Triacetylene linkers and methods of use

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 631,683 filed on February 17, 2018, U.S. Provisional Application Serial No. 62 / 646,739 filed on March 22, 2018, U.S. Provisional Application Serial No. 62 / 663,763 filed on April 27, 2018, and U.S. Provisional Application Serial No. 62 / 790,300 filed on January 9, 2019, all of which are incorporated herein by reference in their entirety. Field of the Invention

[0003] The present disclosure relates to triyne linkers suitable for use with synthetic oligonucleotides, such as RNA interference (RNAi) agents.

[0004] background

[0005] Synthetic oligonucleotides, such as antisense compounds, aptamers, ribozymes and RNA interference (RNAi) agents or molecules are increasingly used in biomedical research, diagnosis and therapy. These synthetic oligonucleotides have been used to inhibit or knock down gene expression in vitro, in situ and in vivo in a sequence-dependent manner.

[0006] It is usually useful to attach or connect a targeting ligand or other pharmacological or pharmacokinetic enhancer or modifier to a synthetic oligonucleotide, particularly for therapeutic in vivo delivery. In order to be useful, the connection chemistry should be modular, so that it is easily adapted to different synthetic oligonucleotides and different targeting ligands and pharmacological modifiers. In addition, the connection chemistry should have simple reaction conditions, high efficiency (i.e., providing high chemical yields), no toxic or other harmful products are required and no toxic or other harmful by-products are produced. The connection chemistry should also be stable outside the target cell, such as in the circulation, subcutaneous space or extracellular space, but at the final site of action, such as in the target cell, it is easy to break. In addition, particularly for oligonucleotide-based therapies, the length of the linker and flexibility are known to significantly affect the in vivo efficacy of the therapeutic compound, particularly by changing cellular uptake in some cases.

[0007] There is a need for linking agents of suitable properties for linking oligonucleotide-based compounds, such as RNAi agents, to targeting ligands.

[0008] Overview

[0009] In one aspect, the invention provides compounds according to the structure of Formula I:

[0010]

[0011] or a pharmaceutically acceptable salt thereof,

[0012] Where L1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0013] Q is a tetravalent carbon, a tetrasubstituted phenyl group or an optionally substituted alkylene group;

[0014] R comprises a conjugation moiety or an RNAi agent; and

[0015] X is NR x or key, and R x is H or optionally substituted C1-C6 alkyl.

[0016] In one aspect, the present invention provides compounds according to the structure of Formula II:

[0017]

[0018] or a pharmaceutically acceptable salt thereof,

[0019] in,

[0020] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0021] L 4 is a linker comprising an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group;

[0022] R 1 Each instance of is optionally substituted alkyl;

[0023] R 2 is optionally substituted alkyl; and

[0024] R 4 is H or optionally substituted alkyl.

[0025] Another aspect of the invention described herein is a compound according to the structure of Formula III:

[0026]

[0027] or a pharmaceutically acceptable salt thereof,

[0028] in

[0029] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0030] L 4is a linker comprising an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group;

[0031] R 4 is H or optionally substituted alkyl;

[0032] X is O or S; and

[0033] The RNA comprises or consists of a RNAi agent.

[0034] In another aspect, described herein are compounds according to the structure of Formula IV:

[0035]

[0036] or a pharmaceutically acceptable salt thereof,

[0037] in,

[0038] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0039] L 4 is a linker comprising an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group;

[0040] R 1 and R 2 are each independently optionally substituted alkyl;

[0041] R 4 is H or optionally substituted alkyl; and

[0042] TL is the targeting ligand.

[0043] Another aspect of the invention described herein is a compound according to the structure of Formula V:

[0044]

[0045] or a pharmaceutically acceptable salt thereof,

[0046] in,

[0047] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0048] L 4 is a linker comprising an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group;

[0049] R 4is H or optionally substituted alkyl;

[0050] TL is the targeting ligand;

[0051] Y is O or S; and

[0052] The RNA comprises or consists of a RNAi agent.

[0053] In another aspect, described herein are compounds according to the structure of Formula VI:

[0054]

[0055] or a pharmaceutically acceptable salt thereof,

[0056] in,

[0057] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0058] L 4 is a linker comprising an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group;

[0059] R 3 is H, optionally substituted alkyl, or optionally substituted aryl; and

[0060] R 4 is H or optionally substituted alkyl.

[0061] Another aspect of the invention described herein is a compound according to the structure of Formula VII:

[0062]

[0063] or a pharmaceutically acceptable salt thereof,

[0064] in,

[0065] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0066] L 4 is a linker comprising an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group;

[0067] R 4 Each instance of is H or optionally substituted alkyl;

[0068] X is O or S; and

[0069] The RNA comprises or consists of a RNAi agent.

[0070] In another aspect, described herein are compounds according to the structure of Formula VIII:

[0071]

[0072] or a pharmaceutically acceptable salt thereof,

[0073] in,

[0074] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0075] L 4 is a linker comprising an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group;

[0076] R 4 is H or optionally substituted alkyl; and

[0077] TL is the targeting ligand.

[0078] Another aspect of the invention described herein is a compound according to the structure of Formula IX:

[0079]

[0080] or a pharmaceutically acceptable salt thereof,

[0081] in,

[0082] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0083] L 4 is a linker comprising an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group;

[0084] R 4 is H or optionally substituted alkyl;

[0085] TL is the targeting ligand;

[0086] X is O or S; and

[0087] The RNA comprises or consists of a RNAi agent.

[0088] Another aspect of the present invention provides a compound of formula II:

[0089]

[0090] Method of reacting with a RNAi agent to form a compound of Formula III:

[0091]

[0092] in,

[0093] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0094] L 4 is a linking group comprising an optionally substituted alkylene group, an optionally substituted arylene group, and an optionally substituted cycloalkylene group;

[0095] R 1 Each instance of is optionally substituted alkyl;

[0096] R 2 is optionally substituted alkyl; and

[0097] R 4 is H or optionally substituted alkyl

[0098] X is O or S; and

[0099] The RNA comprises or consists of a RNAi agent.

[0100] Another aspect of the present invention provides a compound of formula VI

[0101]

[0102] Method of reacting with a RNAi agent comprising a free amine to form a compound of Formula VII:

[0103]

[0104] in,

[0105] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0106] L 4 is a linker comprising an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group;

[0107] R 3 is H, optionally substituted alkyl, or optionally substituted aryl; and

[0108] R 4Each instance of is H or optionally substituted alkyl; and

[0109] The RNA comprises or consists of a RNAi agent.

[0110] Another aspect of the present invention provides a compound of formula III

[0111]

[0112] a method of reacting with a targeting ligand comprising an azide to form a compound of formula V,

[0113]

[0114] in,

[0115] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0116] L 4 is a linker comprising an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group;

[0117] R 4 is H or optionally substituted alkyl;

[0118] TL is the targeting ligand;

[0119] Y is O or S; and

[0120] The RNA comprises or consists of a RNAi agent.

[0121] Another aspect of the present invention provides a compound of formula VII

[0122]

[0123] a method of reacting with a targeting ligand comprising an azide to form a compound of formula IX,

[0124]

[0125] in,

[0126] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0127] L 4 is a linker comprising an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group;

[0128] R 4 Each instance of is H or optionally substituted alkyl; and

[0129] The RNA comprises or consists of a RNAi agent.

[0130] Details

[0131] Disclosed herein are novel compounds containing phosphoramidite triynes, their synthesis and methods of use thereof. The novel compounds disclosed herein exhibit improved reaction yields, stability and biological activity when used to conjugate synthetic oligonucleotides such as RNAi agents to targeting ligands or other pharmacokinetic (PK) enhancers or modifiers.

[0132] Disclosed herein are triacetylene linkers, their synthesis, and methods of use thereof. The triacetylene linkers disclosed herein can be linked to an oligonucleotide, which can then be readily linked to a related compound, such as a targeting ligand, a lipid, cholesterol, a presenting agent (such as an endosomolytic polymer), or a pharmacological modifier. The triacetylene linkers disclosed herein can facilitate the synthesis of oligonucleotide conjugates with improved yields and fewer impurities compared to the use of other known linkers, while maintaining or even improving, in some embodiments, the efficacy of an oligonucleotide conjugate, such as an RNAi agent linked to one or more targeting ligands and / or pharmacokinetic modifiers.

[0133] As used herein, the term "linker" refers to an organic moiety that connects two parts of a compound. Linkers typically contain direct bonds or atoms, such as oxygen or sulfur, units, such as NR L (where R Lwherein the alkyl radical is hydrogen, acyl, aliphatic or substituted aliphatic), C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylaryl The present invention relates to a alkyl group comprising a alkyl heteroarylalkyl group, a alkyl heteroarylalkyl group, a alkyl heteroarylalkenyl group, a alkyl heteroarylalkynyl group, a alkenyl heteroarylalkyl group, a alkenyl heteroarylalkenyl group, a alkenyl heteroarylalkynyl group, a alkynyl heteroarylalkyl group, a alkynyl heteroarylalkenyl group, a alkynyl heteroarylalkynyl group, a alkyl heterocyclylalkyl group, a alkyl heterocyclylalkenyl group, a alkyl heterocyclylalkynyl group, a alkenyl heterocyclylalkyl group, a alkenyl heterocyclylalkenyl group, a alkenyl heterocyclylalkynyl group, a alkynyl heterocyclylalkyl group, a alkynyl heterocyclylalkenyl group, a alkynyl heterocyclylalkynyl group, a alkyl aryl group, a alkenyl aryl group, a alkynyl aryl group, a alkyl heteroaryl group, a alkenyl heteroaryl group, a alkynyl heteroaryl group, and the one or more methylene groups may be replaced by O, S, S(O), SO2, N(R L ) (where R L is hydrogen, acyl, aliphatic or substituted aliphatic), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle;

[0134]

[0135]

[0136] Insertion or termination.

[0137] Reactive groups are those commonly available in the art and include, but are not limited to, activated esters, NHS, TFP, PFP, tetrazine, norbornene, trans-cyclooctene, hydrazines (eg, HYNIC), aminooxy reagents, and aldehydes (eg, 4-formylbenzoic acid).

[0138] Targeting ligands (which may sometimes be referred to as targeting groups in the art) are used to target or improve compound presentation to target cells or tissues, or specific cell types. Targeting ligands enhance the association of molecules with target cells. Therefore, targeting ligands can enhance the pharmacokinetics or biodistribution properties of the conjugates to which they are attached to improve the cellular distribution and cellular uptake of the conjugates. Targeting groups bind to cells or cell receptors and can trigger endocytosis. Targeting groups can be monovalent, divalent, trivalent, tetravalent or have higher valence. Targeting groups can be, but are not limited to, compounds, cell receptor ligands, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics, hydrophobic groups, cholesterol, cholesteryl or steroids that have affinity for cell surface molecules. In some embodiments, targeting groups include cell receptor ligands. Various targeting groups have been used to target drugs and genes to cells and specific cell receptors. Cell receptor ligands may include, but are not limited to, carbohydrates, polysaccharides, sugars (including but not limited to galactose, galactose derivatives (such as N-acetylgalactosamine), mannose and mannose derivatives), haptens, vitamins, folic acid, biotin, aptamers and peptides (including but not limited to RGD-containing peptides, RGD mimetics, insulin, EGF and transferrin).

[0139] Unless otherwise specified, the term "alkyl" as used herein refers to a saturated aliphatic straight or branched hydrocarbon group having from 1 to 10 carbon atoms. For example, "C1-C6 alkyl" includes alkyl groups having 1, 2, 3, 4, 5, or 6 carbons in a straight or branched arrangement. Non-limiting examples of alkyl groups include methyl, ethyl, Isopropyl , Tert-butyl , n-hexyl. The term "aminoalkyl" as used herein refers to an alkyl group as defined above substituted at any position with one or more amino groups as allowed by normal valence. The amino group may be unsubstituted, monosubstituted or disubstituted. Non-limiting examples of aminoalkyl groups include aminomethyl, dimethylaminomethyl and 2-aminopropyl-1-yl.

[0140] The term "alkylene" as used herein refers to a divalent radical of an alkyl group as described herein. Alkylene is a subset of alkyl and refers to the same residue as alkyl, but with two substitution points. Examples of alkylene are methylene, -CH2- or , ethylene, -CH2CH2- or and propylene, -CH2CH2CH2- .

[0141] Unless otherwise specified, the term "cycloalkyl" as used herein refers to a saturated or unsaturated non-aromatic hydrocarbon ring group having 3 to 14 carbon atoms. Non-limiting examples of cycloalkyl include, but are not limited to, cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl and cyclohexyl. Cycloalkyl may include multiple spiro rings or condensed rings. Cycloalkyl is optionally substituted by mono-, di-, tri-, tetra- or penta- as normal valence allows in any position.

[0142] The term "cycloalkylene" as used herein refers to a divalent radical of a cycloalkyl group as described herein. Cycloalkylene is a subset of cycloalkyl and refers to the same residues as cycloalkyl, but with two substitution points. Examples of cycloalkylene include cyclopropylene. 、1,4-cyclohexylene and 1,5-cyclooxylene The cycloalkylene group is optionally mono-, di-, tri-, tetra- or penta-substituted at any position as normal valence permits. The cycloalkylene group may be monocyclic, bicyclic or tricyclic.

[0143] Unless otherwise specified, the term "alkenyl" as used herein refers to a non-aromatic straight or branched hydrocarbon group containing at least one carbon-carbon double bond and having 2 to 10 carbon atoms. Up to 5 carbon-carbon double bonds may be present in such groups. For example, a "C2-C6" alkenyl group is defined as an alkenyl group having 2 to 6 carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, and cyclohexenyl. The straight, branched, or cyclic portion of the alkenyl group may contain a double bond and may be optionally substituted by a single-, two-, three-, four-, or five-substituted group at any position as permitted by normal valence. The term "cycloalkenyl" refers to a monocyclic hydrocarbon group having a specified number of carbon atoms and at least one carbon-carbon double bond.

[0144] Unless otherwise specified, the term "alkynyl" as used herein refers to a straight or branched hydrocarbon group containing 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond. There may be up to 5 carbon-carbon triple bonds. Thus, "C2-C6 alkynyl" refers to an alkynyl group having 2 to 6 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 2-propynyl, and 2-butynyl. The straight or branched portion of the alkynyl group may optionally be mono-, di-, tri-, tetra- or penta-substituted at any position as normal valence permits.

[0145] As used herein, "alkoxyl" or "alkoxy" refers to an -O-alkyl group having the indicated number of carbon atoms. For example, C1-6 alkoxy is intended to include C1, C2, C3, C4, C5, and C6 alkoxy. For example, C1-8 alkoxy is intended to include C1, C2, C3, C4, C5, C6, C7, and C8 alkoxy. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, sec-pentoxy, n-heptoxy, and n-octoxy.

[0146] As used herein, "keto" refers to any alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl or aryl as defined herein connected via a carbonyl bridge. Examples of keto include, but are not limited to, alkanoyl (e.g., acetyl, propionyl, butyryl, pentanoyl or hexanoyl), alkenoyl (e.g., acryloyl), alkynoyl (e.g., ethynyl, propynyl, butynoyl, pentynyl or hexynyl), aroyl (e.g., benzoyl), heteroaroyl (e.g., pyrroloyl, imidazolyl, quinolinyl or pyridinoyl).

[0147] As used herein, "alkoxycarbonyl" refers to any alkoxy group as defined above (i.e., -C(O)O-alkyl) attached via a carbonyl bridge. Examples of alkoxycarbonyl include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-propoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, or n-pentyloxycarbonyl.

[0148] As used herein, "aryloxycarbonyl" refers to any aryl group as defined herein attached through an oxycarbonyl bridge (ie, -C(O)O-aryl). Examples of aryloxycarbonyl include, but are not limited to, phenoxycarbonyl and naphthoxycarbonyl.

[0149] As used herein, "heteroaryloxycarbonyl" refers to any heteroaryl group as defined herein attached via an oxycarbonyl bridge (i.e., -C(O)O-heteroaryl). Examples of heteroaryloxycarbonyl include, but are not limited to, 2-pyridyloxycarbonyl, 2-oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl, or pyrimidinyloxycarbonyl.

[0150] As used herein, "aryl" or "aromatic" refers to any stable monocyclic or polycyclic carbocyclic ring having up to 6 atoms in each ring, wherein at least one ring is aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, tetrahydronaphthyl, indanyl, and biphenyl. In the case where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring. Aryl groups are optionally mono-, di-, tri-, tetra-, or pentasubstituted at any position as normal valence permits.

