV Equation IV is the structure:

[0140] [ka]

[0141] (In the above formula, L 1 , L 2 and L 3 These are linkers, each independently and optionally containing substituted alkylenes. L 4 This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, and optionally a substituted cycloalkylene. R 1 and R 2Each of these is an alkyl group that is independently and optionally substituted. R 4 is H or optionally a substituted alkyl, and TL is represented by the target ligand, or a pharmaceutically acceptable salt thereof.

[0142] In some embodiments of formula IV, L 1 , L 2 and L 3 Each [ka] In some embodiments of formula IV, L 1 , L 2 and L 3 Each [ka] In some embodiments of formula IV, L 1 L 2 and L 3 Each [ka] That is the case.

[0143] In some embodiments of formula IV, L 4 teeth: [ka] Selected from the group consisting of, [ka] The symbol indicates a connection point.

[0144] In some embodiments of formula IV, R in each case 1 It is isopropyl.

[0145] In some embodiments of formula IV, R 2 teeth [ka] That is the case.

[0146] The example compounds of formula IV are shown in Table 3 below.

[0147] [Table 3-1]

[0148] [Table 3-2]

[0149] [Table 3-3]

[0150] [Table 3-4]

[0151] Formula V Formula V is structure:

[0152] [ka]

[0153] (In the above formula, L 1 , L 2 and L 3 These are linkers, each independently and optionally containing substituted alkylenes. L 4 This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, or optionally a substituted cycloalkylene. R 4 is H or optionally a substituted alkyl, TL is the target ligand, Y is either O or S, and RNA is represented by (containing or consisting of) an RNAi agent, or a pharmaceutically acceptable salt thereof.

[0154] In some embodiments of formula V, L 1 , L 2 and L 3 Each

[0155] [ka] In some embodiments of formula IV, L 1 , L 2 and L 3 Each [ka] In some embodiments of formula V, L 1 L 2 and L 3 Each [ka] That is the case.

[0156] In some embodiments of formula V, L 4 teeth: [ka] Selected from the group consisting of, [ka] The symbol indicates a connection point.

[0157] Examples of compounds representing formula V are shown in Table 4 below.

[0158] [Table 4-1]

[0159] [Table 4-2]

[0160] [Table 4-3]

[0161] [Table 4-4]

[0162] [Table 4-5]

[0163] [Table 4-6]

[0164] [Table 4-7]

[0165] [Table 4-8]

[0166] [Table 4-9]

[0167] Equation VI Equation VI is structure

[0168] [ka]

[0169] (In the above formula, L 1 , L 2 and L 3 These are linkers, each independently and optionally containing substituted alkylenes. L 4This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, or optionally a substituted cycloalkylene. R 3 is H, optionally a substituted alkyl, or optionally a substituted aryl, and R 4 A is represented by (where is H or optionally a substituted alkyl group), or a pharmaceutically acceptable salt thereof.

[0170] In some embodiments of formula VI, L 1 , L 2 and L 3 Each

[0171] [ka] In some embodiments of formula VI, L 1 , L 2 and L 3 Each [ka] In some embodiments of formula VI, L 1 L 2 and L 3 Each [ka] That is the case.

[0172] In some embodiments of formula VI, L 4 teeth: [ka] Selected from the group consisting of, [ka] The symbol indicates a connection point.

[0173] In some embodiments of formula VI, R 3R is an aryl that may be substituted depending on the case. In some embodiments of formula VI, R 3 It is para-nitrophenyl.

[0174] In some embodiments of formula VI, R 4 H is H.

[0175] Formula VII Formula VII is structure:

[0176] [ka]

[0177] (In the above formula, L 1 , L 2 and L 3 These are linkers, each independently and optionally containing substituted alkylenes. L 4 This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, or optionally a substituted cycloalkylene. R in each case 4 is H or optionally a substituted alkyl, and RNA is represented by (containing or consisting of) an RNAi agent, or a pharmaceutically acceptable salt thereof.

[0178] In some embodiments of formula VII, L 1 , L 2 and L 3 Each

[0179] [ka] In some embodiments of formula VII, L 1 , L 2 and L 3 Each [ka] In some embodiments of formula VII, L1 L 2 and L 3 Each [ka] That is the case.

[0180] In some embodiments of formula VII, L 4 teeth: [ka] Selected from the group consisting of, [ka] The symbol indicates a connection point.

[0181] The example compounds for formula VII are shown in Table 5 below.

[0182] [Table 5-1]

[0183] [Table 5-2]

[0184] [Table 5-3]

[0185] Formula VIII Formula VIII is structure:

[0186] [ka]

[0187] (In the above formula, L 1 , L 2 and L 3 These are linkers, each independently and optionally containing substituted alkylenes. L4 This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, or optionally a substituted cycloalkylene. R 3 is H, optionally substituted alkyl, and optionally substituted aryl. R 4 is H or optionally a substituted alkyl, and TL is represented by the target ligand, or a pharmaceutically acceptable salt thereof.

[0188] In some embodiments of formula VIII, L 1 , L 2 and L 3 Each [ka] In some embodiments of formula VIII, L 1 , L 2 and L 3 Each [ka] In some embodiments of formula VIII, L 1 L 2 and L 3 Each [ka] That is the case.

[0189] In some embodiments of formula VIII, L 4 teeth: [ka] Selected from the group consisting of, [ka] The symbol indicates a connection point.

[0190] In some embodiments of formula VIII, R3 R is a substituted aryl in some cases. In some embodiments of formula VIII, R 3 It is para-nitrophenyl.

[0191] Examples of compounds representing formula VIII are shown in Table 6 below.

[0192] [Table 6-1]

[0193] [Table 6-2]

[0194] [Table 6-3]

[0195] Formula IX Formula IX is structure:

[0196] [ka]

[0197] (In the above formula, L 1 , L 2 and L 3 These are linkers, each independently and optionally containing substituted alkylenes. L 4 This is a linker, which optionally comprises a substituted alkylene, optionally a substituted arylene, or optionally a substituted cycloalkylene. TL is the target ligand, R in each case 4 is H or optionally a substituted alkyl, and RNA is represented by (containing or consisting of) an RNAi agent, or a pharmaceutically acceptable salt thereof.

[0198] In some embodiments of formula IX, L 1 , L 2 and L 3 Each [ka] In some embodiments of formula IX, L 1 , L 2 and L 3 Each [ka] In some embodiments of formula IX, L 1 L 2 and L 3 Each [ka] That is the case.

[0199] In some embodiments of formula IX, L 4 teeth: [ka] Selected from the group consisting of, [ka] The symbol indicates a connection point.

[0200] Examples of compounds representing formula IX are shown in Table 7 below.

[0201] [Table 7-1]

[0202] [Table 7-2] Here, TL comprises a target ligand, and RNA comprises or consists of an RNAi agent.

[0203] L 1 , L 2, L 3 In some embodiments of formulas I to IX, L in each case 1 , L 2 or L 3 L is a linker containing alkylenes, which may be substituted in some cases. 1 , L 2 or L 3 This may include any suitable linker known in the art. In some embodiments, L 1 , L 2 or L 3 It contains a chain with a length of 1 to 50 atoms. 1 , L 2 or L 3 The length of the chain indicates the number of direct atoms between the alkyne and the quaternary carbon, but there may be additional atoms branching off from the atoms in the chain. In some embodiments, L 1 , L 2 or L 3 The lengths are 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 It can be 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.

[0204] In some embodiments, L in all cases 1 , L 2 and L 3 They are identical. In other embodiments, each L 1 , L 2 and L 3 These are different parts.

[0205] In some embodiments, L 1 , L 2, L 3 or L 4 It can contain amides.

[0206] In some embodiments, L 1 , L 2 , L 3 or L 4 It may contain polyethylene glycol (PEG) chains.

[0207] In some embodiments, L 1 , L 2 , L 3 or L 4In the case of alkylenes substituted by the above, the alkylenes are amides, ethers, esters, thioethers, thions, ketones, amines, sulfones, sulfonamides, or chains of atoms, for example, but not limited to substituted or unsubstituted alkenyls, arylalkyls, arylalkenyls, heteroarylalkyls, heteroarylalkenyls, heteroarylalkynyls, heterocyclylalkyls, heterocyclylalkenyls, heterocyclylalkynyls, aryls, heteroaryls, heterocyclyl, cycloalkyls, cycloalkenyls, alkylarylalkyls, alkylarylalkenyls, alkylarylalkynyls, alkenylarylalkyls, alkenylarylalkenyls, alkenylarylalkynyls, alkenylarylalkynyls, alkynylarylalkyls, alkynylarylalkenyls, alkynylarylalkynyls, alkylheteroaryl It may be interrupted by alkyl, alkyl heteroaryl alkenyl, alkyl heteroaryl alkynyl, alkenyl heteroaryl alkyl, alkenyl heteroaryl alkenyl, alkenyl heteroaryl alkynyl, alkynyl heteroaryl alkyl, alkynyl heteroaryl alkenyl, alkynyl heteroaryl alkynyl, alkyl heterocyclyl alkyl, alkyl heterocyclyl alkenyl, alkyl heterocyclyl alkynyl, alkenyl heterocyclyl alkyl, alkenyl heterocyclyl alkenyl, alkenyl heterocyclyl alkynyl, alkynyl heterocyclyl alkyl, alkynyl heterocyclyl alkenyl, alkynyl heterocyclyl alkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkyl heteroaryl, alkenyl heteroaryl, or alkynyl heteroaryl.

[0208] In some embodiments, L 1 , L 2 and L 3 Each is independent of the others. [ka] It can be selected from the group consisting of the following:

[0209] L 4 In embodiments of formulas I to IX, L 4 L is a linker containing alkylenes, which may be substituted in some cases. 4 L may include any suitable coupling portion known in the art. In some embodiments, L 4 It contains a chain with a length of 1 to 50 atoms. 2 The length of the chain indicates the number of direct atoms between the alkyne and the quaternary carbon, but there may be additional atoms branching off from the atoms in the chain. In some embodiments, L 2 The lengths are 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 It can be 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.

[0210] In some embodiments, L 4 teeth: [ka] Selected from the group consisting of, [ka] The symbol indicates a connection point.

[0211] R In embodiments of formulas I to IX, R comprises a coupling moiety or an RNAi agent. In some embodiments, R comprises a coupling moiety, and the coupling moiety is a phosphoramidite. In other embodiments, R comprises a coupling moiety, and the coupling moiety is an ester. In other embodiments, R comprises a coupling moiety, and the coupling moiety is a carbonate.

[0212] In some embodiments, R comprises an RNAi agent. When R comprises an RNAi agent, R may contain additional atoms that do not form part of the RNAi sequence. For example, in some embodiments, R [ka] It can be, where RNA includes RNAi agents, where [ka] The symbol indicates a binding site. In some embodiments, the RNAi agent is bound to a compound of formulas I-IX at the 5' end of the sense strand.

[0213] In some embodiments, R is [ka] It is selected from the group consisting of the following.

[0214] Pharmaceutical composition In some embodiments, the present disclosure provides pharmaceutical compositions comprising therapeutic compounds containing one or more trialquine binders disclosed herein.