[0151] The term "arylene" as used herein refers to a divalent radical of an aryl group as described herein. Arylene is a subset of aryl and refers to the same residue as aryl, but with two substitution points. Examples of arylene groups include phenylene, which refers to a divalent phenyl group. Arylene groups are optionally mono-, di-, tri-, tetra- or penta-substituted at any position as normal valence allows.

[0152] As used herein, the term "coupling moiety" refers to a chemical moiety that can be used to couple two molecules together. For example, a "coupling moiety" can refer to a phosphoramidite that reacts with an alcohol on a separate molecule to form an organophosphate. Further examples of coupling agents can include, but are not limited to, esters, carbonates, carboxylic acids, olefins, alcohols, amines, aldehydes, ketones, alkynes, halogens, Grignard reagents, leaving groups, and any other moiety known in the art for coupling two molecules.

[0153] As used herein, the term "halo" refers to a halogen group. For example, "halo" may refer to a fluorine (F), chlorine (Cl), bromine (Br) or iodine (I) group.

[0154] The term "heteroaryl" as used herein represents a stable monocyclic or polycyclic ring having up to 7 atoms in each ring, wherein at least one ring is aromatic and contains 1 to 4 heteroatoms selected from O, N and S. Examples of heteroaryl include, but are not limited to, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, benzimidazolinonyl, benzoxazolinyl, quinolinyl, isoquinolinyl, dihydroisoindolone, imidazopyridinyl, isoindolinone, indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl and tetrahydroquinoline. "Heteroaryl" is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl. In the case where the heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom-containing ring. The heteroaryl group is optionally mono-, di-, tri-, tetra- or penta-substituted at any position as allowed by normal valence.

[0155] As used herein, the term "heteroarylene" refers to a divalent radical of a heteroaryl group as described herein. Heteroarylene is a subset of heteroaryl and refers to the same residue as heteroaryl, but with two substitution points. Examples of heteroaryl groups include pyridylene, pyrimidylene, and pyrrolylene. Heteroarylene is optionally substituted mono-, di-, tri-, tetra-, or penta-substituted at any position as normal valence permits.

[0156] The term "heterocycle", "heterocyclic" or "heterocyclyl" as used herein refers to a 3 to 14-membered aromatic or non-aromatic heterocycle containing 1 to 4 heteroatoms selected from O, N and S, including polycyclic groups. The term "heterocyclic" as used herein is also considered to be synonymous with the terms "heterocycle" and "heterocyclyl" and is understood to also have the same definition as described herein. "Heterocyclyl" includes the heteroaryl mentioned above, and dihydro and tetrahydro analogs thereof. The example of heterocyclyl includes but is not limited to azetidinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothienyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indolizinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthalene naphthpyridinyl, oxadiazolyl, oxooxazolidinyl, oxazolyl, oxazoline, oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyridonyl, pyrimidinyl, pyrimidonyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl, tetrahydrofuranyl, pyranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, tetrazolyl, tetrazolopyridinyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, 1,4-dioxane, hexahydroazepinyl, piperazinyl, piperidinyl, pyridin-2-one, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl , dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothiophenyl, dihydrotriazolyl, dihydroazetidinyl, dioxythiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl and tetrahydrothiophenyl and N-oxide thereof. The connection of heterocyclic substituents can be carried out through carbon atoms or through heteroatoms. Heterocyclic groups are optionally substituted by single-, two-, three-, four- or five-substituted at any position as allowed by normal valence.

[0157] The term "heterocycloalkyl" as used herein refers to a 3- to 14-membered non-aromatic heterocyclic ring containing 1 to 4 heteroatoms selected from O, N and S, including polycyclic groups. Examples of heterocyclyl groups include, but are not limited to, azetidinyl, oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, oxetanyl, pyranyl, pyridonyl, pyrimidonyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrofuranyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothiophenyl, dihydrotriazolyl, dihydrothiomorpholinyl, and tetrahydrothiophenyl, and N-oxides thereof. Attachment of the heterocycloalkyl substituent may be via a carbon atom or via a heteroatom. The heterocycloalkyl group is optionally mono-, di-, tri-, tetra- or pentasubstituted at any position as permitted by normal valence.

[0158] The term "heterocycloalkylene" as used herein refers to a divalent radical of a heterocycloalkyl group as described herein. Heterocycloalkylene is a subset of heterocycloalkyl and refers to the same residue as heterocycloalkyl, but with two substitution points. Examples of heterocycloalkylene include piperidinylene, azetidinylene, and tetrahydrofuranylene. Heterocycloalkylene is optionally substituted by mono-, di-, tri-, tetra-, or penta- as normal valence permits.

[0159] The terms "treatment" and the like as used herein refer to methods or steps taken to provide relief or a reduction in the number, severity and / or frequency of one or more symptoms of a disease in a subject. "Treatment" as used herein may include prevention, management, prophylactic treatment and / or inhibition of the number, severity and / or frequency of one or more symptoms of a disease in a subject.

[0160] As used herein, the phrase "introducing into a cell" when referring to an RNAi agent refers to the functional presentation of the RNAi agent into a cell. The phrase "functional presentation" refers to the presentation of the RNAi agent into a cell in a manner that enables the RNAi agent to have the desired biological activity, such as sequence-specific inhibition of gene expression.

[0161] Unless otherwise specified, the symbols used in this paper are The use of means that any one or more groups may be attached thereto, which is consistent with the scope of the invention described herein. Multiple times in a structure describe the point of attachment of a particular variable in a compound of Formula I. Unless otherwise indicated, the variables shown can be oriented so that any one of the points of attachment on the variable can be attached to any one of the points of attachment on the compound of Formula I. For example, the variable L 1has two points of attachment to the compound of Formula I. Although L 1 One embodiment of can be shown as , but this embodiment should also be understood to refer to L 1 for of compounds.

[0162] The term "isomer" as used herein refers to compounds with the same molecular formula but different bonding properties or order of their atoms or the arrangement of their atoms in space. Isomers with different arrangements of their atoms in space are called "stereoisomers". Stereoisomers that are not mirror images of each other are called "diastereomers", and stereoisomers that are non-overlapping mirror images are called "enantiomers" or sometimes optical isomers. The carbon atom bonded to four non-identical substituents is called a "chiral center". When the compounds described herein contain olefinic double bonds or other geometric asymmetric centers and their isomeric structures are not clearly defined, the compounds are intended to include independent or mixed E and Z geometric isomers. The compounds of formula I or their pharmaceutically acceptable salts are, for example, intended to include all possible isomers, as well as their racemic and optically pure forms. Similarly, unless explicitly stated otherwise, all tautomeric forms are also intended to be included.

[0163] As used herein, a linking group is one or more atoms that connects one molecule or a portion of a molecule to another molecule or a second molecule or a second portion of a molecule. In the art, the terms linking group and spacers are sometimes used interchangeably. Similarly, as used in the art, the term scaffold is sometimes used interchangeably with a linking group. In some embodiments, the linking group may include a peptide cleavable linking group. In some embodiments, the linking group may include or consist of the peptide phenylalanine-citrulline-phenylalanine-proline. In some embodiments, the linking group may include or consist of a PEG group.

[0164] As used herein, the term "linked" when referring to a connection between two molecules refers to the two molecules being linked by a covalent bond or the two molecules being associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond). In some instances, if the term "linked" refers to an association between two molecules via a non-covalent bond, the association between the two different molecules has a molecular mass of less than 1 x 10 -4 M (e.g. less than 1 x 10 -5 M, less than 1 x 10 -6 M or less than 1 x 10 -7 M) K DIf not specified, the term "linked" as used herein may refer to a connection between a first compound and a second compound with or without the presence of any intervening atoms or groups of atoms.

[0165] It is readily understood and appreciated by those of ordinary skill in the art that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending on the environment in which the compound or composition is located. Accordingly, as used herein, the structures disclosed herein contemplate that certain functional groups, such as OH, SH, or NH, may be protonated or deprotonated. As it is readily understood by those of ordinary skill in the art, the disclosure herein is intended to cover the disclosed compounds and compositions regardless of their protonation state based on the pH of the environment.

[0166] Structures may be depicted with bonds "floating" on the ring structure to indicate bonding to any carbon or heteroatom on the ring as valence permits. For example, the structure means that R can replace any hydrogen atom at any of the five available positions on the ring. A "floating" bond can also be used in a bicyclic structure to indicate bonding to any position on either ring of the bicyclic ring as valence permits. In the case of a bicyclic ring, the bond will be shown as "floating" on both rings, for example It means that R can replace any hydrogen atom at any of the seven available positions on the ring.

[0167] As used in the claims herein, the phrase "consisting of excludes any element, step, or ingredient not specified in the claim. When used in the claims herein, the phrase "consisting essentially of limits the scope of the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0168] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of at least one RNAi agent and one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than active pharmaceutical ingredients (APIs, therapeutic products, such as RNAi agents) that have been appropriately evaluated for safety and intentionally included in drug delivery systems. Excipients do not exert or are not intended to exert therapeutic effects at the intended dose. Excipients can be used to a) assist in processing drug delivery systems during manufacturing, b) protect, support or enhance the stability, bioavailability or patient acceptability of APIs, c) assist in product identification, and / or d) enhance any other attributes of overall safety and effectiveness of API presentation during storage or use.

[0169] Excipients include, but are not limited to: absorption enhancers, anti-adherents, anti-foaming agents, antioxidants, binders, buffers, carriers, coatings, colorants, presentation enhancers, dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, tonicity agents, vehicles, water repellents, and wetting agents. Pharmaceutically acceptable excipients may or may not be inert substances.

[0170] The pharmaceutical composition may contain other additional components that are typically present in pharmaceutical compositions. Pharmaceutically active materials may include, but are not limited to, antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.). It is also contemplated that cells, tissues, or ex vivo organs expressing or containing the RNAi agents specified herein may be used as "pharmaceutical compositions". As used herein, "pharmacologically effective amount", "therapeutically effective amount", or simply "effective amount" refers to the amount of RNAi agent that produces the desired pharmacological, therapeutic, or preventive results.

[0171] The term polynucleotide or polynucleic acid refers to a polymer containing at least two nucleotides. Nucleotides are monomeric units of polynucleotide polymers. Polynucleotides with less than 120 monomeric units are often referred to as 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 may contain a different type of backbone than the natural ribose or deoxyribose-phosphate backbone. Synthetic oligonucleotides can be synthesized using any known technology in the art. Polynucleotide backbones known in the art include: PNA (peptide nucleic acid), thiophosphates, phosphorodiamidates, morpholinos, and other variants of the phosphate backbone of natural nucleic acids. Bases include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs. Synthetic derivatives of purines and pyrimidines include, but are not limited to, modifications that place new reactive groups on nucleotides, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides. The term base encompasses any known base analogs of DNA and RNA. The term polynucleotide includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) as well as combinations of DNA, RNA and other natural and synthetic nucleotides.

[0172] The synthetic oligonucleotides of the present invention may be chemically modified. The use of chemically modified polynucleotides may improve various properties of the polynucleotides, including, but not limited to, resistance to in vivo nuclease degradation, cellular uptake, activity, and sequence-specific hybridization. Non-limiting examples of such chemical modifications include: phosphorothioate / salt internucleotide linkages, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, 2′-deoxyribonucleotides, “universal base” nucleotides, 5-C-methyl nucleotides, 2′,3′-seco nucleotide mimics (unlocked nucleobase analogs, denoted herein as N UNA or NUNA), and inverted deoxyabasic residue incorporation. These chemical modifications, when applied to various polynucleotide constructs, have been shown to preserve polynucleotide activity in cells while significantly improving the serum stability of these compounds.

[0173] In some embodiments, the synthetic oligonucleotides of the invention comprise a duplex having two strands, wherein one or both strands may be chemically modified, wherein each strand is about 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides. In some embodiments, the synthetic oligonucleotides of the invention comprise one or more modified nucleotides. The synthetic oligonucleotides of the invention may comprise about 5 to about 100% (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotide positions) of modified nucleotides.

[0174] The synthetic oligonucleotides may contain 5' or 3' terminal modifications. 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.

[0175] "RNAi agent" (also referred to as "RNAi initiator") refers to a composition containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that can reduce or inhibit (e.g., reduce or inhibit under appropriate conditions) the translation of messenger RNA (mRNA) transcription of a target mRNA in a sequence-specific manner. As used herein, RNAi agents can work through an RNA interference mechanism (i.e., inducing RNA interference by interacting with the RNA interference pathway machinery of mammalian cells (RNA-induced silencing complex or RISC)) or through any alternative mechanism or pathway. Although it is believed that RNAi agents, as the term is used herein, work primarily through an RNA interference mechanism, the disclosed RNAi agents are not subject to or limited to any particular pathway or mechanism of action. The RNAi agents disclosed herein consist of a sense strand and an antisense strand, and include, but are not limited to: short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and Dicer substrates. The antisense strand of the RNAi agent described herein is at least partially complementary to the targeted mRNA (i.e., HIF-2 alpha mRNA). The RNAi agent can include one or more modified nucleotides and / or one or more non-phosphodiester bonds.

[0176] As used herein, the terms "silencing," "reducing," "inhibiting," "downregulating," or "knockdown" when referring to the expression of a given gene refer to a decrease in gene expression as measured by the level of RNA transcribed from the gene or the level of a polypeptide, protein, or protein subunit translated from the mRNA in the cell, cell population, tissue, organ, or subject in which the gene is transcribed, when the cell, cell population, tissue, organ, or subject is treated with a RNAi agent described herein, as compared to a second cell, cell population, tissue, organ, or subject that has not been so treated.

[0177] In some embodiments, the RNAi agent comprises at least two partial, substantially or completely complementary sequences. In some embodiments, the two RNAi agent sequences comprise a sense strand containing a first sequence and an antisense strand containing a second sequence. In some embodiments, the two RNAi agent sequences comprise two sense strands comprising a first sequence together and an antisense strand containing a second sequence, wherein the sense strand and the antisense strand form a partial duplex (meroduplex) together. The sense strand can be connected to the antisense strand via a connecting molecule, such as a polynucleotide linker or a non-nucleotide linker.

[0178] The antisense strand comprises a nucleotide sequence complementary to a portion of the mRNA encoded by the target gene, and the complementary region is most preferably less than 30 nucleotides in length. The sense strand of the RNAi agent comprises a sequence with at least 85% identity to at least a portion of the target mRNA. The RNAi agent, after being presented to a cell expressing the target gene, inhibits the expression of the target gene in vitro or in vivo.

[0179] In some embodiments, the RNAi agent may be composed of naturally occurring nucleotides or may be composed of at least one modified nucleotide or nucleotide mimics. The sense strand and antisense strand of the RNAi agent of the present invention may be synthesized and / or modified by methods perfected in the art. The RNAi agent nucleotides or nucleotide bases may be connected by covalent internucleotide linkages containing phosphate (natural) or phosphate-free (non-natural), i.e., the RNAi agent may have a natural or non-natural oligonucleotide backbone. In some embodiments, the RNAi agent contains non-standard (non-phosphate) connections between nucleotide bases.

[0180] In some embodiments, the RNAi agent may comprise a 5' or 3' terminal modification. 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.

[0181] In some embodiments, RNAi agents may comprise overhangs, ie, protruding nucleotides that are generally unpaired and do not directly participate in the double helical structure generally formed by the core sequences of the sense and antisense strands.

[0182] In some embodiments, the RNAi agent may contain 3' and / or 5' overhangs of 1-5 bases independently on each sense strand and antisense strand. In some embodiments, both the sense strand and the antisense strand contain 3' and 5' overhangs. In some embodiments, one or more 3' overhanging nucleotides of one strand form base pairs with one or more 5' overhanging nucleotides of another strand. In some embodiments, the one or more 3' overhanging nucleotides of one strand are not paired with the one or more 5' overhanging nucleotides of another strand. The sense strand and antisense strand of the RNAi agent may or may not contain the same number of nucleotide bases. The antisense strand and the sense strand may form a duplex in which the 5' end has only a blunt end, the 3' end has only a blunt end, both the 5' and 3' ends are blunt ends, or both the 5' end and the 3' end are not blunt ends. In some embodiments, one or more nucleotides in the overhang contain a thiophosphate, a phosphorothioate, a deoxynucleotide inverted (3' to 3' linkage) nucleotide, or are a modified ribonucleotide or deoxynucleotide.

[0183] The list of known mRNA sequences can be found in the databases maintained by various research institutions, including the database GenBank, which is the National Center for Biotechnology Information, a database maintained by a branch under the National Institutes of Health in the United States, as a part of the International Nucleotide Sequence Database Collaboration. Known effective siRNA sequences and homologous binding sites are also fully presented in the relevant literature. RNAi agent molecules are easily designed and produced by techniques known in the art. In addition, there are computational tools that can improve the chance of finding effective and specific sequence motifs (Pei et al. 2006, Reynolds et al. 2004, Khvorova et al. 2003, Schwarz et al. 2003, Ui-Tei et al. 2004, Heale et al. 2005, Chalk et al. 2004, Amarzguioui et al. 2004).

[0184] Formula I

[0185] Formula I is represented by the following structure:

[0186]

[0187] Where L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0188] Q is a tetravalent carbon atom, a tetrasubstituted phenyl group, or an optionally substituted alkylene group;

[0189] R comprises a conjugation moiety or an RNAi agent; and

[0190] X is NR x or key, and R x is H or optionally substituted C1-C6 alkyl.

[0191] In some embodiments of Formula I, Q is a tetravalent carbon. In other embodiments of Formula I, Q is ,in In other embodiments, Q is ,in Indicates the connection point.

[0192] In some embodiments of Formula I, L 1 , L 2 and L3 Each is In some embodiments of Formula I, L 1 , L 2 and L 3 Each is In some embodiments of Formula I, L 1 , L 2 and L 3 Each is .

[0193] In some embodiments of Formula I, X is NH.

[0194] 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 right Nitrophenol esters 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.

[0195] In some embodiments of Formula I, R is selected from

[0196]

[0197] .

[0198] Exemplary compounds of Formula I are shown in Table 1 below:

[0199] Table 1. Compounds of Formula I

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206] Formula II

[0207] Formula II is represented by the following structure:

[0208]

[0209] or a pharmaceutically acceptable salt thereof,

[0210] in,

[0211] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0212] L 4 is a linking group comprising an optionally substituted alkylene group, an optionally substituted arylene group, and an optionally substituted cycloalkylene group;

[0213] R 1 Each instance of is optionally substituted alkyl;

[0214] R 2 is optionally substituted alkyl; and

[0215] R 4 is H or optionally substituted alkyl.

[0216] In some embodiments of Formula II, L 1 , L 2 and L 3 Each is In some embodiments of Formula II, L 1 , L 2 and L 3 Each is In some embodiments of Formula II, L 1 , L 2 and L 3 Each is .

[0217] In some embodiments of Formula II, R 1 isopropyl in each case.

[0218] In some embodiments of Formula II, R 2 yes .

[0219] In some embodiments of Formula II, L 4 Selected from:

[0220]

[0221] in Indicates the connection point.

[0222] Formula III

[0223] Formula III is represented by the following structure:

[0224]

[0225] or a pharmaceutically acceptable salt thereof,

[0226] in,

[0227] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0228] L 4 is a linking group comprising an optionally substituted alkylene group, an optionally substituted arylene group, and an optionally substituted cycloalkylene group;

[0229] R 4 is H or optionally substituted alkyl;

[0230] X is O or S; and

[0231] The RNA comprises or consists of a RNAi agent.

[0232] 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 thiophosphate.

[0233] In some embodiments of Formula III, L 1 , L 2 and L 3 Each is In some embodiments of Formula III, L 1 , L 2 and L 3 Each is In some embodiments of Formula III, L 1 , L 2 and L 3 Each is .

[0234] In some embodiments of Formula III, L 4 Selected from:

[0235]

[0236] in Indicates the connection point.

[0237] Exemplary compounds of Formula III are shown in Table 2 below:

[0238] Table 2. Compounds of Formula III

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246] Formula IV

[0247] Formula IV is represented by the following structure:

[0248]

[0249] or a pharmaceutically acceptable salt thereof,

[0250] in,

[0251] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0252] L 4 is a linking group comprising an optionally substituted alkylene group, an optionally substituted arylene group, and an optionally substituted cycloalkylene group;

[0253] R 1 and R 2 are each independently an optionally substituted alkyl group;

[0254] R 4 is H or optionally substituted alkyl; and

[0255] TL is the targeting ligand.

[0256] In some embodiments of Formula IV, L 1 , L 2 and L 3 Each is In some embodiments of Formula IV, L 1 , L 2 and L 3 Each is In some embodiments of Formula IV, L 1 , L 2 and L 3 Each is .

[0257] In some embodiments of Formula IV, L 4 Selected from:

[0258]

[0259] in Indicates the connection point.

[0260] In some embodiments of Formula IV, R 1 isopropyl in each case.

[0261] In some embodiments of Formula IV, R 2 yes .

[0262] Exemplary compounds of Formula IV are shown in Table 3 below.

[0263] Table 3. Exemplary compounds of Formula IV

[0264]

[0265]

[0266]

[0267]

[0268] Formula V

[0269] Formula V is represented by the following structure:

[0270]

[0271] or a pharmaceutically acceptable salt thereof,

[0272] in,

[0273] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0274] L 4 is a linker comprising an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group;

[0275] R 4 is H or optionally substituted alkyl;

[0276] TL is the targeting ligand;

[0277] Y is O or S; and

[0278] The RNA comprises or consists of a RNAi agent.

[0279] In some embodiments of Formula V, L 1 , L 2 and L 3 Each is In some embodiments of Formula IV, L 1 , L 2 and L 3 Each is In some embodiments of Formula V, L 1 , L 2 and L 3 Each is .

[0280] In some embodiments of Formula V, L 4 Selected from:

[0281]

[0282] in Indicates the connection point.

[0283] Exemplary compounds of Formula V are shown in Table 4 below:

[0284] Table 4. Exemplary compounds of Formula V

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294] Formula VI

[0295] Formula VI is represented by the following structure

[0296]

[0297] or a pharmaceutically acceptable salt thereof,

[0298] in,

[0299] L1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0300] L 4 is a linker comprising an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group;

[0301] R 3 is H, optionally substituted alkyl, or optionally substituted aryl; and

[0302] R 4 is H or optionally substituted alkyl.

[0303] In some embodiments of Formula VI, L 1 , L 2 and L 3 Each is In some embodiments of Formula VI, L 1 , L 2 and L 3 Each is In some embodiments of Formula VI, L 1 , L 2 and L 3 Each is .

[0304] In some embodiments of Formula VI, L 4 Selected from:

[0305]

[0306] in Indicates the connection point.

[0307] In some embodiments of Formula VI, R 3 is an optionally substituted aryl group. In some embodiments of Formula VI, R 3 It is p-nitrophenyl.

[0308] In some embodiments of Formula VI, R 4 It's H.

[0309] Formula VII

[0310] Formula VII is represented by the following structure:

[0311]

[0312] or a pharmaceutically acceptable salt thereof,

[0313] in,

[0314] L1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0315] L 4 is a linker comprising an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group;

[0316] R 4 Each instance of is H or optionally substituted alkyl; and

[0317] The RNA comprises or consists of a RNAi agent.

[0318] In some embodiments of Formula VII, L 1 , L 2 and L 3 Each is In some embodiments of Formula VII, L 1 , L 2 and L 3 Each is In some embodiments of Formula VII, L 1 , L 2 and L 3 Each is .

[0319] In some embodiments of Formula VII, L 4 Selected from:

[0320]

[0321] in Indicates the connection point.

[0322] Exemplary compounds of Formula VII are shown in Table 5 below.

[0323] Table 5. Exemplary compounds of Formula VII

[0324]

[0325]

[0326]

[0327] Formula VIII

[0328] Formula VIII is represented by the following structure:

[0329]

[0330] or a pharmaceutically acceptable salt thereof,

[0331] in,

[0332] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0333] L 4 is a linker comprising an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group;

[0334] R 3 is H, optionally substituted alkyl, and optionally substituted aryl;

[0335] R 4 is H or optionally substituted alkyl; and

[0336] TL is the targeting ligand.

[0337] In some embodiments of Formula VIII, L 1 , L 2 and L 3 Each is In some embodiments of Formula VIII, L 1 , L 2 and L 3 Each is In some embodiments of Formula VIII, L 1 , L 2 and L 3 Each is .

[0338] In some embodiments of Formula VIII, L 4 Selected from:

[0339]

[0340] in Indicates the connection point.

[0341] In some embodiments of Formula VIII, R 3 is an optionally substituted aryl. In some embodiments of Formula VIII, R 3 It is p-nitrophenyl.

[0342] Exemplary compounds of Formula VIII are shown in Table 6 below.

[0343] Table 6. Exemplary compounds of Formula VIII

[0344]

[0345]

[0346]

[0347] Formula IX

[0348] Formula IX is represented by the following structure:

[0349]

[0350] or a pharmaceutically acceptable salt thereof,

[0351] in,

[0352] L 1 , L 2 and L 3 are each independently a linker comprising an optionally substituted alkylene group;

[0353] L 4 is a linker comprising an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group;

[0354] TL is the targeting ligand;

[0355] R 4 Each instance of is H or optionally substituted alkyl; and

[0356] The RNA comprises or consists of a RNAi agent.

[0357] In some embodiments of Formula IX, L 1 , L 2 and L 3 Each is In some embodiments of Formula IX, L 1 , L 2 and L 3 Each is In some embodiments of Formula IX, L 1 , L 2 and L 3 Each is .

[0358] In some embodiments of Formula IX, L 4 Selected from:

[0359]

[0360] in Indicates the connection point.

[0361] Exemplary compounds of Formula IX are shown in Table 7 below.

[0362] Table 7. Exemplary compounds of Formula IX

[0363]

[0364]

[0365] wherein the TL comprises a targeting ligand and the RNA comprises or consists of an RNAi agent.

[0366] L 1 , L 2 , L 3

[0367] In the embodiments of Formula I-IX, L 1 , L 2 or L 3 Each instance of is a linker comprising an optionally substituted alkylene group. 1 , L 2 or L 3 Any suitable linking moiety known in the art may be included. In some embodiments, L 1 , L 2 or L 3 Contains chains between 1 and 50 atoms in length. 1 , L 2 or L 3 The chain length of indicates the number of atoms directly between the alkyne and the quaternary carbon, but additional atoms may branch from the atoms in the chain. 1 , L 2 or L 3 The length can be 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. 43, 44, 45, 46, 47, 48, 49, or 50 atoms.

[0368] In some embodiments, L 1 , L 2 and L 3 In some other embodiments, L 1 , L 2and L 3 Each is a different part.

[0369] In some embodiments, L 1 , L 2 , L 3 or L 4 Amides may be included.

[0370] In some embodiments, L 1 , L 2 , L 3 or L 4 Polyethylene glycol (PEG) chains may be included.

[0371] In some embodiments, L 1 , L 2 , L 3 or L 4 The optionally substituted alkylene may be amide, ether, ester, thioether, thioketone, ketone, amine, sulfone, sulfonamide or a chain of atoms such as, but not limited to, substituted or unsubstituted alkenyl, arylalkyl, arylalkenyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkyne The invention also provides a 3-Hydrogen-based radical, wherein the 3-Hydrogen-based radical is inserted into the radical and the residue may be an alkyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl or alkynylheteroaryl.

[0372] In some embodiments, L 1 , L 2 and L 3 Can be independently selected from: , and .

[0373] L 4

[0374] In the embodiments of Formula I-IX, L 4 is a linker comprising an optionally substituted alkylene group. 4 Any suitable linking moiety known in the art may be included. In some embodiments, L 4 Contains chains between 1 and 50 atoms in length.2 The chain length of indicates the number of atoms directly between the alkyne and the quaternary carbon, but additional atoms may branch from the atoms in the chain. 2 The length can be 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. 43, 44, 45, 46, 47, 48, 49, or 50 atoms.

[0375] In some embodiments, L 4 Selected from:

[0376]

[0377] in Indicates the connection point.

[0378] R

[0379] In embodiments of Formulas I-IX, R comprises a coupling moiety or a 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.

[0380] In some embodiments, R comprises an RNAi agent. When R comprises an RNAi agent, R may include additional atoms that do not form part of the RNAi sequence. For example, in some embodiments, R may be , wherein RNA is an RNAi agent, and The point of attachment is indicated. In some embodiments, the RNAi agent is bonded to the compound of Formula I-IX at the 5' end of the sense strand.

[0381] In some embodiments, R is selected from

[0382]

[0383] .

[0384] Pharmaceutical composition

[0385] In some embodiments, the present disclosure provides pharmaceutical compositions comprising a therapeutic compound comprising one or more triacetylene linkers disclosed herein.

[0386] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of an active pharmaceutical ingredient (API) and optionally one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than active pharmaceutical ingredients (API, therapeutic products) that are intentionally included in a drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dose. Excipients can be used to a) assist in processing drug delivery systems during manufacturing, b) protect, support or enhance the stability, bioavailability or patient acceptability of API, c) assist in product identification, and / or d) enhance any other properties of overall safety and effectiveness of API presentation during storage or use. Pharmaceutically acceptable excipients may or may not be inert substances.

[0387] Excipients include, but are not limited to, absorption enhancers, anti-adherents, anti-foaming agents, antioxidants, binders, buffers, carriers, coatings, colorants, presentation enhancers, presentation polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, water repellents, and wetting agents.

[0388] The pharmaceutical compositions described herein may contain other additional components that are typically present in pharmaceutical compositions. In some embodiments, the additional components are pharmaceutically active materials. Pharmaceutically active materials include, but are not limited to, antipruritic agents, astringents, local anesthetics or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.), small molecule drugs, antibodies, antibody fragments, aptamers, and / or vaccines.

[0389] The pharmaceutical compositions may also contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts for varying the osmotic pressure, buffers, coating agents or antioxidants. They may also contain other agents having known therapeutic benefits.

[0390] The pharmaceutical composition can be administered in many ways depending on whether local or systemic treatment is needed and the area to be treated. Administration can be performed by any means well known in the art, such as, but not limited to, topical (e.g., by transdermal patch), pulmonary (e.g., by inhalation or blowing of powder or aerosol, including by nebulizer, intratracheal, intranasal), epidermal, transdermal, oral or parenteral. Parenteral administration includes, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal (e.g., by means of implanted device), intracranial, intraparenchymal, intrathecal and intraventricular administration. In some embodiments, the pharmaceutical composition described herein is administered by subcutaneous injection. The pharmaceutical composition can be administered orally, for example, in the form of tablets, coated tablets, lozenges, hard or soft gelatin capsules, solutions, emulsions or suspensions. It can also be administered rectally (e.g., using suppositories); topically or transdermally (e.g., using ointments, creams, gels or solutions); or parenterally (e.g., using injections).

[0391] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if soluble in water) or dispersions and sterile powders for preparing sterile injection solutions or dispersions on the spot. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline. It should be stable under the conditions of manufacture and storage and should be free from the contaminating effects of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using a coating agent such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. In many cases, it is preferred to include an isotonic agent in the composition, such as sugar, polyols such as mannitol, sorbitol and sodium chloride. Extended absorption of the injectable composition can be achieved by including an agent that delays absorption in the composition, such as aluminum monostearate and gelatin.

[0392] Sterile injections can be prepared by incorporating the desired amount of active compound into an appropriate solvent, optionally with one or a combination of the above-listed ingredients, followed by filtration sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing an alkaline dispersion medium and other desired ingredients selected from those listed above. In the case of sterile powders for the preparation of sterile injections, preparation methods include vacuum drying and lyophilization, which produce a powder of the active ingredient + any additional desired ingredients from a previously filtration sterilized solution.

[0393] Formulations suitable for intra-articular administration may be in the form of a sterile aqueous preparation of any ligand described herein, the ligand may be in microcrystalline form, for example in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems may also be used to provide any ligand described herein for intra-articular and intraocular administration.

[0394] The active compound can be prepared with a carrier that prevents the compound from being rapidly removed from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The preparation methods of such preparations will be apparent to those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art as described, for example, in U.S. Patent No. 4,522,811.

[0395] The pharmaceutical composition may contain other additional components that are commonly present in pharmaceutical compositions. Such additional components include, but are not limited to, antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.). As used herein, "pharmacologically effective amount," "therapeutically effective amount," or simply "effective amount" refers to the amount of a pharmaceutically active agent that produces a pharmacological, therapeutic, or preventive result.

[0396] Medicaments containing triacetylene linkers are also an object of the present invention, as are processes for the manufacture of such medicaments, which process comprises bringing one or more compounds containing triacetylene linkers and, if desired, one or more other substances of known therapeutic benefit into a pharmaceutically usable form.

[0397] The triacetylene linkers and pharmaceutical compositions comprising the triacetylene linkers disclosed herein may be packaged or included in a kit, container, pack, or dispenser. The triacetylene linkers and pharmaceutical compositions comprising the triacetylene linkers may be packaged in pre-filled syringes or vials.