[0215] When used herein, “pharmaceutical composition” comprises a pharmacologically effective amount of an active pharmaceutical ingredient (API) and, optionally, one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance other than the active pharmaceutical ingredient (API, therapeutic product) that is intentionally included in the drug delivery system. Excipients do not exert, or are not intended to exert, a therapeutic effect at the intended dose. Excipients may act to a) assist in the processing of the drug delivery system during manufacturing, b) protect, support or improve the stability, bioavailability or patient acceptability of the API, c) assist in product identification, and / or d) improve the overall safety, efficacy, or other attributes of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0216] Excipients include, but are not limited to, absorption enhancers, anti-adhesion agents, defoamers, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, lubricants, humectants, oils, polymers, preservatives, physiological saline, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, isotonic agents, vehicles, water repellents, and wetting agents.

[0217] The pharmaceutical compositions described herein may include other additional components commonly found in pharmaceutical compositions. In some embodiments, the additional components are pharmaceutically active materials. Examples of pharmaceutically active materials include, but are not limited to, antipruritics, astringents, topical anesthetics or anti-inflammatory drugs (e.g., antihistamines, diphenhydramines, etc.), small molecule drugs, antibodies, antibody fragments, aptamers, and / or vaccines.

[0218] The pharmaceutical composition may also contain preservatives, solubilizers, stabilizers, humectants, emulsifiers, sweeteners, colorants, odorants, salts for osmotic pressure fluctuations, buffers, coatings, or antioxidants. They may also contain other agents with known therapeutic effects.

[0219] The pharmaceutical composition can be administered in several ways, depending on whether topical or systemic treatment is preferred and the area to be treated. Administration can be carried out by any method commonly known in the art, and may include, for example, topical (e.g., by a transdermal patch), transpulmonary (e.g., by inhalation or blowing of powder or aerosol, including by nebulizer, intratracheal, intranasal), epidermal, transdermal, oral, or parenteral administration. Parenteral administration may include, for example, intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, subcutaneous (e.g., via an implanted device), intracranial, intraparenchymal, subarachnoid, 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, sugar-coated tablets, hard or soft gelatin capsules, solutions, emulsions, or suspensions. The drug may also be administered rectally, for example, using suppositories; topically or transdermally, for example, using ointments, creams, gels, or solutions; or parenterally, for example, using injectable solutions.

[0220] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline. They should be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. Carriers can be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to include isotonic agents, such as sugars, polyhydric alcohols, such as mannitol, sorbitol, and sodium chloride, in the composition. Sustained absorption of an injectable composition can be achieved by including absorption-delaying agents in the composition, such as aluminum monostearate and gelatin.

[0221] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent, along with one or a combination of the components listed above, and sterilizing by filtration as necessary. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preparation methods include vacuum drying and freeze-drying, which yield powders of the active ingredient and any additional desired components from a pre-sterile filtered solution.

[0222] Formulations suitable for intra-articular administration may be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension, or in the form of a sterile aqueous preparation of any ligand described herein. Liposome formulations or biodegradable polymer systems may also be used to present any ligand described herein for both intra-articular and intraocular administration.

[0223] The active compound can be prepared with a carrier that protects the compound from rapid elimination from the body, such as in controlled-release formulations including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0224] A pharmaceutical composition may contain other additional components commonly found in pharmaceutical compositions. Such additional components may include, but are not limited to, antipruritics, astringents, topical anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramines, etc.). As used herein, “pharmacologically effective amount,” “therapeutic effective amount,” or simply “effective amount” refers to the amount of a pharmaceutically active agent that produces a pharmacological, therapeutic, or prophylactic effect.

[0225] Pharmaceuticals containing trialquine binders are also subject to the present invention, as are methods for producing such pharmaceuticals, the methods comprising converting one or more compounds containing trialquine binders and, optionally, one or more other substances having known therapeutic effects, into a pharmaceutically acceptable form.

[0226] The trialquine binders and pharmaceutical compositions containing the trialquine binders disclosed herein may be packaged or contained in kits, containers, packs, or dispensers. The trialquine binders and pharmaceutical compositions containing the trialquine binders may be packaged in pre-filled syringes or vials.

[0227] Target ligands, pharmacokinetic (PK) modulators, and delivery vehicles In some embodiments, the Trialquine conjugate is conjugated to one or more non-nucleotide groups, which may include, but are not limited to, target ligands, pharmacokinetic (PK) modulators, delivery polymers, or delivery vehicles. The non-nucleotide groups can enhance the targeting, delivery, or binding of the cargo molecule. The non-nucleotide groups may be covalently bound to the RNAi agent at the 3' or 5' end of the sense strand. In some embodiments, the non-nucleotide groups are bound to the 5' end of the RNAi agent sense strand. In some embodiments, the Trialquine linker of formula I is bound to the RNAi agent via an unstable, cleavable, or reversible bond or linker.

[0228] In some embodiments, non-nucleotide groups enhance the pharmacokinetic or biodistribution properties of the RNAi agent or conjugate to which they are bound, improving cell- or tissue-specific distribution and cell-specific uptake of the conjugate. In some embodiments, non-nucleotide groups enhance endocytosis of the RNAi agent.

[0229] The target ligand or target moiety enhances the pharmacokinetic or biodistribution characteristics of the cargo molecule to which they are bound, thereby improving the cell-specific (and in some cases, organ-specific) distribution and cell-specific (or organ-specific) uptake of the RNAi agent. In some embodiments, the target ligand may be a target compound and a PK enhancer or modulator. In some embodiments, the target ligand is directed to a cell receptor.

[0230] Conjugation to target ligand In some embodiments, the trial quinlinker of formula I can be conjugated to an RNAi agent by a coupling agent. An exemplary scheme for conjugating the trial quinlinker of formula I to an RNAi molecule is shown in the following reaction scheme: [ka] (In the above formula, L 1 , L 2 , L 3 Q and X are all as described in Formula I, R contains the coupling portion, RG is the reactive group, and L 4 (This is a linker containing optionally substituted alkylenes, optionally substituted arylenes, or optionally substituted cycloalkylenes.)

[0231] In some embodiments, the target ligand (TL) may be conjugated to the trialukine moiety before conjugation to the RNAi molecule. An example of this reaction is shown in the scheme below. [ka] (In the above formula, L 1 , L 2 , L 3 Q and X are all as described in Equation I, R includes the coupling part, and L 4 (This is a linker containing optionally substituted alkylenes, optionally substituted arylenes, or optionally substituted cycloalkylenes.)

[0232] RNAi molecules can be synthesized having a reactive group such as an amino group (also referred to herein as an amine). In some embodiments, the reactive group may be attached to the 5′-terminus and / or 3′-terminus of the RNAi agent. In some embodiments, the RNAi agent may be double-stranded. In embodiments where the RNAi agent is double-stranded, the reactive group may be the sense strand or antisense strand of the RNAi agent.

[0233] For example, in some embodiments, the 5' end of the sense strand of the RNAi agent is NH2-C6H 12 RNAi agents having a (hexyleneamine) group are synthesized. Subsequently, the terminal amino group can be reacted to form a conjugate with, for example, the coupling moiety of the compound of formula I. In some embodiments, the coupling moiety is an ester, the reactive group on the RNAi agent is a primary amine, and an amide bond is formed between the RNAi agent and the trialquine linker. An example of this reaction is shown in the following scheme using the compound of formula VI. [ka] (In the above formula, L 1 , L 2 , L 3 , L 4 , R 3 , R 4 (And RNA is as described in formulas VI and VII).

[0234] In other embodiments, the RNAi agent is synthesized having a terminal -CH2OH group. In some embodiments, the coupling agent in R of formula I comprises a phosphoramidite. The RNAi agent containing the terminal alcohol can be reacted with the trialquine of formula II to produce a phosphate as shown in the reaction scheme below. [ka]

[0235] In some embodiments, the target ligand (TL) may be conjugated to a trialakine binder as described herein, after the trialakine binder has been conjugated to the RNAi agent. An example of this reaction is shown in the scheme below. [ka] [Examples]

[0236] example Example 1. Synthesis of compound 1(2-cyanoethyl ((1r,4r)-4-((1,7-dioxo-4-(3-oxo-3-(prop-2-in-1-ylamino)propyl)-1,7-bis(prop-2-in-1-ylamino)heptan-4-yl)carbamoyl)cyclohexyl)diisopropylphosphoramidite) [ka] To a solution of compound 1 (12.00 g, 25.6 mmol) and DIPEA (12.22 g, 16.47 mL, 94.6 mmol) in DMF (50 mL), TBTU (28.72 g, 89.5 mmol) was added at 0°C. The internal temperature rose from 0°C to 16°C. While maintaining an internal temperature below 20°C, propargylamine (4.93 g, 5.73 mL, 89.5 mmol) was added dropwise. 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 cloudy, and was stirred at room temperature. After 1.5 hours, the precipitate was collected by filtration, rinsed with DCM (100 mL), and dried. Yield of compound 2: 10.4 g (70%). 34 H 36 The calculated [M+H] for N4O5 was 581.70, while the measured value was 581.79. [ka]

[0237] To a solution of compound 2 (12.17 g, 21.0 mmol) in DMF (60 mL), triethylamine (10.6 g, 14.7 mL, 105 mmol) was added at room temperature. The reaction mixture was stirred overnight. The reaction mixture was then concentrated and purified using CombiFlash® with silica gel as the stationary phase, and eluted under a gradient of MeOH (0-13%) in DCM containing 1% triethylamine. Yield of compound 3: 6.08 g (81%). 19 H26 The calculated [M+H] for N4O3 was 359.45, while the measured value was 359.35. [ka]

[0238] Compound 4 (2.55 g, 17.69 mmol) in pyridine (26 mL) was treated with acetic anhydride (12.8 mL, 135 mmol) and stirred at room temperature for 4 hours. Upon completion, all volatiles were removed, and compound 5 was isolated by elution with a gradient solution of ethyl acetate in hexane containing 1% acetic acid and separation on silica. Yield: 2.56 g (78%). 1 H NMR (400 MHz, DMSO-d6): δ12.11 (s, br, 1H) 4.56 (m, 1H), 2.21 (m, 1H), 1.97 (s, 3H), 1.90 (m, 4H), 1.38 (m, 4H). [ka]

[0239] To a solution of compound 5 (400 mg, 2.15 mmol) in DCM (5 mL), DMF (16 mg, 17 μL, 0.215 mmol) and oxalyl chloride (1.36 g, 922 μL, 10.74 mmol) were added at 0°C. After 30 minutes, 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. [ka]

[0240] Pyridine (1.88 g, 1.92 mL, 23.7 mmol) was added to a solution of compound 3 (1000 mg, 2.79 mmol) in DCM (10 mL). The reaction mixture was cooled to 0°C, and a solution of compound 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. The reaction was quenched by adding water (10 mL). The mixture was diluted with DCM (30 mL) and washed with saturated aqueous solution NH4Cl (20 mL) and brine (10 mL). The organic phase was dried over Na2SO4, filtered, and concentrated. The residue was purified using CombiFlash® with silica gel as the stationary phase, and eluted using a gradient solution (0-7%) of MeOH in DCM. Yield of compound 7: 630 mg (43%). 28 H 38 The calculated [M+H] for N4O6 was 527.64, while the measured value was 527.69. [ka]

[0241] To a solution of compound 7 (288 mg, 0.55 mmol) in THF (1.75 mL), a 1 M NaOH solution (2.73 mL, 2.73 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 1.5 hours, then heated to 35°C for a further 30 minutes. Upon consumption of the starting material, the reaction mixture was acidified to pH=5 using 2 M HCl and concentrated. The residue was co-evaporated with ACN (20 mL). After drying, the residue was purified using CombiFlash® with silica gel as the stationary phase and eluted using a gradient solution (0-10%) in MeOH DCM. Yield of compound 8: 216 mg (81%). 26 H 36 The calculated [M+H] for N4O5 was 485.61, while the measured value was 485.56. [ka]

[0242] Compound 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′-tetraisopropylphosphodiamidite (199 mg, 0.66 mmol). The reaction mixture was stirred at 0°C for 30 minutes. Upon consumption of the starting material, triethylamine (44 mg, 61 μL, 0.44 mmol) was added, and the reaction mixture was concentrated into oil. The oil was purified using CombiFlash® with silica gel as the stationary phase and eluted with a gradient solution (50–100%) in DCM of siRNA containing 1% triethylamine. Yield of Compound 9 (Compound 1): 174 mg (58%). 35 H 53 The calculated [M+H] for N6O6P was 685.83, while the measured value was 685.94.