[0398] Targeting ligands, pharmacokinetic (PK) modulators, and delivery vehicles

[0399] In some embodiments, the triacetylene linker is conjugated to one or more non-nucleotide groups, including but not limited to targeting ligands, pharmacokinetic (PK) modulators, delivery polymers, or delivery vehicles. Non-nucleotides can enhance the targeting, delivery, or connection of cargo molecules. Non-nucleotides can be covalently linked to the RNAi agent at the 3' or 5' end of the sense strand. In some embodiments, the non-nucleotide is linked to the 5' end of the sense strand of the RNAi agent. In some embodiments, the triacetylene linker of Formula I is linked to the RNAi agent via an unstable, breakable, or reversible bond or linker.

[0400] In some embodiments, the non-nucleotides enhance the pharmacokinetic or biodistribution properties of the RNAi agent or conjugate to which they are attached to improve the cell or tissue-specific distribution and cell-specific uptake of the conjugate. In some embodiments, the non-nucleotides enhance the endocytosis of the RNAi agent.

[0401] Targeting ligands or targeting moieties enhance the pharmacokinetic or biodistribution properties of the cargo molecules to which they are attached to improve the cell-specific (including organ-specific in some cases) distribution and cell-specific (or organ-specific) uptake of RNAi agents. In some embodiments, targeting ligands include targeting compounds and PK enhancers or modulators. In some embodiments, targeting ligands are directed to cell receptors.

[0402] Conjugation to targeting ligands

[0403] In some embodiments, the triacetylene linker of Formula I can be conjugated to the RNAi agent via a coupling agent. An exemplary scheme for conjugating the triacetylene linker of Formula I to the RNAi molecule is shown in the following reaction scheme:

[0404]

[0405] Where L 1 , L 2 , L 3 , Q and X are as described in Formula I, R comprises a coupling portion, RG is a reactive group and L 4 is a linking group containing an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group.

[0406] In some embodiments, the targeting ligand (TL) can be conjugated to the triyne moiety first and then to the RNAi molecule. An example of such a reaction is shown in the following scheme:

[0407]

[0408] Where L 1 , L 2 , L 3 , Q and X are as described in Formula I, R comprises a coupling portion and L 4 is a linking group containing an optionally substituted alkylene group, an optionally substituted arylene group, or an optionally substituted cycloalkylene group.

[0409] RNAi molecules having reactive groups, such as amino groups (also referred to herein as amines) can be synthesized. In some embodiments, the reactive group can be attached to the 5′-end and / or the 3′-end of the RNAi agent. In some embodiments, the RNAi agent can be double-stranded. In embodiments where the RNAi agent is double-stranded, the reactive group can be on the sense strand or the antisense strand of the RNAi agent.

[0410] For example, in some embodiments, the RNAi agent is synthesized to have NH2-C6H 12 The terminal amino group can then react to form a conjugate with a coupling moiety, such as a compound of Formula I. In some embodiments, the coupling moiety is an ester, and the reactive group on the RNAi agent is a primary amine, and an amide bond is formed between the RNAi agent and the triyne linker. An example of such a reaction is shown in the following scheme using a compound of Formula VI:

[0411]

[0412] Where L 1 , L 2 , L 3 , L 4 , R 3 , R 4 and RNA are defined as in Formulae VI and VII.

[0413] In other embodiments, RNAi agents are synthesized with terminal -CH2OH groups. In some embodiments, the coupling agent in R of Formula I comprises a phosphoramidite. As shown in the following reaction scheme, an RNAi agent comprising a terminal alcohol can be reacted with a triyne of Formula II to form a phosphate:

[0414]

[0415] In some embodiments, a targeting ligand (TL) can be conjugated to a triyne linker as described herein after the triyne linker has been conjugated to the RNAi agent. An example of such a reaction is shown in the following scheme:

[0416] . Example

[0417] Example 1. Compound 1 (2-cyanoethyl ((1r, 4r)-4-((1,7-dioxo-4-(3-oxo-3-(propan-2- (1-ylamino)propyl)-1,7-bis(prop-2-yn-1-ylamino)hept-4-yl)carbamoyl)cyclohexyl)diisopropyl Synthesis of phosphoramidites .

[0418] To a solution of 1 (12.00 g, 25.6 mmol) and DIPEA (12.22 g, 16.47 mL, 94.6 mmol) in DMF (50 mL) at 0°C was added TBTU (28.72 g, 89.5 mmol). The internal temperature increased from 0°C to 16°C. Propargylamine (4.93 g, 5.73 mL, 89.5 mmol) was added dropwise while maintaining the internal temperature less than 20°C. The cooling bath was removed and the reaction mixture was stirred at room temperature overnight. 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 h, the precipitate was collected by filtration, rinsed with DCM (100 mL) and dried. Yield of 2: 10.4 g (70%). C 34 H 36 [M+H] calculated for N4O5: 581.70, found: 581.79.

[0419]

[0420] To a solution of 2 (12.17 g, 21.0 mmol) in DMF (60 mL) was added triethylamine (10.6 g, 14.7 mL, 105 mmol) at room temperature. The reaction mixture was stirred overnight. The reaction mixture was then concentrated and purified by CombiFlash® using silica gel as stationary phase and gradient elution with MeOH / DCM containing 1% triethylamine (0-13%). Yield of 3: 6.08 g (81%). 19 H 26 Calculated value of [M+H] for N4O3: 359.45, found: 359.35.

[0421]

[0422] 4 (2.55 g, 17.69 mmol) was treated with acetic anhydride (12.8 mL, 135 mmol) in pyridine (26 mL) and stirred at room temperature for 4 hours. After completion all volatiles were removed and 5 was isolated as follows: separation on silica with a gradient of ethyl acetate / hexanes containing 1% acetic acid. Yield: 2.56 g (78%). 1H 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).

[0423]

[0424] To a solution of 5 (400 mg, 2.15 mmol) in DCM (5 mL) at 0 °C was added DMF (16 mg, 17 µL, 0.215 mmol) and oxalyl chloride (1.36 g, 922 µL, 10.74 mmol). After 30 m, the cooling bath was removed and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated and the product was used in the next step without further purification.

[0425]

[0426] To a solution of 3 (1000 mg, 2.79 mmol) in DCM (10 mL) was added pyridine (1.88 g, 1.92 mL, 23.7 mmol). The reaction mixture was cooled to 0 °C and a solution of 6 (398 mg, 1.95 mmol) in DCM (5 mL) was added dropwise. The cooling bath was removed and the mixture was stirred at room temperature overnight. Water (10 mL) was added to quench the reaction. The mixture was diluted with DCM (30 mL) and washed with saturated aqueous NH4Cl (20 mL) and brine (10 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash® using silica gel as the stationary phase and eluted with a gradient of MeOH / DCM (0-7%). Yield of 7: 630 mg (43%). C 28 H 38 Calculated value of [M+H] for N4O6: 527.64, found: 527.69.

[0427]

[0428] To a solution of 7 (288 mg, 0.55 mmol) in THF (1.75 mL) was added 1 M NaOH solution (2.73 mL, 2.73 mmol) at room temperature. The reaction mixture was stirred at room temperature for 1.5 h and then heated to 35 °C for another 30 m. After the starting material was consumed, 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 by CombiFlash® using silica gel as stationary phase and eluted with a MeOH / DCM (0-10%) gradient. Yield of 8: 216 mg (81%). 26 H 36 Calculated [M+H] for N4O5: 485.61, found: 485.56.

[0429]

[0430] 8 (213 mg, 0.44 mmol) was azeotropically dried from anhydrous ACN (2 x 10 mL) and then dissolved in ACN (4 mL). The reaction mixture was cooled to 0 °C. 4,5-Dicyanoimidazole (26 mg, 0.22 mmol) was added, followed by 2-cyanoethyl N , N , N′ , N′ -Tetraisopropylphosphorodiamidite (199 mg, 0.66 mmol). The reaction mixture was stirred at 0 °C for 30 min. After the starting material was consumed, triethylamine (44 mg, 61 µL, 0.44 mmol) was added and the reaction mixture was concentrated to an oil. The oil was purified by CombiFlash® using silica gel as stationary phase and gradient elution with EtOAc / DCM containing 1% triethylamine (50-100%). Yield of 9 (Compound 1): 174 mg (58%). C 35 H 53 Calculated value of [M+H] for N6O6P: 685.83, found: 685.94.

[0431] Example 2. Compound 2 ( 2-Cyanoethyl((1s,4s)-4-((1,7-dioxo-4-(3-oxo-3-(propan-2- (1-ylamino)propyl)-1,7-bis(prop-2-yn-1-ylamino)hept-4-yl)carbamoyl)cyclohexyl)diisopropyl Phosphoramidite )

[0432]

[0433] To a solution of 10 (cis-4-hydroxycyclohexanecarboxylic acid, 2.00 g, 13.87 mmol) in pyridine (19.75 g, 20.20 mL, 250 mmol) at 0°C was added acetic anhydride (10.83 g, 10.03 mL, 106 mmol). The cooling bath was removed and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated and the residue was purified by CombiFlash® using silica gel as stationary phase and eluted with a gradient of EtOAc / hexane (0-30%). Yield of 11: 1.75 g (68%). C9H 14 Calculated [MH] for O4: 185.20, Found: 185.35.

[0434]

[0435] To a solution of 11 (420 mg, 2.26 mmol) in DCM (5 mL) was added DMF (16.5 mg, 17.4 µL, 0.226 mmol) and oxalyl chloride (1.43 g, 0.97 mL, 11.3 mmol) at 0 °C. After 30 m, the cooling bath was removed and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated, co-evaporated with toluene, and the product 12 was used in the next step without further purification.

[0436]

[0437] To a solution of 3 (400 mg, 1.12 mmol) in DMF (2 mL) was added pyridine (750 mg, 767 µL, 9.50 mmol). The reaction mixture was cooled to 0 °C and a solution of 12 (457 mg, 2.23 mmol) in DCM (2 mL) was added dropwise. The cooling bath was removed and the mixture was stirred at room temperature for 1.5 h. The mixture was diluted with DCM (20 mL) and quenched with saturated aqueous NH4Cl (10 mL). The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase and gradient elution with MeOH / DCM (0-10%). Yield of 13: 365 mg (62%). 1H NMR (400 MHz, DMSO-d6): δ 8.21 (t, 3H), 7.07 (s, 1H), 4.84 (m, 1H), 3.81 (dd, 6H), 3.07 (t, 3H), 2.18 (m, 1H), 1.99 (m, 9H), 1.80-1.72 (m, 8H),1.64-1.42 (m, 6H).

[0438]

[0439] To a solution of 13 (360 mg, 0.68 mmol) in THF (2.2 mL) was added 1 M NaOH solution (3.42 mL, 3.42 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 h and then heated to 35 °C for another 1.5 h. After the starting material was consumed, 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 by CombiFlash® using silica gel as stationary phase and eluted with a MeOH / DCM (0-12%) gradient. Yield of 8: 250 mg (75 %). 1 H NMR (400 MHz, DMSO-d6): δ 8.22 (t, 3H), 6.96 (s, 1H), 4.24 (d, 1H), 3.81 (dd, 6H), 3.75 (s, br, 1H), 3.07 (t,3H), 2.10 (m,1H), 1.99 (m, 6H), 1.82-1.58 (m, 10H), 1.36 (m, 4H).

[0440]

[0441] 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′-Tetraisopropylphosphorodiamidite (229 mg, 0.76 mmol). The reaction mixture was stirred at 0 °C for 30 m and then at room temperature for 1.5 h. The reaction mixture was concentrated to an oil and then dissolved in DCM (15 mL). The mixture was washed with saturated aqueous NaHCO3 (2 x 5 mL) and brine (5 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash® using silica gel as the stationary phase and eluted with a gradient of EtOAc / DCM containing 1% triethylamine (50-100%). Yield of 15 (Compound 2): 204 mg (59%). C 35 H 53 Calculated [M+H] for N6O6P: 683.81, found: 684.14.

[0442] Example 3. Compound 3 ( 2-Cyanoethyl(5-((1,7-dioxo-4-(3-oxo-3-(prop-2-yn-1-ylamino (1,7-bis(prop-2-yn-1-ylamino)hept-4-ylamino)-5-oxopentyl)diisopropylphosphoramidite )

[0443]

[0444] To a solution of 3 (475 mg, 1.33 mmol) in DMF (5 mL) was added triethylamine (402 mg, 555 µL, 3.98 mmol) and glutaric anhydride (190 mg, 1.65 mmol) at room temperature. The reaction mixture was stirred for 1 h, and then DMAP (8.1 mg, 0.066 mmol), MeOH (424 mg, 536 µL, 13.25 mmol) and N -(3-Dimethylaminopropyl)- N '-Ethylcarbodiimide hydrochloride (508 mg, 2.65 mmol). The mixture was stirred at room temperature overnight. The reaction mixture was diluted with DCM (20 mL) and washed with saturated aqueous NaHCO3 (10 mL). The aqueous layer was back-extracted with DCM (2 x 5 mL). The combined organic phases were dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase and gradient elution with MeOH / DCM (0-7.5%). Yield of 16: 273 mg (42%). C 25 H 34 Calculated value of [M+H] for N4O6: 487.58, found: 487.61.

[0445]

[0446] To a solution of 16 (173 mg, 0.36 mmol) in MeOH (0.87 mL) and iPrOH (1.74 mL) was added sodium borohydride (54 mg, 1.42 mmol) at 0 °C. After 30 m, the cooling bath was removed and lithium chloride (10 mg) was added. The reaction mixture was stirred at room temperature overnight. The next day, another portion of sodium borohydride (27 mg, 0.71 mmol) was added and the reaction was continued for 1 h. The reaction mixture was concentrated and purified by CombiFlash® using silica gel as stationary phase and gradient elution with MeOH / DCM (0-12%). Yield of 17: 93 mg. C 24 H 34 Calculated [M+H] for N4O5: 459.57, found: 459.63.

[0447]

[0448] Compound 17 (175 mg, 0.38 mmol) was azeotropically dried from anhydrous ACN (2 x 5 mL) and then dissolved in ACN (3 mL). The reaction mixture was cooled to 0 °C. 4,5-Dicyanoimidazole (22.5 mg, 0.19 mmol) was added, followed by 2-cyanoethyl N , N , N′ , N′ -Tetraisopropylphosphorodiamidite (173 mg, 0.57 mmol). The reaction mixture was stirred at 0 °C for 30 min and then at room temperature for 30 min. The reaction mixture was concentrated to an oil and then dissolved in DCM (15 mL). The mixture was washed with saturated aqueous NaHCO3 (5 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase and gradient elution with EtOAc / DCM containing 1% triethylamine (50-100%). Yield of 18 (Compound 3): 132 mg (53%). C 33 H 51 Calculated [M+H] for N6O6P: 659.79, found: 659.93.

[0449] Example 4. Compound 4 ( 2-Cyanoethyl((1r,4r)-4-((11,17-dioxo-14-(3-oxo-3-((2- (2-(prop-2-yn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaicosine Heptadecan-1,26-diyn-14-yl)carbamoyl)cyclohexyl)diisopropylphosphoramidite )

[0450]

[0451] To a solution of 19 (4.42 g, 5.23 mmol) in DMF (25 mL) was added triethylamine (3.63 g, 5.00 mL. 35.9 mmol) at room temperature. The reaction mixture was stirred overnight. The reaction mixture was then concentrated and purified by CombiFlash® using silica gel as stationary phase and gradient elution with MeOH / DCM (0-20%). Yield of 20: 3.08 g (95%). 1 H NMR(400 MHz, DMSO-d6): δ 7.82 (t, 3H), 4.14 (d, 6H), 3.58-3.49 (m,12H), 3.42-3.36 (m, 9H), 3.17 (q, 6H), 2.05 (m, 6H), 1.41 (m, 6H).

[0452]

[0453] To a solution of 20 (900 mg, 1.45 mmol) and compound 5 (404 mg, 2.17 mmol) in DMF (7 mL) was added O-(7-azabenzotriazol-1-yl)-hexafluorophosphate at 0 °C. N , N , N' , N' -Tetramethyluronium salt (HATU, 550 mg, 2.17 mmol) followed by DIEA (374 mg, 503 µL, 2.90 mmol). The cooling bath was removed and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated to an orange oil, which was dissolved in DCM (25 mL). The mixture was washed with 1M HCl (2 x 10 mL) and saturated aqueous NaHCO3 (10 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash® using silica gel as the stationary phase and eluted with a gradient of MeOH / DCM (0-8%). Yield of compound 27: 880 mg (77%). C 40 H 62 N4O 12 [M+H] calcd: 791.96, found: 792.08.

[0454]

[0455] To a solution of compound 27 (925 mg, 1.17 mmol) in THF (6 mL) was added 1 M NaOH (5.85 mL, 5.85 mmol) at room temperature. The mixture was heated to 35 °C for 2 h. The reaction mixture was neutralized to pH = 6 using 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 phases were dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash® using silica gel as the stationary phase and gradient elution with MeOH / DCM (0-12%). Yield of compound 28: 580 mg (66%). 1 H NMR(400 MHz, DMSO-d6): δ 7.82 (t, 3H),7.04 (s, 1H), 4.51 (d, 1H), 4.14 (d, 6H), 3.58-3.49 (m,12H), 3.42-3.36 (m, 9H), 3.18 (q, 6H), 2.06-1.92 (m, 7H), 1.88-1.62 (m, 10H), 1.35 (m, 2H), 1.10 (m, 2H).

[0456]

[0457] 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′ -Tetraisopropylphosphorodiamidite (348 mg, 1.12 mmol). The cooling bath was removed and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated to an oil and then dissolved in DCM (30 mL). The mixture was washed with saturated aqueous NaHCO3 (2 x 10 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash® using silica gel as the stationary phase and eluted with a gradient of MeOH / DCM containing 1% triethylamine (0-2%). Yield of 29 (Compound 4): 610 mg (83%). 1H NMR(400 MHz, DMSO-d6): δ 7.82(t, 3H), 7.07(s, 1H), 4.14 (d, 6H), 3.76-3.60 (m, 2H), 3.58-3.48 (m, 14H), 3.42-3.36 (m,9H), 3.18 (q, 6H), 2.74 (t, 2H), 2.12-2.04 (m, 1H), 2.02-1.89 (m, 8H), 1.83-1.67 (m, 8H), 1.45-1.31 (m, 2H),1.30-1.21 (m, 2H), 1.13 (dd, 12H). 31 P NMR(400MHz, DMSO-d6): δ 144.6.

[0458] Example 5. Compound 5 ( 2-Cyanoethyl((1s,4s)-4-((11,17-dioxo-14-(3-oxo-3-((2- (2-(prop-2-yn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaicosine Synthesis of Heptac-1,26-diyne-14-yl)carbamoyl)cyclohexyl)diisopropylphosphoramidite

[0459]

[0460] To a solution of 20 (1070 mg, 1.72 mmol) in DCM (7 mL) was added pyridine (1.22 g, 1.25 mL, 15.5 mmol). The reaction mixture was cooled to 0 °C and a solution of 12 (1.06 g, 5.15 mmol) in DCM (3.5 mL) was added dropwise. The cooling bath was removed and the mixture was stirred at room temperature for 2 h. The mixture was diluted with DCM (20 mL) and quenched with saturated aqueous NH4Cl (10 mL). The layers were separated and the organic phase was washed with saturated aqueous NaHCO3 (10 mL) and brine (10 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash® using silica gel as the stationary phase and gradient elution with MeOH / DCM (0-7%). Yield of compound 24: 295 mg (22%). C 40 H 62 N4O 12 [M+H] calcd: 791.96, found: 792.08.

[0461]

[0462] To a solution of compound 24 (290 mg, 0.37 mmol) in THF (2 mL) was added 1 M NaOH (1.83 mL, 1.83 mmol) at room temperature. The mixture was heated to 35 °C for 3 h. The reaction mixture was quenched with saturated aqueous NH4Cl (8 mL) and further acidified to pH = 6 using 2 M HCl. The mixture was extracted with DCM (3 x 15 mL). The combined organic phases were dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash using silica gel as the stationary phase and gradient eluted with MeOH / DCM (0-12%). Yield of compound 25: 183 mg (67%). 1 H NMR(400 MHz, DMSO-d6): δ 7.83 (t, 3H),6.97 (s, 1H), 4.24 (d, 1H), 4.14 (d, 6H), 3.75 (s, br,1H), 3.58-3.49 (m, 12H), 3.42-3.36 (m, 9H), 3.18 (q, 6H), 2.10 (m, 1H), 1.97(m, 6H), 1.82-1.60 (m,10H), 1.38 (m, 4H).

[0463]

[0464] Compound 25 (180 mg, 0.24 mmol) was azeotropically dried from anhydrous ACN (2 x 5 mL) and then dissolved in ACN (2 mL). The reaction mixture was cooled to 0 °C. 4,5-Dicyanoimidazole (14.2 mg, 0.12 mmol) was added, followed by 2-cyanoethyl N , N , N′ , N′ -Tetraisopropylphosphorodiamidite (109 mg, 0.36 mmol). The reaction mixture was stirred at 0 °C for 30 min and then at room temperature for 1.5 h. Another portion of 2-cyanoethyl N , N , N′ , N′-Tetraisopropylphosphorodiamidite (36 mg, 0.12 mmol) was added and the reaction mixture was stirred for another 3 h. The reaction mixture was concentrated to an oil and then dissolved in DCM (15 mL). The mixture was washed with a mixture of saturated aqueous NaHCO3 (2.5 mL) and water (2.5 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase and gradient elution with MeOH / DCM containing 1% triethylamine (0-2%). Yield of 26 (Compound 5): 116 mg (51%). C 47 H 77 N6O 12 [M+H] calcd. for P: 950.15, found: 950.18.

[0465] Example 6. Compound 6 ( 2-Cyanoethyl(11,16-dioxo-14,14-bis(3-oxo-3-((2-(2-(propano- 2-Alkyn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7-dioxa-10,15-diazaeicos-1-yn-20- (2-(2-(2-propyl)phosphoramidite) )

[0466]

[0467] To a solution of 1 (3.00 g, 6.39 mmol) and DIPEA (2.89 g, 3.89 mL 16.47 mL, 22.4 mmol) in DMF (50 mL) at 0 °C was added TBTU (6.77 g, 21.1 mmol). Then a solution of propargyl-PEG2-amine (3.02 g, 21.1 mmol) in DMF (5 mL) was added dropwise. The cooling bath was removed and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with DCM (30 mL) and washed with 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 by CombiFlash® using silica gel as stationary phase and gradient elution with MeOH / DCM (0-10%). Yield of 19: 4.42 g (82%).

[0468]

[0469] To a solution of 20 (960 mg, 1.54 mmol) in DCM (8 mL) was added triethylamine (468 mg, 645 µL, 4.62 mmol) and glutaric anhydride (220 mg, 1.93 mmol). The reaction mixture was stirred at room temperature overnight. The next day, DMAP (9.4 mg, 0.077 mmol), MeOH (494 mg, 624 µL, 15.42 mmol) and N -(3-Dimethylaminopropyl)- N '-Ethylcarbodiimide hydrochloride (591 mg, 3.08 mmol). The reaction mixture was stirred for 5 h. The reaction mixture was concentrated to an oil, which was dissolved in DCM (45 mL) and then washed with saturated aqueous NaHCO3 (10 mL) and saturated aqueous NH4Cl (10 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash® using silica gel as the stationary phase and eluted with a gradient of MeOH / DCM (0-7.5%). Yield of 21: 880 mg (76%). C 37 H 58 N4O 12 [M+H] calcd: 751.90, found: 751.90.

[0470]

[0471] To a solution of 21 (877 mg, 1.17 mmol) in THF (4 mL) and MeOH (1.75 mL) was added a solution of lithium chloride (25 mg, 0.58 mmol) in water (1.75 mL). The mixture was cooled to 0 °C and sodium borohydride (265 mg, 7.01 mmol) was added in one portion. The cooling bath was removed and the reaction mixture was stirred at room temperature overnight. The reaction mixture was quenched by the addition of saturated aqueous NH4Cl (5 mL). After stirring for 10 m, the mixture was concentrated to remove THF and MeOH. The remaining aqueous phase was diluted with water (5 mL) and extracted with DCM (3 x 20 mL). The combined organic phases were dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash® using silica gel as the stationary phase and eluted with a gradient of MeOH / DCM (0-12%). Yield of 22: 562 mg (66%). C 36 H 58 N4O 11 [M+H] calcd: 723.19, found: 723.81.

[0472]

[0473] 22 (560 mg, 0.77 mmol) was azeotropically dried from anhydrous ACN (2 x 10 mL) and then dissolved in ACN (5 mL). The reaction mixture was cooled to 0 °C. 4,5-Dicyanoimidazole (45.7 mg, 0.39 mmol) was added, followed by 2-cyanoethyl N , N , N′ , N′ -Tetraisopropylphosphorodiamidite (350 mg, 1.16 mmol). The reaction mixture was stirred at 0 °C for 30 m and then at room temperature for 30 m. The reaction mixture was concentrated to an oil and then dissolved in DCM (30 mL). The mixture was washed with saturated aqueous NaHCO3 (2 x 10 mL) and brine (10 mL). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by CombiFlash® using silica gel as the stationary phase and eluted with a gradient of MeOH / DCM containing 1% triethylamine (0-2%). Yield of compound 6: 434 mg (61%). 1 H NMR(400 MHz, DMSO-d6): δ 7.82(t, 3H),7.13(s, 1H), 4.14 (d, 6H), 3.72-3.65 (m, 2H), 3.58-3.48 (m, 16H), 3.42-3.36 (m,9H), 3.17 (q, 6H), 2.75 (t, 2H), 2.09-1.92 (m, 8H), 1.83-1.72 (m, 6H), 1.52(m, 4H), 1.13 (dd, 12H). 31 P NMR (400 MHz, DMSO-d6): δ 146.3.

[0474] Example 7. Compound 7 ( 2-Cyanoethyl(4-((11,17-dioxo-14-(3-oxo-3-((2-(2-(propano- 2-Alkyn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacos-1, 26-Diyn-14-yl)carbamoyl)phenyl)diisopropylphosphoramidite )

[0475]

[0476] Step 1. Reagent 1 (200 mg, 0.32 mmol), 4-acetoxybenzoic acid (86.4 mg, 0.48 mmol), N, N-To a solution of diisopropylethylamine (123.8 mg, 0.17 mL, d = 0.742 g / mL, 0.96 mmol) in DMF (2 mL) was added HATU (243.2 mg, 0.64 mmol). The reaction mixture was stirred at room temperature. After all the starting materials were consumed as confirmed by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL×3). The combined organic layers were washed successively with HCl (aq) and brine. The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA: DCM, MPB: 20% MeOH / DCM, 0-50% ramp in 30 min) to provide the product. Yield: 133 mg.

[0477] Step 2. The amide product from step 1 was dissolved in 2 mL MeOH and 100 mg 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% for two steps. 38 H 53 N4O 11 MS (ESI) m / z calculated for [MH]: 741.37, found: 741.67.

[0478]

[0479] To a solution of 2 (100 mg, 0.1346 mmol), diisopropylammonium tetrazolium salt (11.5 mg, 0.0673 mmol) and 3Å molecular sieves (20 mg) in DCM (2 mL) was added 2-cyanoethyl Ν,Ν,Ν',Ν' -Tetraisopropylphosphorodiamidite (60.9 mg, 0.064 mL, 0.2019 mmol, 1.5 eq). The reaction mixture was stirred at room temperature. After all the starting materials were consumed by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL×3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA: 1% TEA / DCM, MPB: 1% TEA and 4% MeOH in DCM, 0-50% ramp in 30 min) to provide compound 7. Yield: 105 mg (83%). C 47 H 70 N6O 12 MS (ESI) m / z calculated for P [MH]: 941.48, found: 941.88.

[0480] Example 8. Compound 8 ( 2-Cyanoethyl(3-((11,17-dioxo-14-(3-oxo-3-((2-(2-(propano- 2-Alkyn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacos-1, 26-Diyn-14-yl)carbamoyl)phenyl)diisopropylphosphoramidite )

[0481]

[0482] Step 1. Reagent 1 (200 mg, 0.32 mmol), 3-acetoxybenzoic acid (86.7 mg, 0.48 mmol), N, N- To a solution of diisopropylethylamine (123.8 mg, 0.17 mL, d = 0.742 g / mL, 0.96 mmol) in DMF (2 mL) was added HATU (243.2 mg, 0.64 mmol). The reaction mixture was stirred at room temperature. After all the starting materials were consumed as confirmed by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL×3). The combined organic layers were washed successively with HCl (aq) and brine. The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA: DCM, MPB: 10% MeOH / DCM, 0-40% ramp in 30 min) to provide the product. Yield: 148 mg.

[0483] Step 2. The amide product from step 1 was dissolved in 2 mL MeOH and 100 mg 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% for two steps. 38 H 55 N4O 11 MS (ESI) m / z calculated for [M+H]: 743.39, found: 743.65.

[0484]

[0485] To a solution of 3 (125 mg, 0.1683 mmol), diisopropylammonium tetrazolium salt (14.4 mg, 0.0841 mmol) and 3Å molecular sieves (20 mg) in DCM (2 mL) was added 2-cyanoethyl Ν,Ν,Ν',Ν'-Tetraisopropylphosphorodiamidite (76.1 mg, 0.08 mL, 0.2524 mmol, 1.5 eq). The reaction mixture was stirred at room temperature. After all the starting materials were consumed by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL×3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA: 1% TEA / DCM, MPB: 1% TEA and 4% MeOH in DCM, 0-50% ramp in 30 min) to provide compound 8. Yield: 130 mg (82%). C 47 H 70 N6O 12 MS (ESI) m / z calcd for P[MH]: 941.48, found: 941.79.

[0486] Example 9. Compound 9 ( 2-Cyanoethyl(2-((11,17-dioxo-14-(3-oxo-3-((2-(2-(propano- 2-Alkyn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptacos-1, 26-Diyn-14-yl)carbamoyl)phenyl)diisopropylphosphoramidite )

[0487]

[0488] Step 1. To a solution of 1 (200 mg, 0.32 mmol), triethylamine (97.3 mg, 0.134 mL, d = 0.726 g / mL, 0.96 mmol) in DCM (2 mL) was added o-acetylsalicylic acid chloride (127.6 mg, 0.6423 mmol, 1.2 eq, CAS Registry Number: 5538-51-2). The reaction mixture was stirred at room temperature. After all the starting materials were consumed by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL×3). The combined organic layers were washed successively with HCl (aq) and brine. The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA: DCM, MPB: 10% MeOH / DCM, 0-50% ramp in 30 min) to provide the product. Yield: 177.8 mg.

[0489] Step 2. The amide product from step 1 was dissolved in 2 mL MeOH and 100 mg 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% for two steps. 38 H 53 N4O 11MS (ESI) m / z calculated for [MH]: 741.39, found: 741.67.

[0490]

[0491] To a solution of 4 (105 mg, 0.1457 mmol), diisopropylammonium tetrazolium salt (12.5 mg, 0.0728 mmol) and 3Å molecular sieves (20 mg) in DCM (2 mL) was added 2-cyanoethyl Ν,Ν,Ν',Ν' -Tetraisopropylphosphorodiamidite (66 mg, 0.069 mL, 0.2185 mmol, 1.5 eq). The reaction mixture was stirred at room temperature. After all the starting materials were exhausted by LC-MS, the reaction mixture was quenched with 2 mL of saturated NaHCO3 aqueous solution and extracted with ethyl acetate (10 mL×3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude material was loaded onto a silica gel column and purified (MPA: 1% TEA / DCM, MPB: 1% TEA and 4% MeOH in DCM, 0-50% ramp in 30 min) to provide compound 9. Yield: 181 mg (83%). C 47 H 70 N6O 12 MS (ESI) m / z calculated for P [MH]: 941.48, found: 941.79.

[0492] Example 10. Compound 10 ( 2-Cyanoethyl(4'-((11,17-dioxo-14-(3-oxo-3-((2-(2- (prop-2-yn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptazone Carbon-1,26-diyn-14-yl)carbamoyl)-[1,1'-biphenyl]-4-yl)diisopropylphosphoramidite )

[0493]

[0494] 1 (200 mg, 0.3212 mmol), 4′-hydroxy-4-biphenylcarboxylic acid (103.2 mg, 0.4817 mmol) and N,N-To a solution of diisopropylethylamine (124.5 mg, 0.17 mL, d = 0.742 g / mL, 0.96 mmol) in DMF (2 mL) was added HATU (244.2 mg, 0.64 mmol). The reaction mixture was stirred at room temperature. After all the starting materials were consumed by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL×3). The combined organic layers were washed successively with HCl (aq) and brine. The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA: DCM, MPB: 10% MeOH / DCM, 0-50% ramp in 30 min) to provide the product. Yield: 138 mg, 52%. C 44 H 59 N4O 11 MS (ESI) m / z calcd. for [M+H]: 819.42, found: 819.90.