[0243] Example 2. Synthesis of Compound 2 (2-cyanoethyl ((1s,4s)-4-((1,7-dioxo-4-(3-oxo-3-(prop-2-in-1-ylamino)propyl)-1,7-bis(prop-2-in-1-ylamino)heptan-4-yl)carbamoyl)cyclohexyl)diisopropylphosphoramidite) [ka] To a solution of compound 10 (cis-4-hydroxycyclohexanecarboxylic acid, 2.00 g, 13.87 mmol) in pyridine (19.75 g, 20.20 mL, 250 mmol), acetic anhydride (10.83 g, 10.03 mL, 106 mmol) was added at 0°C. 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 using CombiFlash® with silica gel as the stationary phase, and eluted using a gradient solution (0-30%) of butyl in hexane. Yield of compound 11: 1.75 g (68%). C9H 14 The calculated [MH] for O4 was 185.20, and the measured value was 185.35. [ka]

[0244] To a solution of compound 11 (420 mg, 2.26 mmol) in DCM (5 mL), DMF (16.5 mg, 17.4 μL, 0.226 mmol) and oxalyl chloride (1.43 g, 0.97 mL, 11.3 mmol) were added at 0°C. After 30 minutes, the cooling bath was removed, and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated and co-evaporated with toluene, and product 12 was used in the next step without purification. [ka]

[0245] Pyridine (750 mg, 767 μL, 9.50 mmol) was added to a solution of compound 3 (400 mg, 1.12 mmol) in DMF (2 mL). The reaction mixture was cooled to 0°C, and a solution of compound 12 (457 mg, 2.23 mmol) in DCM (2 mL) was added dropwise. The cooling bath was removed, and the reaction mixture was stirred at room temperature for 1.5 hours. The mixture was diluted with DCM (20 mL) and quenched with saturated aqueous solution NH4Cl (10 mL). The organic phase was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified using CombiFlash® with silica gel as the stationary phase, and eluted with a gradient solution (0-10%) of MeOH in DCM. Yield of compound 13: 365 mg (62%). 1 H NMR(400 MHz, DMSO-d6): δ8.21 (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). [ka]

[0246] To a solution of compound 13 (360 mg, 0.68 mmol) in THF (2.2 mL), a 1 M NaOH solution (3.42 mL, 3.42 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 2 hours, and then heated to 35°C for a further 1.5 hours. Upon consumption of the starting material, 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 using CombiFlash® with silica gel as the stationary phase and eluted with a gradient solution (0-12%) in MeOH DCM. Yield of compound 8: 250 mg (75%). 1H NMR(400 MHz, DMSO-d6): δ8.22 (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). [ka]

[0247] Compound 14 (245 mg, 0.51 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 (30 mg, 0.25 mmol) was added, followed by 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphodiamidite (229 mg, 0.76 mmol). The reaction mixture was stirred at 0°C for 30 minutes and then stirred at room temperature for 1.5 hours. The reaction mixture was concentrated in 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 using CombiFlash® with silica gel as the stationary phase, and then eluted in a gradient solution (50-100%) of ethylamine-containing butyl in DCM. Yield of compound 15 (compound 2): 204 mg (59%). 35 H 53 The calculated [MH] for N6O6P was 683.81, and the measured value was 684.14.

[0248] Example 3. Synthesis of Compound 3 (2-cyanoethyl (5-((1,7-dioxo-4-(3-oxo-3-(prop-2-in-1-ylamino)propyl)-1,7-bis(prop-2-in-1-ylamino)heptan-4-yl)amino)-5-oxopentyl)diisopropylphosphoramidite) [ka] To a solution of compound 3 (475 mg, 1.33 mmol) in DMF (5 mL), triethylamine (402 mg, 555 μL, 3.98 mmol) and glutaric anhydride (190 mg, 1.65 mmol) were added at room temperature. The reaction mixture was stirred for 1 hour, 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 phase was dried over Na2SO4, filtered, and concentrated. The residue was purified using CombiFlash® with silica gel as the stationary phase, and then eluted in a gradient solution (0-7.5%) of MeOH in DCM. Yield of compound 16: 273 mg (42%). 25 H 34 The calculated [M+H] for N4O6 was 487.58, while the measured value was 487.61. [ka]

[0249] To a solution of compound 16 (173 mg, 0.36 mmol) in MeOH (0.87 mL) and iPrOH (1.74 mL), sodium borohydride (54 mg, 1.42 mmol) was added at 0°C. After 30 minutes, the cooling bath was removed and lithium chloride (10 mg) was added. The reaction mixture was stirred overnight at room temperature. The following day, the additional sodium borohydride (27 mg, 0.71 mmol) was added, and the reaction was continued for 1 hour. The reaction mixture was concentrated and purified using CombiFlash® with silica gel as the stationary phase, and eluted with a gradient solution (0-12%) in DCM of MeOH. Yield of compound 17: 93 mg. 24 H 34 The calculated [M+H] for N4O5 was 459.57, while the measured value was 459.63. [ka]

[0250] 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′-tetraisopropylphosphodiamidite (173 mg, 0.57 mmol). The reaction mixture was stirred at 0°C for 30 minutes and then at room temperature for 30 minutes. The reaction mixture was concentrated in 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 using CombiFlash® with silica gel as the stationary phase and eluted with a gradient (50–100%) of siRNA in DCM containing 1% triethylamine. Yield of compound 18 (compound 3): 132 mg (53%). 33 H 51 The calculated [M+H] for N6O6P was 659.79, while the measured value was 659.93.

[0251] Example 4. Compound 4 (2-cyanoethyl ((1r,4r)-4-((11,17-dioxo-14-(3-oxo-3-((2-(2-(prop-2-in-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacosa-1,26-diin-14-yl)carbamoyl)cyclohexyl) diisopropylphosphoramidite) [ka] To a solution of compound 19 (4.42 g, 5.23 mmol) in DMF (25 mL), triethylamine (3.63 g, 5.00 mL, 35.9 mmol) was added at room temperature. The reaction mixture was stirred overnight. The reaction mixture was then concentrated and purified using CombiFlash® with silica gel as the stationary phase, and eluted with a gradient solution (0-20%) in MeOH DCM. Yield of compound 20: 3.08 g (95%). 1 H NMR(400 MHz, DMSO-d6): δ7.82 (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). [ka]

[0252] To a solution of compound 20 (900 mg, 1.45 mmol) and compound 5 (404 mg, 2.17 mmol) in DMF (7 mL), 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) was added at 0°C. The cooling bath was removed, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated in orange oil and 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 using CombiFlash® with silica gel as the stationary phase, and eluted with a gradient solution (0-8%) of MeOH in DCM. Yield of compound 27: 880 mg (77%). 40 H 62 N4O 12 The calculated [M+H] for this was 791.96, and the measured value was 792.08. [ka]

[0253] To a solution of compound 27 (925 mg, 1.17 mmol) in THF (6 mL), 1 M NaOH (5.85 mL, 5.85 mmol) was added at room temperature. The mixture was heated at 35°C for 2 hours. The reaction mixture was acidified to pH = 6 with 2 M HCl. Sodium chloride (approximately 3 g) was added to the aqueous phase, and the mixture was extracted with DCM (3 x 40 mL). The combined organic phase was dried over Na2SO4, filtered, and concentrated. The residue was purified using CombiFlash® with silica gel as the stationary phase, and eluted with a gradient solution (0-12%) of MeOH in DCM. Yield of compound 28: 580 mg (66%). 1H NMR(400 MHz, DMSO-d6): δ7.82 (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). [ka]

[0254] Compound 28 (577 mg, 0.77 mmol) was azeotropically dried from anhydrous ACN (2 x 20 mL) 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′-tetraisopropylphosphodiamidite (348 mg, 1.12 mmol). The cooling bath was removed, and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated in 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 using CombiFlash® with silica gel as the stationary phase, and eluted with a gradient solution (0-2%) in DCM containing 1% triethylamine MeOH. Yield of compound 29 (compound 4): 610 mg (83%). 1H NMR(400 MHz, DMSO-d6): δ7.82(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 P NMR (400 MHz, DMSO-d6): δ144.6.

[0255] Example 5. Synthesis of Compound 5 (2-cyanoethyl ((1s,4s)-4-((11,17-dioxo-14-(3-oxo-3-((2-(2-(prop-2-in-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacosa-1,26-diin-14-yl)carbamoyl)cyclohexyl)diisopropylphosphoramidite) [ka] Pyridine (1.22 g, 1.25 mL, 15.5 mmol) was added to a solution of compound 20 (1070 mg, 1.72 mmol) in DCM (7 mL). The reaction mixture was cooled to 0°C, and a solution of compound 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 hours. The mixture was diluted with DCM (20 mL) and quenched with saturated aqueous solution NH4Cl (10 mL). The layers were separated, and the organic phase was washed with saturated aqueous solution NaHCO3 (10 mL) and brine (10 mL). The organic phase was dried over Na2SO4, filtered, and concentrated. The residue was purified using CombiFlash® with silica gel as the stationary phase, and eluted with a gradient solution (0-7%) of MeOH in DCM. Yield of compound 24: 295 mg (22%). 40H 62 N4O 12 The calculated [M+H] for this was 791.96, and the measured value was 792.08. [ka]

[0256] To a solution of compound 24 (290 mg, 0.37 mmol) in THF (2 mL), 1 M NaOH (1.83 mL, 1.83 mmol) was added at room temperature. The mixture was heated at 35°C for 3 hours. The reaction mixture was quenched with saturated aqueous solution 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 phase was dried over Na2SO4, filtered, and concentrated. The residue was purified using CombiFlash® with silica gel as the stationary phase and eluted with a gradient solution (0-12%) of MeOH in DCM. Yield of compound 25: 183 mg (67%). 1 H NMR(400 MHz, DMSO-d6): δ7.83 (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). [ka]

[0257] 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′-tetraisopropylphosphodiamidite (109 mg, 0.36 mmol). The reaction mixture was stirred at 0°C for 30 minutes, and then stirred at room temperature for 1.5 hours. The additional portion of 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphodiamidite (36 mg, 0.12 mmol) was added, and the reaction mixture was stirred for a further 3 hours. The reaction mixture was concentrated in 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 using CombiFlash® with silica gel as the stationary phase and eluted in a gradient solution (0-2%) in MeOH containing 1% triethylamine. Yield of compound 26 (compound 5): 116 mg (51%). 47 H 77 N6O 12 The calculated [M+H] for P was 950.15, while the measured value was 950.18.