[0495]

[0496] To a solution of 5 (138 mg, 0.1685 mmol), diisopropylammonium tetrazolium salt (14.4 mg, 0.0843 mmol) and 3Å molecular sieves (20 mg) in DCM (2 mL) was added 2-cyanoethyl Ν,Ν,Ν',Ν' -Tetraisopropylphosphorodiamidite (76.2 mg, 0.08 mL, 0.2528 mmol, 1.5 eq). The reaction mixture was stirred at room temperature. After all the starting materials were exhausted by LC-MS, the reaction mixture was quenched with 2 mL of saturated NaHCO3 aqueous solution and extracted with ethyl acetate (10 mL×3). It was dried over Na2SO4 and concentrated under high vacuum. The crude material was loaded onto a silica gel column and purified (MPA: 1% TEA / DCM, MPB: 1% TEA and 4% MeOH in DCM, 0-50% ramp in 30 min) to provide compound 10. Yield: 171 mg (99%). C 53 H 74 N6O 12 MS (ESI) m / z calcd for P [MH] 1017.51, found 1017.99.

[0497] Example 11. Compound 11 ( 2-Cyanoethyl((1r,3r)-3-((11,17-dioxo-14-(3-oxo-3- ((2-(2-(Prop-2-yn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazepine Heptacosa-1,26-diyn-14-yl)carbamoyl)cyclobutyl)diisopropylphosphoramidite )

[0498]

[0499] 1 (300 mg, 0.4817 mmol), trans-3-hydroxycyclobutanecarboxylic acid (83.9 mg, 0.7226 mmol, CAS No.: 1268521-85-2) and N,N- To a solution of diisopropylethylamine (186.8 mg, 0.252 mL, d = 0.742 g / mL, 1.4452 mmol) in DMF (3 mL) was added HATU (366.3 mg, 0.9635 mmol). The reaction mixture was stirred at room temperature. After all the starting materials were consumed as confirmed by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL×3). The combined organic layers were washed successively with HCl (aq) and brine. The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA:DCM, MPB: 10% MeOH / DCM, 0-100% ramp in 30 min) to provide the product. Yield: 333.2 mg, 88%. C 36 H 57 N4O 11 MS (ESI) m / z calcd. for [M+H]: 721.40, found: 721.96.

[0500]

[0501] To a solution of 6 (166.5 mg, 0.2310 mmol), diisopropylammonium tetrazolium salt (19.8 mg, 0.1155 mmol) and 3Å molecular sieves (20 mg) in DCM (2 mL) was added 2-cyanoethyl Ν,Ν,Ν',Ν' - Tetraisopropylphosphorodiamidite (104.4 mg, 0.11 mL, 0.3465 mmol, 1.5 eq). The reaction mixture was stirred at room temperature. After all the starting materials were consumed by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL×3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA: 1% TEA / DCM, MPB: 1% TEA and 4% MeOH in DCM, 0-50% ramp in 30 min) to provide compound 11. Yield: 200 mg (94%). C 45 H 72 N6O 12 MS (ESI) m / z calculated for P [MH]: 919.50, found: 919.73.

[0502] Example 12. Compound 12 ( 2-Cyanoethyl(4-((11,17-dioxo-14-(3-oxo-3-((2-(2- (prop-2-yn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptazone (1,26-diyn-14-yl)carbamoyl)bicyclo[2.2.2]octan-1-yl)diisopropylphosphoramidite )

[0503]

[0504] 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- To a solution of diisopropylethylamine (373.6 mg, 0.503 mL, d =0.742 g / mL, 2.8904 mmol) in DMF (5 mL) was added HATU (732.7 mg, 1.9269 mmol). The reaction mixture was stirred at room temperature. After all the starting materials were consumed by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL×3). The combined organic layers were washed successively with HCl (aq) and brine. The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA: DCM, MPB: 10% MeOH / DCM, 0-100% ramp in 30 min) to provide the product. Yield: 398 mg, 54%.C 40 H 61 N4O 11 MS (ESI) m / z calcd for [MH]: 773.45, found: 773.80.

[0505]

[0506] To a solution of 7 (200 mg, 0.2581 mmol), diisopropylammonium tetrazolium salt (22.1 mg, 0.1290 mmol) and 3Å molecular sieves (20 mg) in DCM (2 mL) was added 2-cyanoethyl Ν,Ν,Ν',Ν'-Tetraisopropylphosphorodiamidite (116.7 mg, 0.123 mL, 0.3871 mmol, 1.5 eq). The reaction mixture was stirred at room temperature. After all the starting materials were exhausted by LC-MS, the reaction mixture was quenched with 2 mL of saturated NaHCO3 aqueous solution and extracted with ethyl acetate (10 mL×3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA: 1% TEA / DCM, MPB: 1% TEA and 4% MeOH in DCM, 0-50% ramp in 30 min) to provide compound 12. Yield: 40 mg (16%). C 49 H 78 N6O 12 MS (ESI) m / z calcd for P[MH]: 973.54, found: 973.75.

[0507] Example 13. Compound 13 ( 2-cyanoethyl(3-((11,17-dioxo-14-(3-oxo-3-((2-(2- (prop-2-yn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7,21,24-tetraoxa-10,18-diazaheptazone (1,26-diyn-14-yl)carbamoyl)bicyclo[1.1.1]pentan-1-yl)diisopropylphosphoramidite )

[0508]

[0509] 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- To a solution of diisopropylethylamine (249.1 mg, 0.336 mL, d = 0.742 g / mL, 1.9269 mmol) in DMF / DCM (10 mL, 1:1 v / v) was added HATU (488.4 mg, 1.2846 mmol). The reaction mixture was stirred at room temperature. After all the starting materials were consumed by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL×3). The combined organic layers were washed successively with HCl (aq) and brine. The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA:DCM, MPB: 10% MeOH / DCM, 0-100% ramp in 30 min) to provide 8. Yield: 387.6 mg, 82%. C 37 H 57 N4O 11 MS (ESI) m / z calculated for [M+H]: 733.40, found: 733.66.

[0510]

[0511] To a solution of 8 (387.6 mg, 0.5289 mmol), diisopropylammonium tetrazolium salt (45.3 mg, 0.2644 mmol) and 3Å molecular sieves (20 mg) in DCM (2 mL) was added 2-cyanoethyl Ν,Ν,Ν',Ν' -Tetraisopropylphosphorodiamidite (239.1 mg, 0.252 mL, 0.7933 mmol, 1.5 eq). The reaction mixture was stirred at room temperature. After all the starting materials were consumed by LC-MS, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL×3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude material was loaded onto a silica gel column and purified (MPA: 1% TEA / DCM, MPB: 1% TEA and 4% MeOH in DCM, 0-50% ramp in 30 min) to provide a pure phosphoramidite product. Yield: 206.7 mg (42%). C 46 H 72 N6O 12 MS (ESI) m / z calculated for P [MH]: 931.50, found: 931.71.

[0512] Example 14. Compound 14 ( 2-Cyanoethyl(11,16,20-trioxo-14,14-bis(3-oxo-3-((2- (2-(prop-2-yn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7-dioxa-10,15,21-triazaheptazoline (Carbon-1-yn-27yl)diisopropylphosphoramidite ) and compound 22 ( 11,16-dioxo-14,14-bis(3-oxo-3-((2- (2-(prop-2-yn-1-yloxy)ethoxy)ethyl)amino)propyl)-4,7-dioxa-10,15-diazaeicosane-1- Alkyne-20-oic acid 4-nitrophenyl ester )

[0513]

[0514] To a 3 liter jacketed reactor was added 500 mL of DCM and 4 (75.0 g, 0.16 mol). The internal temperature of the reaction was cooled to 0°C and TBTU (170.0 g, 0.53 mol) was added. The suspension was then treated dropwise with amine 5 (75.5 g, 0.53 mol), keeping the internal temperature less than 5°C. The reaction was then treated slowly with DIPEA (72.3 g, 0.56 mol), keeping the internal temperature less than 5°C. After the addition was complete, the reaction was warmed to 23°C over 1 hour and allowed to stir for 3 hours. A 10% kicker charge of all three reagents was added and allowed to stir for an additional 3 hours. The reaction was considered complete when <1% of 4 remained. The reaction mixture was washed with saturated ammonium chloride solution (2 x 500 mL) and once with saturated sodium bicarbonate solution (500 mL). The organic layer was then dried over sodium sulfate and concentrated to an oil. The mass of the crude oil was 188 g and it contained 72% 6 as determined by QNMR. The crude oil was sent to the next step. 46 H 60 N4O 11 Calculated mass = 845.0 m / z. Found [M+H] = 846.0.

[0515]

[0516] 121.2 grams 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%), keeping the internal temperature below 23 °C. The formation of dibenzofulvene (DBF) relative to the consumption of Fmoc-amine 6 was monitored by HPLC method 1 and the reaction was complete within 10 hours. Glutaric anhydride (12.8 g, 0.11 mol) was added to the solution and the intermediate amine 7 was converted to compound 8 within 2 hours. After completion, DMF and TEA were removed under reduced pressure at 30 °C to produce 100 grams of crude oil. Due to the high solubility of compound 7 in water, aqueous workup could not be used and chromatography was the only way to remove DBF, TMU and glutaric anhydride. The crude oil (75 g) was purified in three portions on a Teledyne ISCO Combi-flash® purification system. The crude oil (25 g) was loaded onto a 330 g silica gel column and eluted with 0-20% methanol / DCM over 30 minutes to yield 42 g of compound 8 (54% yield over 3 steps). 36 H 55 N4O 12 Calculated mass = 736.4 m / z. Found [M+H] = 737.0.

[0517]

[0518] Compound 8 (42.0 g, 0.057 mol) was co-stripped with 10 volumes of acetonitrile before use to remove any residual methanol from the chromatographic solvent. The oil was redissolved in DMF (210 mL) and cooled to 0°C. The solution was treated with 4-nitrophenol (8.7 g, 0.063 mol) followed by EDC hydrochloride (12.0 g, 0.063 mol) and found to reach completion within 10 hours. The solution was cooled to 0°C and 10 volumes of ethyl acetate were added followed by 10 volumes of saturated ammonium chloride solution, keeping the internal temperature below 15°C. The layers were allowed to separate and the ethyl acetate layer was washed with brine. The combined aqueous layers were extracted twice with 5 volumes of ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated to an oil. The crude oil (55 g) was purified in three portions on a Teledyne ISCO Combi-flash® purification system. The crude oil (25 g) was loaded onto a 330 g silica gel column and eluted from 0 - 10% methanol / DCM over 30 min to give 22 g of pure 9 (Compound 22) (50% yield). 42 H 59 N5O 14 Calculated mass = 857.4 m / z. Found [M+H] = 858.0.

[0519]

[0520] A solution of ester 9 (49.0 g, 57.1 mmol) and 6-amino-1-hexanol (7.36 g, 6.28 mmol) in dichloromethane (3 volumes) was treated dropwise with triethylamine (11.56 g, 111.4 mmol). The reaction was monitored by observing the disappearance of compound 9 on HPLC method 1 and was found to be complete within 10 minutes. The crude reaction mixture was diluted with 5 volumes of dichloromethane and washed with saturated ammonium chloride (5 volumes) and brine (5 volumes). The organic layer was dried over sodium sulfate and concentrated to an oil. The crude oil was purified on a Teledyne ISCO Combi-flash® purification system using a 330 g silica gel column. 4-Nitrophenol was eluted with 100% ethyl acetate and washed from the column with 20% methanol / DCM for 10 to produce a colorless oil (39 g, 81% yield). 42 H 69 N5O 12 Calculated mass = 836.0 m / z. Found [M+H] = 837.0.

[0521]

[0522] Alcohol 10 was co-stripped twice with 10 volumes of acetonitrile to remove any residual methanol from the chromatographic solvent and once more with anhydrous dichloromethane (KF < 60 ppm) to remove traces of water. Alcohol 10 (2.30 g, 2.8 mmol) was dissolved in 5 volumes of anhydrous dichloromethane (KF < 50 ppm) and treated with diisopropylammonium tetrazolium salt (188 mg, 1.1 mmol). The solution was cooled to 0°C and treated dropwise with 2-cyanoethyl N,N,N',N'-tetraisopropylphosphoramidite (1.00 g, 3.3 mmol). The solution was removed from the ice bath and stirred at 20°C. The reaction was found to be complete in 3-6 hours. The reaction mixture was cooled to 0°C and treated with 10 volumes of 1:1 saturated ammonium bicarbonate / brine solution, then warmed to ambient temperature over 1 minute and allowed to stir at 20°C for an additional 3 minutes. The two-phase mixture was transferred to a separatory funnel and 10 volumes of dichloromethane were added. The organic layer was separated and washed with 10 volumes of saturated sodium bicarbonate solution to hydrolyze the unreacted bisphosphorus reagent. The organic layer was dried over sodium sulfate and concentrated to an oil to yield 3.08 g of 94 wt % compound 14. 51 H 86 N7O 13 Calculated mass for P = 1035.6 m / z. Found [M+H] = 1036.

[0523] Example 15. Compound 15 ( 5-((1,3-bis(prop-2-yn-1-yloxy)-2-((prop-2-yn-1-yloxy 4-nitrophenyl (2-nitrophenyl)-5-oxopentanoate )

[0524]

[0525] Step 1. Di-tert-butyl dicarbonate (2.35 g, 10.7 mmol) was added t A solution in BuOH (10 mL) was added to a suspension of tris(hydroxymethyl)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 h. The solvent was removed under reduced pressure to provide a residue, which was purified by precipitation with cold EtOAc. Vacuum filtration provided the pure compound (1.4449, 80% yield) as a white solid. C9H 20 MS (ESI) m / z calculated for NO5[M+H]: 222.13, found: 222.24.

[0526] Step 2. A solution of triol-NHBoc 10 (500 mg, 2.26 mmol) in anhydrous DMF (6 mL) was stirred at 0°C with propargyl bromide (80 wt% in toluene, 1.46 mL, 13.6 mmol). Several portions of finely ground KOH (951 mg, 13.6 mmol) were added over a period of 15 min. The mixture was then heated to 35°C and stirred under nitrogen atmosphere for 24 h. 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 was loaded onto a silica gel column and purified (MPA: hexane, MPB: EA, 0-10% ramp in 30 min) to provide the pure product 11. Yield: 483 mg (64%).

[0527] Steps 3 and 4. To a solution of trialk-NHBoc 11 (483 mg, 1.44 mmol) in anhydrous DCM (5.6 mL) was added TFA (2.3 mL) dropwise at 0°C. The brown mixture was then stirred at room temperature for 2 h. Concentration under high vacuum afforded a solid without further purification. The crude was dissolved into 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 stationary phase afforded 0.9357 g of product 14. (MPA: DCM, MPB: 20% MeOH / DCM, 0-50% ramp in 30 min). 18 H 22 MS (ESI) m / z calculated for NO6[MH]: 348.15, found: 348.28.

[0528]

[0529] At 0 °C, 14 (470 mg, 1.3 mmol) and p-Nitrophenol (936 mg, 6.7 mmol, 5 eq) in DCM (10 mL) was added EDC HCl salt (1.28 g, 6.7 mmol, 5 eq). The reaction mixture was then stirred at room temperature. After all the starting materials were consumed by LC-MS, 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 was loaded onto a silica gel column and purified (MPA: hexane, MPB: EA, 0-60% ramp in 30 min) to provide the pure product in the form of a yellow oil. Yield: 471 mg (77%). C 24 H 27 MS (ESI) m / z calculated for N2O8[M+H]: 471.18, found: 471.33.

[0530] Example 16. Compound 16 ( 5-(((S)-1-(((R)-1,5-dioxo-1,5-bis(prop-2-yn-1-ylamino 4-nitro-5-((1,5-dioxo-5-(prop-2-yn-1-ylamino)pentan-2-yl)amino)-1,5-dioxo-5-(prop-2-yn-1-ylamino)pentan-2-yl)amino)-5-oxopentanoic acid Phenyl ester )

[0531]

[0532] Step 1. To a solution of methyl 3,4,5-trihydroxybenzoate 15 (4.6 g, 25 mmol, CAS No. 99-24-1) and propargyl bromide (11.9 g, 11.1 mL, d = 1.57 g / mL, 100 mmol, 4 eq) in DMF (50 mL) was added K2CO3 (13.8 g, 100 mmol, 4 eq). The reaction mixture was then stirred at room temperature overnight. After the starting material was consumed as confirmed by TLC, the reaction mixture was filtered and concentrated under reduced pressure.

[0533] Step 2. The above crude was dissolved in EtOH / H2O (200 mL, 1:1 v / v), and then 90 mL of 4 M NaOH aqueous solution was added to the reaction. After all starting materials were consumed as confirmed by TLC, the reaction mixture was concentrated under reduced pressure to remove EtOH and filtered to provide 17 (6.18 g), a white solid. The solid was used in the next step without further purification.

[0534] Step 3. To a solution of 17 (73 mg, 0.35 mmol) and PNP (139 mg, 1 mmol, 3 eq) in DCM (5 mL) was added EDC HCl salt (191 mg, 1 mmol, 3 eq) at 0°C. The reaction mixture was then stirred at room temperature. After all the starting material was consumed as confirmed by TLC, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL×3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA: hexane, MPB: EA, 0-40% ramp in 30 min) to provide compound 16. C 22 H 14 MS (ESI) m / z calculated for NO7[MH]: 404.08, found: 404.48.

[0535] Example 17. Compound 17 ( 5-(((S)-1-(((R)-1,5-dioxo-1,5-bis(prop-2-yn-1-ylamino 4-nitro-5-((1,5-dioxo-5-(prop-2-yn-1-ylamino)pentan-2-yl)amino)-1,5-dioxo-5-(prop-2-yn-1-ylamino)pentan-2-yl)amino)-5-oxopentanoic acid Phenyl ester )

[0536]

[0537] Step 1. At 0°C, acid 18 (4.225 g, 10 mmol), amine 19 (2.959 g, 10 mmol), N,N- To a solution of diisopropylethylamine (3.87 g, 0.52 mL, d = 0.742 g / mL, 30 mmol) in DMF (20 mL) was added HBTU (5.685 g, 15 mmol). The reaction mixture was stirred at room temperature. After all starting materials were consumed as confirmed by LC-MS, the reaction was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (20 mL×3). The combined organic layers were washed with brine. The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA: hexane, MPB: EA, 0-33% ramp in 30 min) to provide the product 20, which was used in the next step. C 37 H 51 MS (ESI) m / z calculated for N2O9[M+H]: 667.36, found: 667.49.

[0538] Step 2. The product from step 1 was dissolved in TFA / DCM (20 mL, 1:1 v / v). The reaction was stirred at room temperature for 3 h. After all starting materials were consumed as confirmed by LC-MS, the mixture was concentrated under reduced pressure overnight. Yield 3.4 g. C25 H 25 MS (ESI) m / z calculated for N2O9[MH]: 497.16, found: 497.35.

[0539] Step 3. To a flame-dried round-bottom flask were added 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). Cooled to 0 °C and TBTU (2.256 g, 7.0281 mmol. 3.5 eq) was added. After all starting materials were consumed as confirmed by LC-MS, the reaction mixture was concentrated under reduced pressure. The product was obtained by filtration and washed with DCM (5 mL) and H2O (5 mL). Lyophilized overnight to provide 0.8818 g of 22 as a white solid. 34 H 36 MS (ESI) m / z calculated for N5O6[M+H]: 610.27, found: 610.41.

[0540] Steps 4, 5 and 6. 22 (100 mg, 0.1642 mmol) in DMF (1 mL) was added to triethylamine (0.1658 g, 0.228 mL, d = 0.726 g / mL, 1.6420 mmol) at room temperature. The reaction mixture was stirred overnight. After all starting material was consumed as confirmed by LC-MS, glutaric anhydride (28.1 mg, 0.2463 mmol) and DMAP (2.0 mg, 0.0164 mmol) were added. The reaction mixture was stirred overnight. PNP (114.1 mg, 0.821 mmol) and EDC-HCl (156.8 mg, 0.8210 mmol) were added. After the starting material was consumed, the reaction mixture was concentrated and purified by CombiFlash® using silica gel as stationary phase and gradient elution with MeOH / DCM (0-20%). Yield: 34 mg, 34%. C 30 H 35 MS (ESI) m / z calculated for N6O9[M+H]: 623.25, found: 623.38.

[0541] Example 18 Compound 18 ( 5-((1,7-dioxo-4-(3-oxo-3-(prop-2-yn-1-ylamino)propane 4-nitrophenyl 1,7-bis(prop-2-yn-1-ylamino)hept-4-yl)amino)-5-oxopentanoate )

[0542]

[0543] To a solution of 3 (1.00 g, 2.79 mmol) in DMF (5 mL) was added triethylamine (0.847 g, 1.17 mL, 8.37 mmol) and glutaric anhydride (493 mg, 4.32 mmol) at room temperature. The reaction mixture was stirred overnight. The next day, 4-nitrophenol (896 mg, 6.44 mmol) and N -(3-Dimethylaminopropyl)- N '-Ethylcarbodiimide hydrochloride (1.23 g, 6.44 mmol) and the reaction mixture was stirred overnight. The reaction mixture was concentrated. The residue was purified by CombiFlash® using silica gel as stationary phase and gradient elution with MeOH / DCM (0-6%). Yield of 31 (Compound 18): 1.13 g (74%). 30 H 35 [M+H] calculated for N5O8: 594.65, found: 594.39.

[0544] Example 19. Compound 19 ( 3-((1,7-dioxo-4-(3-oxo-3-(prop-2-yn-1-ylamino)propane 2,3,5,6-bis(prop-2-yn-1-ylamino)hept-4-yl)carbamoyl)bicyclo[1.1.1]pentane-1-carboxylic acid Tetrafluorophenyl ester )

[0545]

[0546] Step 1. To a solution of acid 26 (52.2 mg, 0.3073 mmol, 1.1 eq), TBTU (134.5 mg, 0.4190 mmol, 1.5 eq) and DIEA (108.1 mg, 0.1457 mL, d = 0.742 g / mL, 0.8380 mmol) in DMF (0.5 mL) was added amine 25 (100 mg, 0.2793 mmol). The reaction was stirred at room temperature. After all starting material was consumed as confirmed by LC-MS, the reaction mixture was concentrated under reduced pressure. Purification on Combiflash® (MPA: DCM, MPB: 20% MeOH / DCM, 0-50% ramp in 30 min) afforded the pure product 27. Yield: 114 mg, 80%. C 27 H 35 MS (ESI) m / z calculated for N4O6[M+H]: 511.26, found: 511.75.

[0547] Step 2. The above product was dissolved in THF / H2O (0.6 mL, 2:1 v / v), and then LiOH (16 mg, 0.66 mmol, 3 eq) was added to the reaction. After all the starting material was consumed as confirmed by LC-MS, the reaction mixture was neutralized by adding 0.66 mmol HCl (aq). The mixture was concentrated under reduced pressure and lyophilized over the weekend. The crude material was used in the next step without further purification.

[0548] Step 3. To a solution of 28, TFP (182.6 mg, 1.1 mmol, 5 eq) and DIEA (179.6 mg, 0.242 mL, d = 0.742 g / mL, 1.39 mmol) in DCM (5 mL) was added EDC HCl salt (210.1 mg, 1.1 mmol, 5 eq) at 0°C. The reaction mixture was then stirred at room temperature. After all starting materials were consumed as confirmed by TLC, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL×3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA: hexane, MPB: EA, 0-50% ramp in 30 min) to provide compound 19. Yield: 89 mg (63%). C 32 H 33 MS (ESI) m / z calculated for F4N4O6[M+H]: 645.23, found: 645.79.

[0549] Example 20. Compound 20 ( 4'-((1,7-dioxo-4-(3-oxo-3-(prop-2-yn-1-ylamino)propane 2,3,5,6-(1,7-bis(prop-2-yn-1-ylamino)hept-4-yl)carbamoyl)-[1,1'-biphenyl]-4-carboxylic acid Tetrafluorophenyl ester )

[0550]

[0551] Step 1. To a solution of acid 29 (78.7 mg, 0.3073 mmol, 1.1 eq), TBTU (134.5 mg, 0.4190 mmol, 1.5 eq) and DIEA (108.1 mg, 0.1457 mL, d = 0.742 g / mL, 0.8380 mmol) in DMF (0.5 mL) was added 25 (100 mg, 0.2793 mmol). The reaction was stirred at room temperature. After all starting material was consumed as confirmed by LC-MS, the reaction mixture was concentrated under reduced pressure. Purification on Combiflash® (MPA: DCM, MPB: 20% MeOH / DCM, 0-50% ramp in 30 min) afforded the pure product 30. Yield: 165 mg, 99%. C 34 H 37 MS (ESI) m / z calculated for N4O6[M+H]: 597.27, found: 597.81.

[0552] Step 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 all starting material was consumed as confirmed by LC-MS, 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 was used in the next step without further purification.

[0553] Step 3. To a solution of 31, TFP (231 mg, 1.39 mmol, 5 eq) and DIEA (179.6 mg, 0.242 mL, d = 0.742 g / mL, 1.39 mmol) in DCM (5 mL) was added EDC HCl salt (266 mg, 1.39 mmol, 5 eq) at 0°C. The reaction mixture was then stirred at room temperature. After all the starting material was consumed as confirmed by TLC, the reaction mixture was quenched with 2 mL of saturated aqueous NaHCO3 and extracted with ethyl acetate (10 mL×3). The organic layer was dried over Na2SO4 and concentrated under high vacuum. The crude was loaded onto a silica gel column and purified (MPA: hexane, MPB: EA, 0-50% ramp in 30 min) to provide the pure product compound 20. Yield: 87 mg (42%). C 39 H 35 F4N4O6[M+H] + MS (ESI) m / z calculated value: 731.25, found value: 731.85.

[0554] Example 21. Compound 21 ( (4-nitrophenyl)carbonic acid (1r,4r)-4-((1,7-dioxo-4-(3-oxo- 3-(prop-2-yn-1-ylamino)propyl)-1,7-bis(prop-2-yn-1-ylamino)hept-4-yl)carbamoyl)cyclohexyl ester )

[0555]

[0556] To compound 8 (see Example 1) (0.048 g, 0.10 mmol) and DIEA (0.18 mL, 1.0 mmol) in THF (0.5 mL) was added 4-nitrophenyl chloroformate (0.044 g, 0.22 mmol) and the reaction was stirred at 50°C. After completion all volatiles were removed and compound 21 was isolated by separation over silica eluting with a MeOH / DCM gradient. Yield: 0.035 g (54%).

[0557] Example 22. Synthesis of tridentate ligands and conjugation of targeting ligands to RNAi agents

[0558] Targeting ligands can be conjugated to one or more RNAi agents, which can be used to inhibit the expression of one or more targeted genes. Targeting ligands promote the presentation of RNAi agents to targeted cells and / or tissues. Targeting ligands may include certain portions that interact with cell surface receptors, thereby introducing RNAi agents into cells. The general procedure for synthesizing certain targeting ligand-RNAi agent conjugates using the triacetylene linkers described herein illustrated in the non-limiting examples given herein is described below.

[0559] A. Synthesis of RNAi Agents

[0560] RNAi agents can be synthesized using methods known in the art. In order to synthesize the RNAi agents exemplified in the embodiments given herein, the sense and antisense strands of the RNAi agents are synthesized according to the phosphoramidite technology on a solid phase for oligonucleotide synthesis. Depending on the scale, MerMade96E® (Bioautomation), MerMade12® (Bioautomation) or OPPilot 100 (GE Healthcare) are used. Synthesis is performed on a solid support made of controlled pore glass (CPG, 500 Å or 600 Å, obtained from PrimeSynthesis, Aston, PA, USA). All RNA and 2′-modified RNA phosphoramidites are purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the following 2′-O-methyl phosphoramidites are used: (5′-O-dimethoxytrityl-N 6-(Benzoyl)-2′-O-methyl-adenosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, 5′-O-dimethoxy-trityl-N 4 -(acetyl)-2′-O-methyl-cytidine-3′-O-(2-cyanoethyl-N,N-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-phosphoramidites carry the same protecting groups as the 2′-O-methyl RNA amidites. 5′-Dimethoxytrityl-2′-O-methyl-inosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite was purchased from Glen Research (Virginia). Reverse abasic (3′-O-dimethoxytrityl-2′-deoxyribose-5′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite was purchased from ChemGenes (Wilmington, MA, USA). USA). The following UNA phosphoramidites were used: 5′-(4,4′-dimethoxytrityl)-N6-(benzoyl)-2′,3′-sec-adenosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′(4,4′-dimethoxytrityl)-N-acetyl-2′,3′-sec-cytosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite Phosphoramidites, 5′-(4,4′-dimethoxytrityl)-N-isobutyryl-2′,3′-sec-guanosine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and 5′-(4,4′-dimethoxytrityl)-2′,3′-sec-uridine, 2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. TFA aminolinkphosphoramidites are also available (ThermoFisher).

[0561] Alternatively, the triyne moiety was introduced after solid support synthesis (see section F below). For this route, the sense strand was functionalized with 5′ and / or 3′ terminal nucleotides containing primary amines. The TFA amino linker phosphoramidite was dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3Å) were added. As activator solution, 5-benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used. Coupling times were 10 min (RNA), 90 s (2′ O-Me), and 60 s (2′ F). To introduce phosphorothioate bonds, a 100 mM solution of 3-phenyl-1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was used.

[0562] In some embodiments, the compound of formula III is synthesized by reacting a compound of formula II that can be added to the end of the RNAi agent. In some embodiments, a triacetylene linker of formula II is added to the 5' end of the sense strand of a double-stranded RNAi agent. In some embodiments, a triacetylene linker of formula II is added to the 3' end of the sense strand of a double-stranded RNAi agent. In some embodiments, a compound of formula II is added to the 5' end of the antisense strand of a double-stranded RNAi agent. In some embodiments, a compound of formula II is added to the 3' end of the antisense strand of a double-stranded RNAi agent. An exemplary reaction of this type is shown in the following scheme:

[0563]

[0564] When used in conjunction with the RNAi agents given in certain embodiments herein, the triyne-containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other amidites were dissolved in anhydrous acetonitrile (50 mM), and molecular sieves (3Å) 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-phenyl-1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous acetonitrile was used.

[0565] B. Cleavage and deprotection of support bound oligomers.After completion of the solid phase synthesis, the dried solid support was treated with a 1:1 volume solution of 40 wt % methylamine in water and a 28% to 31% ammonium hydroxide solution (Aldrich) for 1.5 hours at 30° C. The solution was evaporated and the solid residue was reconstituted in water (see below).

[0566] C. Purification. The crude oligomers were purified by anion exchange HPLC using a TSKgel SuperQ-5PW 13µm column and a Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0 and contained 20% acetonitrile, and buffer B was the same as buffer A with the addition of 1.5 M sodium chloride. UV traces were recorded at 260 nm. Appropriate fractions were pooled and run on size exclusion HPLC using a GE Healthcare XK 16 / 40 column packed with Sephadex G25 fine using a running buffer of 100 mM ammonium bicarbonate, pH 6.7 and 20% acetonitrile or filtered water.

[0567] D. Annealing. Complementary strands were mixed to form RNAi agents by combining equimolar RNA solutions (sense and antisense) in 1× PBS (phosphate buffered saline, 1×, Corning, Cellgro). Some RNAi agents were lyophilized and stored at −15 to −25°C. Duplex concentration was determined by measuring the absorbance of the solution in 1× PBS on a UV-Vis spectrometer. The absorbance of the solution at 260 nm was then multiplied by the conversion factor and the dilution factor to determine the duplex concentration. The conversion factor used was 0.037 mg / (mL∙cm) or, for some experiments, the conversion factor was calculated from the experimentally determined extinction coefficient.

[0568] E. Conjugation of Targeting Ligands

[0569] Compounds of Formula IV, V, VIII and IX can be synthesized by conjugating a targeting ligand to a triyne compound as described herein. An exemplary reaction is shown in the following scheme:

[0570]

[0571] wherein the variables are as described in Formula I, and TL is the targeting ligand.

[0572] In some embodiments, targeting ligand conjugation can be performed using the following procedure. The following procedure describes the conjugation of targeting ligands to compounds of Formula I where R comprises an RNAi agent, but targeting ligand conjugation can also be performed on compounds of Formula I where R does not contain an RNAi agent.

[0573] Before or after annealing, the 5' or 3' tridentate alkyne functionalized sense strand is conjugated to a targeting ligand. The following example describes the conjugation of a targeting ligand to an annealed duplex: Prepare stock solutions of 0.5M tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 0.5M Cu(II) sulfate pentahydrate (Cu(II)SO4· 5 H2O), and 2M sodium ascorbate solution in deionized water. Make a 75 mg / mL solution of the targeting ligand in DMSO. In a 1.5 mL centrifuge tube containing the triyne functionalized duplex (3 mg, 75 µL, 40 mg / mL in deionized water, ~15,000 g / mol), add 25 µL of 1M Hepes pH 8.5 buffer. After vortexing, add 35 µL of DMSO and vortex the solution. Add the targeting ligand to the reaction (6 eq / duplex, 2 eq / alkyne, ~15 µL) and vortex the solution. Using pH paper, check the pH and verify pH ~8. In a separate 1.5 mL centrifuge tube, mix 50 µL of 0.5M THPTA with 10uL of 0.5M Cu(II)SO4· 5 H2O, vortex and incubate at room temperature for 5 min. After 5 min, add the THPTA / Cu solution (7.2 µL, 6 eq 5:1 THPTA:Cu) to the reaction vial and vortex. Immediately thereafter, add 2M ascorbate (5 µL, 50 eq / duplex, 16.7 / alkyne) to the reaction vial and vortex. Once the reaction is complete (usually complete within 0.5-1h), purify the reaction by native anion exchange chromatography.