[0258] Example 6. Synthesis of Compound 6 (2-cyanoethyl (11,16-dioxo-14,14-bis(3-oxo-3-((2-(2-(prop-2-in-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7-dioxa-10,15-diazaicos-1-in-20-yl)diisopropylphosphoramidite) [ka] To a solution of compound 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), TBTU (6.77 g, 21.1 mmol) was added at 0°C. A solution of propargyl-PEG2-amine (3.02 g, 21.1 mmol) in DMF (5 mL) was then added dropwise. The cooling bath was removed, and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with DCM (30 mL) and washed with 1 N 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 using CombiFlash® with silica gel as the stationary phase, and eluted with a gradient solution (0-10%) of MeOH in DCM. Yield of compound 19: 4.42 g (82%). [ka]

[0259] To a solution of compound 20 (960 mg, 1.54 mmol) in DCM (8 mL), triethylamine (468 mg, 645 μL, 4.62 mmol) and glutaric anhydride (220 mg, 1.93 mmol) were added. The reaction mixture was stirred overnight at room temperature. The following 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 hours. The reaction mixture was concentrated in oil, 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 using CombiFlash® with silica gel as the stationary phase, and then eluted in a gradient solution (0-7.5%) of MeOH in DCM. Yield of compound 21: 880 mg (76%). 37 H58 N4O 12 The calculated [M+H] for this was 751.90, and the measured value was 751.90. [ka]

[0260] A solution of compound 21 (877 mg, 1.17 mmol) in THF (4 mL) and MeOH (1.75 mL) was added to 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 a single addition. The cooling bath was removed, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was quenched by adding saturated aqueous solution NH4Cl (5 mL). After stirring for 10 minutes, the mixture was concentrated to remove THF and MeOH. The residue was diluted with water (5 mL) and extracted with DCM (3 x 20 mL). The combined organic phase was dried over Na2SO4, filtered, and concentrated. The residue was purified using CombiFlash® with silica gel as the stationary phase, and eluted with a gradient solution (0-12%) of MeOH in DCM. Yield of compound 22: 562 mg (66%). 36 H 58 N4O 11 The calculated [M+H] for this was 723.19, while the measured value was 723.81. [ka]

[0261] Compound 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′-tetraisopropylphosphodiamidite (350 mg, 1.16 mmol). The reaction mixture was stirred at 0°C for 30 minutes and then stirred at room temperature for 30 minutes. The reaction mixture was concentrated in 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 using CombiFlash® with silica gel as the stationary phase, and then eluted in a gradient solution (0-2%) of MeOH containing 1% triethylamine in a DCM. Yield of compound 6: 434 mg (61%). 1 H NMR(400 MHz, DMSO-d6): δ7.82(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 P NMR (400 MHz, DMSO-d6): δ146.3.

[0262] Example 7. Synthesis of Compound 7 (2-cyanoethyl (4-((11,17-dioxo-14-(3-oxo-3-((2-(2-(prop-2-in-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacosa-1,26-diin-14-yl)carbamoyl)phenyl)diisopropylphosphoramidite) [ka] Process 1. HATU (243.2 mg, 0.64 mmol) was added to a solution of Compound 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). The reaction mixture was stirred at room temperature. After confirming by LC-MS that all starting materials had been consumed, the reaction mixture was quenched with 2 mL of saturated NaHCO3 aqueous solution and extracted with ethyl acetate (10 mL × 3). The combined organic phase was sequentially 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 column and purified (MPA: DCM, MPB: 20% MeOH in DCM, 0-50% gradient over 30 mins) to provide the product. Yield: 133 mg.

[0263] Process 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 pad of silica gel. The filtrate was collected and concentrated under reduced pressure. Yield: 115 mg, 48% in two steps. 38 H 53 N4O 11 The calculated MS (ESI) m / z [MH] was 741.37, while the measured value was 741.67. [ka]

[0264] Compound 2 (100 mg, 0.1346 mmol), diisopropylammonium tetrazolide (11.5 mg, 0.0673 mmol), and 3Å molecular sieve (20 mg) were dissolved in DCM (2 mL) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (60.9 mg, 0.064 mL, 0.2019 mmol, 1.5 eq) was added. The reaction mixture was stirred at room temperature. After confirming by LC-MS that all starting materials had been consumed, the reaction mixture was quenched with 2 mL of saturated NaHCO3 aqueous solution 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 column and purified (MPA: 1% TEA in DCM, MPB: 1% TEA and 4% MeOH in DCM, 0-50% gradient over 30 minutes) to obtain compound 7. Yield: 105 mg (83%). 47 H 70 N6O 12 The calculated MS (ESI) m / z [MH] for P was 941.48, while the measured value was 941.88.

[0265] Example 8. Synthesis of Compound 8 (2-cyanoethyl (3-((11,17-dioxo-14-(3-oxo-3-((2-(2-(prop-2-in-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacosa-1,26-diin-14-yl)carbamoyl)phenyl)diisopropylphosphoramidite) [ka] Process 1. HATU (243.2 mg, 0.64 mmol) was added to a solution of Compound 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). The reaction mixture was stirred at room temperature. After confirming by LC-MS that all starting materials had been consumed, the reaction mixture was quenched with 2 mL of saturated NaHCO3 aqueous solution and extracted with ethyl acetate (10 mL × 3). The combined organic layer was sequentially 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 column and purified (MPA: DCM, MPB: 10% MeOH in DCM, 0-40% gradient over 30 minutes) to provide the product. Yield: 148 mg.

[0266] Process 2. The amide product from step 1 was dissolved in 2 mL of MeOH, and 100 mg of K2CO3 was added to the reaction mixture. After stirring overnight at room temperature, the reaction mixture was filtered through a short pad of silica gel. The filtrate was collected and concentrated under reduced pressure. Yield: 126 mg, 53% in two steps. 38 H 55 N4O 11 The calculated MS (ESI) m / z [M+H] for this was 743.39, while the measured value was 743.65. [ka]

[0267] Compound 3 (125 mg, 0.1683 mmol), diisopropylammonium tetrazolide (14.4 mg, 0.0841 mmol), and 3Å molecular sieve (20 mg) were dissolved in DCM (2 mL) to which 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (76.1 mg, 0.08 mL, 0.2524 mmol, 1.5 eq) was added. The reaction mixture was stirred at room temperature. After confirming by LC-MS that all starting materials had been consumed, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 solution 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 column and purified (MPA: 1% TEA in DCM, MPB: 1% TEA and 4% MeOH in DCM, 0-50% gradient over 30 minutes) to obtain compound 8. Yield: 130 mg (82%). 47 H 70 N6O 12 The calculated MS (ESI) m / z [MH] for P was 941.48, while the measured value was 941.79.

[0268] Example 9. Synthesis of Compound 9 (2-cyanoethyl (2-((11,17-dioxo-14-(3-oxo-3-((2-(2-(prop-2-in-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacosa-1,26-diin-14-yl)carbamoyl)phenyl)diisopropylphosphoramidite) [ka] Process 1. To a solution of Compound 1 (200 mg, 0.32 mmol) and triethylamine (97.3 mg, 0.134 mL, d = 0.726 g / mL, 0.96 mmol) in DCM (2 mL), O-acetylsalicyroyl chloride (127.6 mg, 0.6423 mmol, 1.2 eq, CAS Registry No.: 5538-51-2) was added. The reaction mixture was stirred at room temperature. After confirming by LC-MS that all starting materials had been consumed, 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 sequentially washed with HCl (aq) and brine. The organic layers were dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica column and purified (MPA: DCM, MPB: 10% MeOH in DCM, 0-50% gradient for 30 minutes) to provide the product. Yield: 177.8 mg.

[0269] Process 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 pad of silica gel. The filtrate was collected and concentrated under reduced pressure. Yield: 126 mg, 53% in two steps. 38 H 53 N4O 11 The calculated MS (ESI) m / z [MH] was 741.39, while the measured value was 741.67. [ka]

[0270] Compound 4 (105 mg, 0.1457 mmol), diisopropylammonium tetrazolide (12.5 mg, 0.0728 mmol), and 3Å molecular sieve (20 mg) were dissolved in DCM (2 mL) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (66 mg, 0.069 mL, 0.2185 mmol, 1.5 eq) was added. The reaction mixture was stirred at room temperature. After confirming that all starting materials had been consumed by monitoring with LC-MS, the reaction mixture was quenched with 2 mL of saturated NaHCO3 aqueous solution 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 column and purified (MPA: 1% TEA in DCM, MPB: 1% TEA and 4% MeOH in DCM, 0-50% gradient over 30 minutes) to obtain compound 9. Yield: 181 mg (83%). 47 H 70 N6O 12 The calculated MS (ESI) m / z [MH] for P was 941.48, while the measured value was 941.79.

[0271] Example 10. Synthesis of Compound 10 (2-cyanoethyl (4'-((11,17-dioxo-14-(3-oxo-3-((2-(2-(prop-2-in-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacosa-1,26-diin-14-yl)carbamoyl)-[1,1'-biphenyl]-4-yl)diisopropylphosphoramidite) [ka] HATU (244.2 mg, 0.64 mmol) was added to a solution of Compound 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). The reaction mixture was stirred at room temperature. After confirming by LC-MS that all starting materials had been consumed, 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 sequentially washed with HCl (aq) and brine. The organic layers were dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica column and purified (MPA: DCM, MPB: 10% MeOH in DCM, 0-50% gradient over 30 minutes) to provide the product. Yield: 138 mg, 52%. 44 H 59 N4O 11 The calculated MS (ESI) m / z [M+H] for this was 819.42, while the measured value was 819.90. [ka]

[0272] Compound 5 (138 mg, 0.1685 mmol), diisopropylammonium tetrazolide (14.4 mg, 0.0843 mmol), and 3Å molecular sieve (20 mg) were dissolved in DCM (2 mL) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (76.2 mg, 0.08 mL, 0.2528 mmol, 1.5 eq) was added. The reaction mixture was stirred at room temperature. After confirming by LC-MS that all starting materials had been consumed, the reaction mixture was quenched with 2 mL of saturated NaHCO3 aqueous solution and extracted with ethyl acetate (10 mL × 3). It was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica column and purified (MPA: 1% TEA in DCM, MPB: 1% TEA and 4% MeOH in DCM, 0-50% gradient over 30 minutes) to obtain compound 10. Yield: 171 mg (99%). 53 H 74 N6O 12 The calculated MS (ESI) m / z [MH] for P was 1017.51, while the measured value was 1017.99.

[0273] Example 11. Synthesis of Compound 11 (2-cyanoethyl ((1r,3r)-3-((11,17-dioxo-14-(3-oxo-3-((2-(2-(prop-2-in-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacosa-1,26-diin-14-yl)carbamoyl)cyclobutyl)diisopropylphosphoramidite) [ka] Compound 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) were dissolved in DMF (3 mL), to which HATU (366.3 mg, 0.9635 mmol) was added. The reaction mixture was stirred at room temperature. After confirming by LC-MS that all starting materials had been consumed, 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 sequentially washed with HCl (aq) and brine. The organic layers were dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica column and purified (MPA: DCM, MPB: 10% MeOH in DCM, 0-100% gradient over 30 minutes) to provide the product. Yield: 333.2 mg, 88%. 36 H 57 N4O 11 The calculated MS (ESI) m / z [M+H] for this was 721.40, while the measured value was 721.96. [ka]

[0274] Compound 6 (166.5 mg, 0.2310 mmol), diisopropylammonium tetrazolide (19.8 mg, 0.1155 mmol), and 3Å molecular sieve (20 mg) were dissolved in DCM (2 mL) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (104.4 mg, 0.11 mL, 0.3465 mmol, 1.5 eq) was added. The reaction mixture was stirred at room temperature. After confirming by LC-MS that all starting materials had been consumed, the reaction mixture was quenched with 2 mL of saturated NaHCO3 aqueous solution 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 column and purified (MPA: 1% TEA in DCM, MPB: 1% TEA and 4% MeOH in DCM, 0-50% gradient over 30 minutes) to obtain compound 11. Yield: 200 mg (94%). 45 H 72 N6O 12 The calculated MS (ESI) m / z [MH] for P was 919.50, while the measured value was 919.73.

[0275] Example 12. Synthesis of Compound 12 (2-cyanoethyl (4-((11,17-dioxo-14-(3-oxo-3-((2-(2-(prop-2-in-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacosa-1,26-diin-14-yl)carbamoyl)bicyclo[2.2.2]octan-1-yl)diisopropylphosphoramidite) [ka] Compound 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) were dissolved in DMF (5 mL) to which HATU (732.7 mg, 1.9269 mmol) was added. The reaction mixture was stirred at room temperature. After confirming by LC-MS that all starting materials had been consumed, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 solution and extracted with ethyl acetate (10 mL × 3). The combined organic layers were sequentially washed with HCl (aq) and brine. The organic layers were dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica column and purified (MPA: DCM, MPB: 10% MeOH in DCM, 0-100% gradient over 30 minutes) to provide the product. Yield: 398 mg, 54%. 40 H 61 N4O 11 The calculated MS (ESI) m / z [MH] was 773.45, while the measured value was 773.80. [ka]

[0276] Compound 7 (200 mg, 0.2581 mmol), diisopropylammonium tetrazolide (22.1 mg, 0.1290 mmol), and 3Å molecular sieve (20 mg) were dissolved in DCM (2 mL) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (116.7 mg, 0.123 mL, 0.3871 mmol, 1.5 eq) was added. The reaction mixture was stirred at room temperature. After confirming by LC-MS that all starting materials had been consumed, the reaction mixture was quenched with 2 mL of saturated NaHCO3 aqueous solution 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 column and purified (MPA: 1% TEA in DCM, MPB: 1% TEA and 4% MeOH in DCM, 0-50% gradient over 30 minutes) to obtain compound 12. Yield: 40 mg (16%). 49 H 78 N6O 12 The calculated MS (ESI) m / z [MH] for P was 973.54, while the measured value was 973.75.

[0277] Example 13. Synthesis of Compound 13 (2-cyanoethyl (3-((11,17-dioxo-14-(3-oxo-3-((2-(2-(prop-2-in-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacosa-1,26-diin-14-yl)carbamoyl)bicyclo[1.1.1]pentan-1-yl) diisopropylphosphoramidite) [ka] Compound 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) were dissolved in DMF / DCM (10 mL, 1:1 v / v) and HATU (488.4 mg, 1.2846 mmol) was added. The reaction mixture was stirred at room temperature. After confirming by LC-MS that all starting materials had been consumed, 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 sequentially washed with HCl (aq) and brine. The organic layers were dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica column and purified (MPA: DCM, MPB: 10% MeOH in DCM, 0-100% gradient over 30 minutes) to yield compound 8. Yield: 387.6 mg, 82%. 37 H 57 N4O 11 The calculated MS (ESI) m / z [M+H] for this was 733.40, while the measured value was 733.66. [ka]

[0278] Compound 8 (387.6 mg, 0.5289 mmol), diisopropylammonium tetrazolide (45.3 mg, 0.2644 mmol), and 3Å molecular sieve (20 mg) were dissolved in DCM (2 mL) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (239.1 mg, 0.252 mL, 0.7933 mmol, 1.5 eq) was added. The reaction mixture was stirred at room temperature. After confirming by LC-MS that all starting materials had been consumed, the reaction mixture was quenched with 2 mL of saturated NaHCO3 aqueous solution 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 column and purified (MPA: 1% TEA in DCM, MPB: 1% TEA and 4% MeOH in DCM, 0-50% gradient over 30 minutes) to obtain a pure phosphoramidite product. Yield: 206.7 mg (42%). 46 H 72 N6O 12 The calculated MS (ESI) m / z [MH] for P was 931.50, while the measured value was 931.71.

[0279] Example 14. Synthesis of Compound 14 (2-cyanoethyl (11,16,20-trioxo-14,14-bis(3-oxo-3-((2-(2-(prop-2-in-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7-dioxa-10,15,21-triazaheptacos-1-in-27-yl)diisopropylphosphoramidite) and Compound 22 (4-nitrophenyl 11,16-dioxo-14,14-bis(3-oxo-3-((2-(2-(prop-2-in-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7-dioxa-10,15-diazycos-1-in-20-oate) [ka] 500 mL of DCM and compound 4 (75.0 g, 0.16 mol) were added to a 3-L jacketed reactor. The reactor was cooled to 0°C, and TBTU (170.0 g, 0.53 mol) was added. The suspension was then treated by adding amine 5 (75.5 g, 0.53 mol) dropwise while maintaining the temperature below 5°C. The reaction mixture was then slowly treated with DIPEA (72.3 g, 0.56 mol) while maintaining the temperature below 5°C. After the additions were complete, the reaction mixture was heated to 23°C over 1 hour and stirred for 3 hours. 10% kicker charges for all three reagents were added, and the mixture was stirred for a further 3 hours. The reaction was considered complete when compound 4 <1%. The reaction mixture was washed with saturated ammonium chloride solution (2 x 500 mL) and then washed once with saturated sodium bicarbonate solution (500 mL). Next, the organic layer was dried over sodium sulfate and concentrated into oil. The mass of the crude oil was 188 g and contained 72% compound 6 by QNMR. The crude oil was then transported to the next step. 46 H 60 N4O 11 Calculated mass = 845.0 m / z. Measured value [M+H] = 846.0. [ka]

[0280] 121.2 g of 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 formation of dibenzofluben (DBF) in response to the consumption of Fmoc-amine 6 was monitored by HPLC (Figure 2), and the reaction was completed within 10 hours. In the solution, glutaric anhydride (12.8 g, 0.11 mol) and intermediate amine 7 were converted to compound 8 within 2 hours. Upon completion, DMF and TEA were removed under reduced pressure at 30°C to obtain 100 g of crude oil. Due to the high solubility of compound 7 in water, an aqueous workup could not be used, and chromatography was the only method for removing DBF, TMU, and glutaric anhydride. Crude oil (75 g) was purified in three steps using a Teledyne ISCO Combi-flash® purification system. Crude oil (25 g) was loaded onto a 330 g silica column and eluted with 0-20% methanol / DCM for 30 minutes to obtain 42 g of compound 8 (54% yield in three steps). 36 H 55 N4O 12 Calculated mass = 736.4 m / z. Measured value [M+H] = 737.0. [ka]

[0281] Compound 8 (42.0 g, 0.057 mol) was co-stripped with 10 volumes of acetonitrile, which was then used to remove residual methanol from the chromatography solvent. This 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), which was found to be complete within 10 hours. The solution was cooled to 0°C, and 10 volumes of ethyl acetate, followed by 10 volumes of saturated ammonium chloride solution, were added while maintaining the internal temperature below 15°C. The layers were separated, 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 into oil. Crude oil (55 g) was purified in three steps using a Teledyne ISCO Combi-Flash® purification system. Crude oil (25 g) was loaded onto a 330 g silica column and eluted with 0-10% methanol / DCM for 30 minutes to obtain 22 g of pure compound 9 (compound 22) (50% yield). 42 H 59 N5O 14 Calculated mass = 857.4 m / z. Measured value [M+H] = 858.0. [ka]

[0282] 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 vols) was treated by dropwise addition of triethylamine (11.56 g, 111.4 mmol). The reaction was monitored by observing the disappearance of compound 9 by HPLC method 1, and it was found to be completed in 10 minutes. The crude reaction mixture was diluted with 5 vols of dichloromethane and washed with saturated ammonium chloride (5 vols) and brine (5 vols). The organic layer was dried over sodium sulfate and concentrated into oil. The crude oil was purified using a Teledyne ISCO Combi-flash® purification system with a 330 g silica column. 4-nitrophenol was eluted with 100% ethyl acetate, and compound 10 was flushed from the column with 20% methanol / DCM to obtain a colorless oil (39 g, 81% yield). 42 H 69 N5O 12 Calculated mass = 836.0 m / z. Measured value [M+H] = 837.0. [ka]

[0283] Alcohol 10 was stripped twice simultaneously with 10 volumes of acetonitrile to remove residual methanol from the chromatography solvent, and the process was repeated with dry dichloromethane (KF < 60 ppm) to remove trace amounts of water. Alcohol 10 (2.30 g, 2.8 mmol) was dissolved in 5 volumes of dry 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 completed within 3–6 hours. The reaction mixture was cooled to 0°C and treated with 10 volumes of saturated ammonium bicarbonate / brine 1:1 solution, then warmed to ambient temperature for 1 minute and stirred at 20°C for 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 any unreacted bis-phosphorus reagent. The organic layer was dried over sodium sulfate and concentrated into oil to obtain 3.08 g of 94 wt% compound 14. 51 H 86 N7O 13 The calculated mass of P is 1035.6 m / z. The measured value [M+H] is 1036.

[0284] Example 15. Synthesis of Compound 15 (4-nitrophenyl 5-((1,3-bis(prop-2-in-1-yloxy)-2-((prop-2-in-1-yloxy)methyl)propan-2-yl)amino)-5-oxopentanoate) [ka] Process 1. Di-tert-butyl dicarbonate (2.35 g, 10.7 mmol) tA solution in BuOH (10 mL) was added to a suspension of tris(hydroxylmethyl)aminomethane (1.00 g, 8.20 mmol, CAS No.: 77-86-1) in a 1:1 MeOH / tBuOH mixture (15 mL), and the mixture was stirred at room temperature for 18 hours. The solvent was removed under reduced pressure to provide the residue, which was purified by precipitation with cold SiO2. By vacuum filtration, the pure compound was provided as a white solid (1.4449, 80% yield). C9H 20 For NO5, the calculated MS (ESI) m / z [M+H] was 222.13, while the measured value was 222.24.

[0285] Process 2. A solution of triol-NHBoc 10 (500 mg, 2.26 mmol) in dry DMF (6 mL) was stirred at 0°C with propargyl bromide (80 wt% in toluene, 1.46 mL, 13.6 mmol). The finely ground KOH portion (951 mg, 13.6 mmol) was added over 15 minutes. The mixture was then heated to 35°C and stirred under a nitrogen atmosphere for 24 hours. The reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 solution and extracted with ethyl acetate (20 mL × 3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica column and purified (MPA: hexane, MPB: EA, 0–10% gradient over 30 minutes) to provide pure product 11. Yield: 483 mg (64%).

[0286] Steps 3 and 4. To a solution of Trialuc-NHBoc 11 (483 mg, 1.44 mmol) in dry DCM (5.6 mL), TFA (2.3 mL) was added dropwise at 0°C. The brown mixture was then stirred at room temperature for 2 hours. Solids were obtained by concentration under high vacuum without further purification. The crude product 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 by Combiflash® using silica gel as the stationary phase yielded 0.9357 g of product 14. (MPA: DCM, MPB: 20% MeOH in DCM, 0-50% gradient over 30 mins). 18 H 22 The calculated MS (ESI) m / z [MH] for NO6 was 348.15, while the measured value was 348.28. [ka]

[0287] To a solution of compound 14 (470 mg, 1.3 mmol) and p-nitrophenol (936 mg, 6.7 mmol, 5 eq) in DCM (10 mL), EDC HCl salt (1.28 g, 6.7 mmol, 5 eq) was added at 0°C. The reaction mixture was then stirred at room temperature. After confirming by LC-MS that all starting materials had been consumed, the reaction mixture was quenched with 2 mL of saturated NaHCO3 aqueous solution and extracted with ethyl acetate (10 mL × 3). It was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica column and purified (MPA: hexane, MPB: EA, 0–60% gradient over 30 mins), and the pure product was provided as yellow oil. Yield: 471 mg (77%). 24 H 27 The calculated MS (ESI) m / z [M+H] for N2O8 was 471.18, while the measured value was 471.33.

[0288] Example 16. Synthesis of Compound 16 (4-nitrophenyl 5-(((S)-1-(((R)-1,5-dioxo-1,5-bis(prop-2-in-1-ylamino)pentan-2-yl)amino)-1,5-dioxo-5-(prop-2-in-1-ylamino)pentan-2-yl)amino)-5-oxopentanoate) [ka] Process 1. To a solution of methyl 3,4,5-trihydroxylbenzoate 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), K2CO3 (13.8 g, 100 mmol, 4 eq) was added. The reaction mixture was then stirred overnight at room temperature. After confirming that the starting materials had been consumed by TLC, the reaction mixture was filtered and concentrated under reduced pressure.

[0289] Process 2. The crude product was dissolved in EtOH / H2O (200 mL, 1:1 v / v), and then 90 mL of 4 M NaOH aq was added to the reaction. After confirming that the starting material had been consumed by TLC, the reaction mixture was concentrated under reduced pressure to remove EtOH, and then filtered to provide a white solid of 17 (6.18 g). The solid was used in the next step without further purification.

[0290] Process 3. To a solution of compound 17 (73 mg, 0.35 mmol) and PNP (139 mg, 1 mmol, 3 eq) in DCM (5 mL), EDC HCl salt (191 mg, 1 mmol, 3 eq) was added at 0°C. The reaction mixture was then stirred at room temperature. After confirming by TLC that all starting materials had been consumed, the reaction mixture was quenched with 2 mL of saturated NaHCO3 aqueous solution 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 column and purified (MPA: hexane, MPB: EA, 0–40% gradient over 30 mins) to provide compound 16. 22 H 14 The calculated MS (ESI) m / z [MH] for NO7 was 404.08, while the measured value was 404.48.

[0291] Example 17. Synthesis of Compound 17 (4-nitrophenyl 5-(((S)-1-(((R)-1,5-dioxo-1,5-bis(prop-2-in-1-ylamino)pentan-2-yl)amino)-1,5-dioxo-5-(prop-2-in-1-ylamino)pentan-2-yl)amino)-5-oxopentanoate) [ka] Process 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), HBTU (5.685 g, 15 mmol) was added at 0°C. The reaction mixture was stirred at room temperature. After confirming by LC-MS that all starting materials had been consumed, the reaction mixture was quenched with 2 mL of saturated NaHCO3 aqueous solution and extracted with ethyl acetate (20 mL × 3). The combined organic layer was washed with brine. The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude product was loaded onto a silica column and purified (MPA: hexane, MPB: EA, 0–33% gradient over 30 mins) to provide product 20, which was used in the next step. 37 H 51 The calculated MS (ESI) m / z [M+H] for N2O9 was 667.36, while the measured value was 667.49.

[0292] Process 2. The product from step 1 was dissolved in TFA / DCM (20 mL, 1:1 v / v). The reaction mixture was stirred at room temperature for 3 hours. After confirming by LC-MS that all starting materials had been consumed, the mixture was concentrated overnight under reduced pressure. Yield: 3.4 g. 25 H 25 The calculated MS (ESI) m / z [MH] for N2O9 was 497.16, while the measured value was 497.35.

[0293] Process 3. Triacid 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) were added to a flame-dried round-bottom flask. The mixture was cooled to 0°C, and TBTU (2.256 g, 7.0281 mmol, 3.5 eq) was added. After confirming by LC-MS that all starting materials had been consumed, 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 provide 0.8818 g of white solids 22. 34 H 36 The calculated MS (ESI) m / z [M+H] for N5O6 was 610.27, while the measured value was 610.41.

[0294] Steps 4, 5, and 6. Compound 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 by LC-MS that all starting materials had been consumed, 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. Upon consumption of the starting materials, the reaction mixture was concentrated and purified using CombiFlash® with silica gel as the stationary phase, and eluted with a gradient of MeOH in DCM (0-20%). Yield: 34 mg, 34%. 30 H 35 The calculated MS (ESI) m / z [M+H] for N6O9 was 623.25, while the measured value was 623.38.

[0295] Example 18 Synthesis of Compound 18 (4-nitrophenyl 5-((1,7-dioxo-4-(3-oxo-3-(prop-2-in-1-ylamino)propyl)-1,7-bis(prop-2-in-1-ylamino)heptan-4-yl)amino)-5-oxopentanoate) [ka] To a solution of compound 3 (1.00 g, 2.79 mmol) in DMF (5 mL), triethylamine (0.847 g, 1.17 mL, 8.37 mmol) and glutaric anhydride (493 mg, 4.32 mmol) were added at room temperature. The reaction mixture was stirred overnight. The following 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 using CombiFlash® with silica gel as the stationary phase, and eluted with a gradient of MeOH in DCM (0-6%). Yield of compound 31 (compound 18): 1.13 g (74%). 30 H 35 The calculated [M+H] for N5O8 was 594.65, while the measured value was 594.39.

[0296] Example 19. Synthesis of Compound 19 (2,3,5,6-tetrafluorophenyl 3-((1,7-dioxo-4-(3-oxo-3-(prop-2-in-1-ylamino)propyl)-1,7-bis(prop-2-in-1-ylamino)heptan-4-yl)carbamoyl)bicyclo[1.1.1]pentane-1-carboxylate) [ka] Process 1. Amine 25 (100 mg, 0.2793 mmol) was added 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) of the above. The reaction mixture was stirred at room temperature. After confirming that all starting materials had been consumed by LC-MS, the reaction mixture was concentrated under reduced pressure. Pure product 27 was obtained by purification using Combiflash® (MPA: DCM, MPB: 20% MeOH in DCM, 0-50% gradient over 30 minutes). Yield: 114 mg, 80%. 27 H 35 The calculated MS (ESI) m / z [M+H] for N4O6 was 511.26, while the measured value was 511.75.

[0297] Process 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 by LC-MS that all starting materials had been consumed, 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.

[0298] Process 3. Compound 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) were dissolved in DCM (5 mL) to which EDC HCl salt (210.1 mg, 1.1 mmol, 5 eq) was added at 0°C. The reaction mixture was then stirred at room temperature. After confirming by TLC that all starting materials had been consumed, the reaction mixture was quenched with 2 mL of saturated NaHCO3 aqueous solution 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 column and purified (MPA: hexane, MPB: EA, 0–50% gradient over 30 min) to provide compound 19. Yield: 89 mg (63%). 32 H 33 The calculated MS (ESI) m / z [M+H] for F4N4O6 was 645.23, while the measured value was 645.79.

[0299] Example 20. Synthesis of Compound 20 (2,3,5,6-tetrafluorophenyl 4'-((1,7-dioxo-4-(3-oxo-3-(prop-2-in-1-ylamino)propyl)-1,7-bis(prop-2-in-1-ylamino)heptan-4-yl)carbamoyl)-[1,1'-biphenyl]-4-carboxylate) [ka] Process 1. Compound 25 (100 mg, 0.2793 mmol) was added 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) of the above. The reaction mixture was stirred at room temperature. After confirming by LC-MS that all starting materials had been consumed, the reaction mixture was concentrated under reduced pressure. Purification with Combiflash® (MPA: DCM, MPB: 20% MeOH in DCM, 0-50% gradient over 30 minutes) provided pure product 30. Yield: 165 mg, 99%. 34 H 37 The calculated MS (ESI) m / z [M+H] for N4O6 was 597.27, while the measured value was 597.81.

[0300] Process 2. The product from step 1 was dissolved in THF / H2O (0.6 mL, 2:1 v / v), and then LiOH (20 mg, 0.83 mmol, 3 eq) was added. After confirming by LC-MS that all starting materials had been consumed, 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.

[0301] Process 3. Compound 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) were dissolved in DCM (5 mL) to which EDC HCl salt (266 mg, 1.39 mmol, 5 eq) was added at 0°C. The reaction mixture was then stirred at room temperature. After confirming by TLC that all starting materials had been consumed, the reaction mixture was quenched with 2 mL of saturated NaHCO3 aqueous solution 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 column and purified (MPA: hexane, MPB: EA, 0-50% gradient over 30 mins) to provide the pure product, compound 20. Yield: 87 mg (42%). 39 H 35 MS (ESI) m / z [M+H] calculated for F4N4O6 + 731.25, measured value: 731.85.

[0302] Example 21. Synthesis of Compound 21 ((1r,4r)-4-((1,7-dioxo-4-(3-oxo-3-(prop-2-in-1-ylamino)propyl)-1,7-bis(prop-2-in-1-ylamino)heptan-4-yl)carbamoyl)cyclohexyl (4-nitrophenyl)carbonate) [ka] Compound 8 (see Example 1) (0.048 g, 0.10 mmol) and DIEA (0.18 mL, 1.0 mmol) in THF (0.5 mL) were mixed with 4-nitrophenyl chloroformate (0.044 g, 0.22 mmol), and the reaction was stirred at 50°C. After completion, all volatile components were removed, and compound 21 was isolated by elution with a gradient solution of MeOH in DCM and separation on silica. Yield: 0.035 g (54%).

[0303] Example 22. Synthesis of tridentate ligands and conjugation of target ligands into RNAi agents. A target ligand can be conjugated to one or more RNAi agents useful for inhibiting the expression of one or more targeted genes. The target ligand facilitates the delivery of the RNAi agent to targeted cells and / or tissues. The target ligand may contain a specific moiety that interacts with cell surface receptors to result in the introduction of the RNAi agent into the cell. The following describes a general procedure for synthesizing a specific target ligand-RNAi agent conjugate using the trialakine conjugates described herein, as illustrated in the non-limiting examples described herein.

[0304] A. Synthesis of RNAi agents RNAi agents can be synthesized using methods commonly known in the art. For the synthesis of RNAi agents shown in the examples herein, the sense and antisense strands of the RNAi agents were synthesized according to solid-phase phosphoramidite techniques used in oligonucleotide synthesis. Depending on the scale, MerMade96E® (Bioautomation), MerMade12® (Bioautomation), or OP Pilot 100 (GE Healthcare) were used. Synthesis was carried out on solid supports made of controlled-pore glass (CPG, 500 Å or 600 Å, available from Prime Synthesis, Aston, PA, USA). All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the following 2'-O-methylphosphoramidite was 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-methylcytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino)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-phosphoramidite had the same protecting group as 2'-O-methylRNAamidite. 5'-dimethoxytrityl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite was purchased from Glen Research (Virginia). The inverted abasic (3'-O-dimethoxytrityl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino)) phosphoramidite was purchased from ChemGenes (Wilmington, MA, USA). The following UNA phosphoramidites are: 5'-(4,4'-dimethoxytrityl)-N6-(benzoyl)-2',3'-seco-adenosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-acetyl-2',3'-seco-cytosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-acetyl-2',3'-seco-cytosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. Sphoramidite, 5'-(4,4'-dimethoxytrityl)-N-isobutyryl-2',3'-seco-guanosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-(4,4'-dimethoxytrityl)-2',3'-seco-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite were used. TFA amino-linked phosphoramidite was also commercially purchased (ThermoFisher).

[0305] Alternatively, the trialquine moiety was introduced after solid support synthesis (see Section F below). In this pathway, the sense strand was functionalized with 5' and / or 3' terminal nucleotides containing primary amines. TFA amino-linked phosphoramidite was dissolved in anhydrous acetonitrile (50 mM), and molecular sieves (3 Å) were added. 5-benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) were used as activator solutions. Coupling times were 10 minutes (RNA), 90 seconds (2'O-Me), and 60 seconds (2'F). To introduce phosphorothioate bonds, a 100 mM solution of 3-phenyl1,2,4-dithiazolin-5-one (POS, PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was used.

[0306] In some embodiments, the compound of formula III is synthesized by reacting it with the compound of formula II, which can be attached to the end of an RNAi agent. In some embodiments, the trialakine binder of formula II is attached to the 5' end of the sense strand of a double-stranded RNAi agent. In some embodiments, the trialakine binder of formula II is attached to the 3' end of the sense strand of a double-stranded RNAi agent. In some embodiments, the compound of formula II is attached to the 5' end of the antisense strand of a double-stranded RNAi agent. In some embodiments, the compound of formula II is attached to the 3' end of the antisense strand of a double-stranded RNAi agent. An example of this type of reaction is shown in the following scheme. [ka]

[0307] When used in combination with RNAi agents as shown in the specific examples herein, the trialquine-containing phosphoramidite was dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), all other amidites were dissolved in anhydrous acetonitrile (50 mM), and molecular sieves (3 Å) were added. 5-benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) were used as the activator solution. Binding times were 10 minutes (RNA), 90 seconds (2'O-Me), and 60 seconds (2'F). To introduce the phosphorothioate bond, a 100 mM solution of 3-phenyl1,2,4-dithiazolin-5-one (obtained from POS, PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was used.

[0308] B. Cleavage and deprotection of support-bound oligomers After the completion of solid-phase synthesis, the dried solid support was treated with a 1:1 volume solution of 40 wt% methylamine and 28-31% ammonium hydroxide solution (Aldrich) in water at 30°C for 1.5 hours. The solution was evaporated, and the solid residue was reconstituted in water (see below).

[0309] C. Purification Crude oligomers were purified by anion exchange HPLC using a TSKgel SuperQ-5PW 13 μm column and a Shimadzu LC-8 system. Buffer A consisted of 20 mM Tris, 5 mM EDTA, pH 9.0, and 20% acetonitrile, while Buffer B was the same as Buffer A, with the addition of 1.5 M sodium chloride. UV traces were recorded at 260 nm. Appropriate fractions were pooled and size exclusion HPLC was performed using a GE Healthcare XK 16 / 40 column packed with Sephadex G-25 Fine, with running buffers of 100 M ammonium bicarbonate, pH 6.7, and 20% acetonitrile or filtered water.

[0310] D. Annealing Equimolar RNA solutions (sense and antisense) were combined in 1× PBS (phosphate-buffered saline, 1×, Corning, Cellgro) to mix complementary strands and form RNAi agents. Several RNAi agents were lyophilized and stored at -15 to -25°C. The double-stranded concentration was determined by measuring the absorbance of the solution in 1× PBS using a UV-Vis spectrometer. Next, the double-stranded concentration was determined by multiplying the absorbance of the solution at 260 nm by a conversion factor and a dilution factor. The conversion factor used was 0.037 mg / (mL·cm), or, in some experiments, the conversion factor was calculated from the experimentally determined extinction coefficient.

[0311] E. Conjugation of target ligands

[0312] Compounds of formulas IV, V, VIII, and IX can be synthesized by conjugating a target ligand to a trialukine compound described herein. An example of the reaction is shown in the scheme. [ka] (In the above equation, each variable is as described in Equation I, and TL is the target ligand.)

[0313] In some embodiments, target ligand conjugation may be performed using the following procedure. The procedure describes the conjugation of a target ligand to a compound of formula I in which R is an RNAi agent, but target ligand conjugation can also be performed to a compound of formula I in which R is not an RNAi agent.

[0314] Either before or after annealing, the 5' or 3' tridentate alkyne-functionalized sense chain is conjugated with the target ligand. The following example describes the conjugation of a target ligand to an annealed double chain: Starting solution 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 was prepared in deionized water. A 75 mg / mL solution of the target ligand in DMSO was prepared. In a 1.5 mL centrifuge tube containing trialkhine-functionalized double chain (3 mg, 75 μL, 40 mg / mL in deionized water, approximately 15,000 g / mol), 25 μL of 1 M Hepes pH 8.5 buffer was added. After vortexing, 35 μL of DMSO was added and the solution was vortexed. The target ligand was added to the reactant (6 eq / double-stranded, 2 eq / alkyne, approximately 15 μL), and the solution was vortexed. The pH was checked using pH paper and confirmed to be approximately 8. In a separate 1.5 mL centrifuge tube, 50 μL of 0.5 M THPTA was mixed with 10 μL of 0.5 M Cu(II)SO4·5H2O, vortexed, and incubated at room temperature for 5 minutes. After 5 minutes, the THPTA / Cu solution (7.2 μL, 6 eq 5:1 THPTA:Cu) was added to the reaction vial and vortexed. Immediately thereafter, 2 M ascorbate (5 μL, 50 eq / double-stranded, 16.7 / alkyne) was added to the reaction vial and vortexed. Once the reaction was complete (typically in 0.5–1 hour), the reactant was immediately purified by non-denaturing anion exchange chromatography.

[0315] F. Addition of Triarquine binder after solid support synthesis RNAi molecules can be synthesized using reactive groups such as amino groups (also referred to herein as amines). In some embodiments, the reactive group may be attached to the 5' and / or 3' ends of the RNAi agent. In some embodiments, the RNAi agent may be double-stranded. In embodiments where the RNAi agent is double-stranded, the reactive group may be located on the sense or antisense strand of the RNAi agent.

[0316] For example, in some embodiments, the 5' end of the sense strand of the RNAi agent is NH2-C6H 12 RNAi agents having a (hexyleneamine) group are synthesized. Subsequently, the terminal amino group can be reacted to form a conjugate with, for example, the coupling moiety of a compound of formula I. In some embodiments, the coupling moiety is an ester, the reactive group of the RNAi agent is a primary amine, and an amide bond is formed between the RNAi agent and the trialquine linker. An example of this reaction is shown in the following scheme using a compound of formula VI.

[0317] [ka] (In the above formula, L 1 , L 2 , L 3 , L 4 , R 3 , R 4 (And RNA is as defined in formulas VI and VII).

[0318] When the RNAi molecule is cleaved from the solid support, the addition of the trialukine binder described herein is carried out as follows: The sense strand was functionalized with 5' and / or 3' terminal nucleotides containing a primary amine. The amine-functionalized double strand was dissolved in 90% DMSO / 10% H2O at approximately 50-70 mg / mL. 40 equivalents of triethylamine were added, followed by 3 equivalents of the trialukine ester of formula VI. Upon completion, the conjugate was precipitated twice in a solvent system of 1x phosphate-buffered saline / acetonitrile (1:14 ratio) and dried.

[0319] In vivo example The linkers described herein can be used in combination with various RNAi agents. The following examples illustrate the use of the linkers described herein with RNAi agents directed to Alpha-ENaC and HIF2α mRNA sequences, and are intended to provide examples of the use of the above linkers without limiting the scope of the present invention to specific RNAi agents. The RNAi agents used in the following examples are shown in Table 8 below. The compounds in Table 8 are shown as structures cleaved from a solid support. In some examples, the compounds were further modified before in vivo administration. In the case of AD5614-5617, AD5620, AD5858, AD5860 and AD5919, the trialquine binder was added to the sense strand as a phosphoramidite of formula II as part of the synthesis on a solid support. In the case of AD04546, AD5347 and AD5453, the sense strand was cleaved from the support in the structures shown in Table 8. Each trialquine binder was added as a compound of formula VI in an amide coupling reaction. Since the target ligand was added after cleavage from the resin, the trialquine binder is shown as the compound of formula III in the cases of AD5614-5617, AD5620, AD5858, AD5860, and AD5919. 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. s represents a phosphorothioate bond, and cPrpu represents 5'-cyclopropylphosphonate-2'-O-methyluridine.

[0320] [Table 8-1]

[0321] [Table 8-2]

[0322] Example 23. Mouse model with kidney tumor (orthotopic xenograft) Creation of SEAP-expressing clear cell renal cell carcinoma (ccRCC) A498 cells A pCR3.1 expression vector expressing reporter gene secretory alkaline phosphatase (SEAP) under a CMV promoter was prepared by directional cloning of SEAP coding sequence 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 construct a SEAP-expressing A498 ccRCC cell line. In short, the pCR3-SEAP plasmid was transfected into A498 ccRCC cells by electroporation according to the manufacturer's recommendations. Stable transfectants were selected by G418 resistance. Selected A498-SEAP clones were evaluated for SEAP expression and integrated stability.

[0323] Transplantation of SEAP-expressing clear cell renal cell carcinoma (ccRCC) A498 cells Female nude mice lacking a thymus gland were anesthetized with approximately 3% isoflurane and positioned in a right lateral decubitus position. A small longitudinal abdominal incision of 0.5–1 cm was made in the left flank. The left kidney was lifted from the peritoneum using a moist cotton swab and gently stabilized. Immediately before injection, a 1.0 ml syringe was filled with the cell / Matrigel mixture, and a 27-gauge needle catheter was attached to the tip of the syringe. The filled syringe was then attached to a syringe pump (Harvard Apparatus, model PHD2000), primed, and air removed. The tip of the 27-gauge needle catheter attached to the syringe was inserted just below the renal capsule near the caudal pole, and then the tip of the needle was carefully advanced 3–4 mm cranially along the renal capsule. A 10 μL aliquot of a 2:1 (vol:vol) cell / Matrigel mixture containing approximately 300,000 cells was slowly injected into the renal parenchyma using the syringe pump. To ensure the injection was complete, the needle was left in the kidney for 15–20 seconds. The needle was then removed from the kidney, and a cotton swab was placed over the injection site for 30 seconds to prevent cell leakage or bleeding. The kidney was then gently returned to the abdomen, and the abdominal wall was closed. Serum was collected every 7–14 days post-transplant to monitor tumor growth using a commercially available SEAP assay kit. For most studies, tumor mice were used for 5–6 weeks post-transplant, at which point tumor measurements were typically around 4–8 mm.

[0324] Determination of HIF2 mRNA expression In the studies reported in the examples herein, mice were euthanized on specific days after injection, and total RNA was isolated from renal tumors using Trizol reagent as recommended by the manufacturer. Relative HiF2α mRNA levels were determined by RT-qPCR as described below and compared to mice treated with delivery buffer (isotonic glucose) alone.

[0325] As preparation for quantitative PCR, total RNA was isolated from tissue samples homogenized with TriReagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's protocol.

[0326] Approximately 500 ng of RNA was reverse transcribed using a High Capacity cDNA Reverse Transcription Kit (Life Technologies). For human (tumor) Hif2α (EPAS1) expression, pre-fabricated TaqMan gene expression assays for human Hif2α (catalog no. 4331182) and CycA (PPIA) (catalog no. 4326316E) were used three times in a biplex reaction with either the TaqMan Gene Expression Master Mix (Life Technologies) or the VeriQuest Probe Master Mix (Affymetrix). Quantitative PCR was performed using a 7500 Fast or StepOnePlus Real-Time PCR system (Life Technologies). ΔΔC T The relative gene expression was calculated using the method.

[0327] Example 24. In vivo administration of an integrin target ligand conjugated to an RNAi agent targeting HIF-2α (EPAS1) in mice with renal tumors. RNAi agents containing the sense and antisense strand sequences shown in Table 8 were synthesized in solid phase using phosphoramidite technology following a common procedure commonly used in oligonucleotide synthesis known in the art (see Example 22 herein). The RNAi agents contained an antisense strand having a nucleic acid sequence at least partially complementary to the HIF-2α (Hif2α or EPAS1) gene. EPAS1 is a member of the HIF (hypoxia-inducible factor) gene family and encodes half of the transcription factors involved in the induction of oxygen-regulated genes, which are induced when oxygen levels decrease (a condition known as hypoxia). Hif2α is known to be frequently overexpressed in clear cell renal cancer (ccRCC) cells. The Hif2α RNAi agents were designed to reduce or inhibit the translation of the Hif2α messenger RNA (mRNA) transcript in a sequence-specific manner, thereby inhibiting the expression of the EPAS1 gene.

[0328] On day 1 of the study, mice with renal tumors (see Example 23) were administered the drug by tail vein injection according to the following administration plan:

[0329] [Table 9]

[0330] The RNAi agents in Example 24 were synthesized with a functionalized amine-reactive group (NH2-C6) at the 5' end of the sense strand to facilitate conjugation to the respective trialquine linker compounds shown. In groups 4-8 and 10, the trialquine linkers were added to the RNAi agents using phosphoramidite compounds 1, 2, 3, 4, and 6, respectively. Each integrin target ligand was synthesized with an azide-reactive group (see, e.g., Example 22) and then conjugated to the trialquine component of the linker. A 40 kilodalton (kDa) PEG moiety was attached to function as a pharmacokinetic (PK) modulator, increasing the circulation time of the drug conjugate. The structures of the target ligands αvβ3 integrin ligands 4.1 and 4.5 are shown below.

[0331] [ka]

[0332] In each group (n=3), three mice with tumors were administered the drug. The mice were sacrificed on day 8 after injection, and total RNA was isolated from the kidney tumors according to the procedure described in Example 4. Next, relative human HIF2α mRNA expression was quantified by probe-based quantitative PCR (RT-qPCR), normalized for cyclophyllin A (PPIA) expression, and expressed as a percentage (geometric mean, + / - 95% confidence interval) of the vehicle control group (isotonic glucose), as described in Example 23.

[0333] [Table 10]

[0334] Example 25. In vivo administration of an integrin target ligand conjugated to an RNAi agent targeting HIF-2α (EPAS1) in mice with renal tumors. RNAi agents containing the sense and antisense strand sequences shown in Table 8 were synthesized in solid phase using phosphoramidite techniques, following a common procedure known in the art and commonly used in oligonucleotide synthesis (see Example 22 herein). The RNAi agents contained an antisense strand having a nucleic acid sequence at least partially complementary to the (Hif2α)(EPAS1) gene.

[0335] On day 1 of the study, mice with kidney tumors (see Example 23) were administered the drug via tail vein injection according to the following drug groups.

[0336] [Table 11]

[0337] The RNAi agents in Example 25 were synthesized with nucleotide sequences directed to target the human Hif2α gene, and in the cases of groups 3–6, a functionalized amine-reactive group (NH2-C6) was included at the 5' end of the sense strand to facilitate conjugation to trialquine linker compounds 15–18. In the cases of groups 3 and 7–9, the trialquine linkers were added to the RNAi agents using phosphoramidite compounds 14, 10, 12, and 13, respectively. Each integrin-targeted ligand was synthesized with an azide-reactive group (see, e.g., Example 22) and then conjugated to the trialquine component of the linker. The 40 kDa PEG moiety and the C-18 diacid moiety were conjugated to function as pharmacokinetic (PK) modulators by increasing the circulation time of the drug conjugate. The structure of the C-18 diacid moiety is shown below.

[0338] [ka]

[0339] The C-18 diacid moiety was attached to the 3' end of the sense chain via an amide bond. The structure of the target ligand αvβ3 integrin ligand 2 is shown below.

[0340] [ka] In the above formula,

[0341] [ka] The symbol indicates the binding site of the binder.

[0342] In each group (n=3), three mice with tumors were administered the treatment. The mice were sacrificed on day 8 after injection, and total RNA was isolated from the kidney tumors according to the procedure described in Example 4. Next, relative human HIF2α mRNA expression was quantified by probe-based quantitative PCR (RT-qPCR), normalized to human cyclophyllin A (PPIA) expression, and expressed as a percentage (geometric mean, + / - 95% confidence interval) of the vehicle control group (isotonic glucose), as described in Example 23.

[0343] [Table 12]

[0344] Example 26. In vivo oral-pharyngeal aspiration administration of an α-ENaCRNAi agent conjugated to a rat epithelial cell target ligand. Trialquine conjugates can be used in a variety of RNAi constructs. RNAi constructs containing the conjugate of the present invention can be administered by a variety of different dosing methods, as described in this example. Trialquine conjugates can also be used with a variety of target ligands. In this example, the target ligand conjugated to the Trialquine conjugate is an αvβ6 target ligand.

[0345] In this example, the trialquine binder of compound 22 was added to the sense chain after the synthesis of the solid support using the method described in Example 22.

[0346] On day 1 of the study, male Sprague Dawley rats were administered 200 microliters via oropharyngeal aspiration (OP) using a pipette, according to the following drug groups.

[0347] [Table 13]

[0348] Compound 22 reacted with the amine bond at the 5' end of the sense chain of each group. The structures of the αvβ6 target ligands are shown below. [ka] [ka] In the above formula, [ka] The symbol indicates the binding site of the binder.

[0349] In each group (n=4), four rats were administered the treatment. On day 9 of the study, the rats were euthanized, and after collection and homogenization, total RNA was isolated from both lungs. α-ENaC(SCNN1A) mRNA expression was quantified by probe-based quantitative PCR, normalized to GAPDH expression, and expressed as the proportion (geometric mean, + / - 95% confidence interval) of the vehicle control group.

[0350] [Table 14] As shown in Table 14 above, various different target ligand structures bound to each RNAi agent using the trialquine-binding compounds disclosed herein showed inhibition of gene expression compared to controls.

[0351] Other Embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative and does not limit the scope of the present invention as defined by the attached claims. Other embodiments, advantages and modifications are within the scope of the following claims.

Claims

1. Formula I: 【Chemistry 1】 (In the above formula, L 1 , L 2 and L 3 Each 【Chemistry 2】 And, Q is tetravalent carbon, R contains a phosphoramidite group, or contains an organophosphate and an RNAi agent, and X is NR x or it is a bond, and R x H or C 1 -C 6 A compound (which is alkyl) or a pharmaceutically acceptable salt thereof.

2. R is 【Transformation 3】 A compound according to claim 1 or a pharmaceutically acceptable salt thereof, selected from the group consisting of the following. 【Request Item 3】 【Table 1-1】 Table 1-2 A compound selected from the group consisting of the above, or a pharmaceutically acceptable salt thereof. 【Request Item 4】 【Table 2-1】 Table 2-2 Table 2-3 A compound or a pharmaceutically acceptable salt thereof, selected from the group consisting of, where RNA is a compound containing or consisting of an RNAi agent.

5. Formula V: 【Chemistry 4】 (In the above formula, L 1 , L 2 and L 3 are each 【Transformation 5】 And, L 4 This is a linker, and the linker contains alkylene, arylene, or cycloalkylene. R 4 is H or alkyl, Y is either O or S, RNA contains or consists of RNAi agents, and A compound of the target ligand (TL) or a pharmaceutically acceptable salt thereof. 【Request Item 6】 【Table 3-1】 Table 3-2 Table 3-3 Table 3-4 Table 3-5 A compound or a pharmaceutically acceptable salt thereof, selected from the group consisting of, where TL contains a target ligand and RNA contains or consists of an RNAi agent.

Images