[0574] F. Addition of Triacetylene Linker after Solid Support Synthesis

[0575] RNAi molecules having reactive groups, such as amino groups (also referred to herein as amines) can be synthesized. In some embodiments, the reactive group can be attached to the 5′-end and / or the 3′-end of the RNAi agent. In some embodiments, the RNAi agent can be double-stranded. In embodiments where the RNAi agent is double-stranded, the reactive group can be on the sense strand or the antisense strand of the RNAi agent.

[0576] For example, in some embodiments, the RNAi agent is synthesized to have NH2-C6H 12 The terminal amino group can then react to form a conjugate with a coupling moiety, such as a compound of Formula I. In some embodiments, the coupling moiety is an ester, and the reactive group on the RNAi agent is a primary amine, and an amide bond is formed between the RNAi agent and the triyne linker. An example of such a reaction is shown in the following scheme using a compound of Formula VI:

[0577]

[0578] Where L 1 , L 2 , L 3 , L 4 , R 3 , R 4 and RNA are defined as in Formulae VI and VII.

[0579] When the RNAi molecule has been cleaved from the solid support, the addition of the trialkyne linker described herein can be performed as follows. The sense strand is functionalized with a 5' and / or 3' terminal nucleotide containing a primary amine. The amine-functionalized duplex is dissolved in 90% DMSO / 10% H2O at ~50-70 mg / mL. 40 equivalents of triethylamine are added, followed by 3 equivalents of the trialkyne ester of Formula VI. Once complete, the conjugate is precipitated twice in a solvent system of 1x phosphate buffered saline / acetonitrile (1:14 ratio) and dried.

[0580] In vivo examples

[0581] The joints described herein can be used in combination with various RNAi agents. The following examples demonstrate that the joints described herein are used together with RNAi agents for Alpha-ENaC and HIF2α mRNA sequences and are intended to provide application examples of the joints rather than limit the scope of the present invention to any particular RNAi agent. The RNAi agents used in the following examples are shown in Table 8 below. The compounds of Table 8 are shown as structures cleaved from a solid support. In some cases, the compounds are further modified before in vivo administration. For AD5614-5617, AD5620, AD5858, AD5860 and AD5919, as part of synthesis on a solid support, a triacetylene linker is added to the sense strand as a phosphoramidite of formula II. In the case of AD04546, AD5347 and AD5453, the sense strand is cleaved from the support with a structure as shown in Table 8. Add respective triacetylene linkers as compounds of formula VI in an amide coupling reaction. The targeting ligand was added after cleavage from the resin, so for AD5614-5617, AD5620, AD5858, AD5860 and AD5919, the triyne linker is represented by a compound of formula III. In Table 8 below, a, c, g and u represent 2'-O-methyladenosine, cytidine, guanosine or uridine, respectively; Af, Cf, Gf and Uf represent 2'-fluoroadenosine, cytidine, guanosine or uridine, respectively; and s represents a phosphorothioate bond, and cPrpu represents 5'-cyclopropyl phosphonate-2'-O-methyl uridine:

[0582]

[0583]

[0584] Example 23. Renal tumor-bearing mouse model (orthotopic xenograft).

[0585] Creation of SEAP-expressing clear cell renal cell carcinoma (ccRCC) A498 cells

[0586] The pCR3.1 expression vector expressing the reporter gene secreted alkaline phosphatase (SEAP) under the CMV promoter was prepared by directional cloning of the SEAP coding sequence 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 create an A498 ccRCC cell line expressing SEAP. Briefly, the pCR3-SEAP plasmid was transfected into A498 ccRCC cells by electroporation according to the manufacturer's recommendations. Stable transfectants were selected by G418 resistance. The selected A498-SEAP clones were evaluated for SEAP expression and integration stability.

[0587] Implantation of SEAP-expressing clear cell renal cell carcinoma (ccRCC) A498 cells

[0588] Female athymic nude mice were anesthetized with ~3% isoflurane and placed in the right lateral decubitus position. A small 0.5-1 cm longitudinal abdominal incision was made in the left flank. Using a wet cotton swab, the left kidney was removed from the peritoneum and gently stabilized. Immediately prior to injection, a 1.0 ml syringe was filled with the cell / Matrigel mixture and a 27 gauge needle cannula was attached to the syringe head. The filled syringe was then connected to a syringe pump (Harvard Apparatus, Model PHD2000) and primed to remove air. The tip of the 27-gauge needle cannula attached to the syringe was inserted just below the renal capsule near the caudal pole, and the needle tip was carefully advanced 3-4 mm cranially along the 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 a syringe pump. The needle was left in the kidney for 15-20 seconds to ensure complete injection. The needle was then removed from the kidney and the cotton swab was pressed against the injection site for 30 seconds to prevent cell leakage or bleeding. The kidney was then carefully placed back into the abdomen and the abdominal wall was closed. Serum was collected every 7-14 days after implantation to monitor tumor growth using a commercial SEAP detection kit. For most studies, tumor-bearing mice were used 5-6 weeks after implantation, at which time tumors typically measured approximately 4-8 mm.

[0589] Determination of HIF2 mRNA expression

[0590] For the studies reported in the Examples herein, mice were euthanized on the indicated days after injection and total RNA was isolated from renal tumors using Trizol reagent according to the manufacturer's recommendations. Relative HiF2α mRNA levels were determined by RT-qPCR as described below and compared to mice treated with delivery buffer (isotonic glucose) alone.

[0591] In preparation for quantitative PCR, total RNA was isolated from tissue samples homogenized in TriReagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's protocol.

[0592] Approximately 500 ng of RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Life Technologies). For human (tumor) Hif2α (EPAS1) expression, pre-made TaqMan Gene Expression Assays for human Hif2α (Catalog # 4331182) and CycA (PPIA) Catalog #: 4326316E) were used in triplicate biplex reactions using TaqMan GeneExpression Master Mix (Life Technologies) or VeriQuest Probe Master Mix (Affymetrix). Quantitative PCR was performed using the 7500 Fast or StepOnePlus Real-Time PCR Systems (Life Technologies). ΔΔC T Methods Relative gene expression was calculated.

[0593] Example 24. Integrin targeting ligands conjugated to RNAi agents targeting HIF-2 alpha (EPAS1) in renal tumors In vivo administration in tumor-bearing mice.

[0594] RNAi agents including the sense and antisense strand sequences given in Table 8 were synthesized on a solid phase according to the phosphoramidite technique according to general procedures known in the art and commonly used for oligonucleotide synthesis. (See Example 22 herein). RNAi agents include those with sequences that are specific to HIF-2 alpha (Hif2α or EPAS1) gene. EPAS1 is a member of the HIF (hypoxia-inducible factor) gene family and encodes one half of a transcription factor involved in gene induction regulated by oxygen and induced when oxygen levels drop, a condition known as hypoxia. Hif2α is known to be frequently overexpressed in clear cell renal carcinoma (ccRCC) cells. Hif2α RNAi agents are designed to reduce or inhibit translation of messenger RNA (mRNA) transcripts of Hif2α in a sequence-specific manner, thereby inhibiting expression of the EPAS1 gene.

[0595] On study day 1, mice bearing renal tumors were injected via tail vein according to the dosing regimen consisting of the following groups: See Example 23) Administration:

[0596] Table 9. Dosing groups of renal tumor-bearing mice in Example 5

[0597] Group RNAi Agents and Dosages Dosage regimen 1 Isotonic glucose (5% dextrose in water (D5W)) (without RNAi agent) Single injection on day 1 3 7.5 mg / kg of Hif2α RNAi agent (AD04546, containing the triacetylene linker compound 14-SV) conjugated to a 40 kilodalton (kDa) PEG moiety (with αvβ3 integrin ligand 4.1) formulated in isotonic glucose Single injection on day 1 4 7.5 mg / kg of Hif2α RNAi agent (AD05614, containing the triacetylene linker compound 1-SV) conjugated to a 40 kilodalton (kDa) PEG moiety (with αvβ3 integrin ligand 4.1) formulated in isotonic glucose Single injection on day 1 5 7.5 mg / kg of Hif2α RNAi agent (AD05615, containing the triacetylene linker compound 2-SV) conjugated to a 40 kilodalton (kDa) PEG moiety (with αvβ3 integrin ligand 4.1) formulated in isotonic glucose Single injection on day 1 6 7.5 mg / kg of Hif2α RNAi agent (AD05616, containing the triacetylene linker compound 3-SV) conjugated to a 40 kilodalton (kDa) PEG moiety (with αvβ3 integrin ligand 4.1) formulated in isotonic glucose Single injection on day 1 7 7.5 mg / kg of Hif2α RNAi agent (AD05617, containing the triacetylene linker compound 6-SV) conjugated to a 40 kilodalton (kDa) PEG moiety (with αvβ3 integrin ligand 4.1) formulated in isotonic glucose Single injection on day 1 8 7.5 mg / kg of Hif2α RNAi agent (AD05614, containing the triacetylene linker compound 1-SV) conjugated to a 40 kilodalton (kDa) PEG moiety (with αvβ3 integrin ligand 4.5), formulated in isotonic glucose Single injection on day 1 10 7.5 mg / kg of Hif2α RNAi agent (AD05620, containing the triacetylene linker compound 4-SV) conjugated to a 40 kilodalton (kDa) PEG moiety (with αvβ3 integrin ligand 4.5), formulated in isotonic glucose Single injection on day 1

[0598] 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 triyne linker compounds shown. In the case of Groups 4-8 and 10, the triyne linkers were added to the RNAi agents using phosphoramidite compounds 1, 2, 3, 4, and 6, respectively. The respective integrin targeting ligands were synthesized with an azide reactive group (see, e.g., Example 22), which were subsequently conjugated to the triyne component of the linker. A 40 kilodalton (kDa) PEG moiety was attached to act as a pharmacokinetic (PK) modulator, thereby increasing the circulation time of the drug product-conjugate. The structures of the targeting ligands αvβ3 integrin ligands 4.1 and 4.5 are shown below:

[0599]

[0600] Three (3) tumor-bearing mice were dosed in each group (n=3). Mice were sacrificed on study day 8 after injection and total RNA was isolated from renal tumors according to the procedure described in Example 4. Relative human HIF2α mRNA expression was then quantified by probe-based quantitative PCR (RT-qPCR) as explained in Example 23, normalized to cyclophilin A (PPIA) expression and expressed as a fraction of the vehicle control group (isotonic glucose) (geometric mean, + / - 95% confidence interval).

[0601] Table 10. Average relative huHif2α mRNA expression at sacrifice in Example 24

[0602] Group ID Mean relative huHIF2α mRNA expression Low(error) High (error) Group 1 (isotonic glucose) 1.000 0.069 0.074 Group 3 - Compound 14-SV 0.377 0.071 0.087 Group 4 - Compound 1-SV 0.357 0.028 0.030 Group 5 - Compound 2-SV 0.369 0.029 0.032 Group 6 - Compound 3-SV 0.290 0.035 0.039 Group 7 - Compound 6-SV 0.348 0.023 0.025 Group 8 - Compound 1-SV 0.424 0.034 0.037 Group 10 - Compound 4-SV 0.307 0.053 0.064

[0603] Example 25. Integrin targeting ligands conjugated to RNAi agents targeting HIF-2 alpha (EPAS1) in renal tumors In vivo administration in tumor-bearing mice.

[0604] RNAi agents including the sense and antisense strand sequences given in Table 8 were synthesized on a solid phase according to the phosphoramidite technique according to general procedures known in the art and commonly used for oligonucleotide synthesis. (See Example 22 herein). RNAi agents include those with a sense and antisense strand sequence corresponding to (Hif2α) ( EPAS1 ) gene is at least partially complementary to the antisense strand of the nucleobase sequence.

[0605] On study day 1, mice bearing renal tumors were injected via tail vein according to the following dosing groups: See Example 23) Administration:

[0606] Table 11. Dosing groups of renal tumor-bearing mice in Example 5

[0607] Group RNAi agents and dosage Dosage regimen 1 Isotonic glucose (5% dextrose in water (D5W)) (without RNAi agent) Single injection on day 1 2 7.5 mg / kg of Hif2α RNAi agent (AD04546, containing the triyne linker 14-SV) conjugated to a C-18 diacid moiety (with αvβ3 integrin ligand 2) formulated in isotonic glucose Single injection on day 1 3 7.5 mg / kg of Hif2α RNAi agent (AD04546, containing triyne linker compound 18-IX) conjugated to C-18 diacid moiety (with αvβ3 integrin ligand 2), formulated in isotonic glucose Single injection on day 1 4 7.5 mg / kg of Hif2α RNAi agent (AD04546, containing triyne linker compound 15-IX) conjugated to C-18 diacid (with αvβ3 integrin ligand 2), formulated in isotonic glucose Single injection on day 1 5 7.5 mg / kg of Hif2α RNAi agent (AD04546, containing triyne linker compound 16-IX) conjugated to C-18 diacid moiety (with αvβ3 integrin ligand 2), formulated in isotonic glucose Single injection on day 1 6 7.5 mg / kg of Hif2α RNAi agent (AD04546, containing triyne linker compound 17-IX) conjugated to C-18 diacid moiety (with αvβ3 integrin ligand 2), formulated in isotonic glucose Single injection on day 1 7 7.5 mg / kg of Hif2α RNAi agent (AD05858, containing the triyne linker compound 10-SV) conjugated to a C-18 diacid moiety (with αvβ3 integrin ligand 2), formulated in isotonic glucose Single injection on day 1 8 7.5 mg / kg of Hif2α RNAi agent (AD05860, containing the triyne linker compound 12-SV) conjugated to a C-18 diacid moiety (with αvβ3 integrin ligand 2), formulated in isotonic glucose Single injection on day 1 9 7.5 mg / kg of Hif2α RNAi agent (AD05919, containing the triyne linker compound 13-SV) conjugated to a C-18 diacid moiety (with αvβ3 integrin ligand 2), formulated in isotonic glucose Single injection on day 1

[0608] The RNAi agent in Example 25 is synthesized, which has a nucleotide sequence intended to target the human Hif2α gene and in the case of Groups 3-6,

[0609] A functionalized amine reactive group (NH2-C6) was included at the 5′ end of the sense strand to facilitate conjugation to triyne linker compounds 15-18. In the case of groups 3 and 7-9, triyne linkers were added to the RNAi agents using phosphoramidite compounds 14, 10, 12, and 13, respectively. The respective integrin targeting ligands were synthesized with an azide reactive group (see, e.g., Example 22), which were subsequently conjugated to the triyne component of the linker. A 40 kDa PEG moiety and a C-18 diacid moiety were attached to act as a pharmacokinetic (PK) modulator by increasing the circulation time of the drug product-conjugate. The structure of the C-18 diacid moiety is shown below:

[0610]

[0611] The C-18 diacid moiety is linked to the 3' end of the sense strand via an amide bond. The structure of the targeting ligand αvβ3 integrin ligand 2 is shown below:

[0612]

[0613] in Indicates the point of attachment to the linker.

[0614] Three (3) tumor-bearing mice were dosed in each group (n=3). Mice were sacrificed on study day 8 after injection and total RNA was isolated from renal tumors according to the procedure described in Example 4. Relative human HIF2α mRNA expression was then quantified by probe-based quantitative PCR (RT-qPCR) as explained in Example 23, normalized to human cyclophilin A (PPIA) expression and expressed as a fraction of the vehicle control group (isotonic glucose) (geometric mean, + / - 95% confidence interval).

[0615] Table 12. Average relative huHif2α mRNA expression at sacrifice in Example 25

[0616] Group ID Mean relative huHIF2α mRNA expression Low(error) High (error) Group 1 (isotonic glucose) 1.000 0.093 0.103 Group 2 - Compound 14-SV 0.585 0.095 0.113 Group 3 - Compound 18-IX 0.482 0.053 0.059 Group 4 - Compound 15-IX 0.546 0.063 0.072 Group 5 - Compound 16-IX 0.572 0.030 0.031 Group 6 - Compound 17-IX 0.504 0.133 0.181 Group 7 - Compound 10-SV 0.484 0.107 0.138 Group 8 - Compound 12-SV 0.605 0.120 0.150 Group 9 - Compound 13-SV 0.475 0.070 0.082

[0617] Embodiment 26 In vivo oropharyngeal in vitro translocation of Alpha-ENaC RNAi agents conjugated to epithelial cell targeting ligands in rats Suction medication

[0618] Triacetylene linkers can be used in various RNAi constructs. RNAi constructs comprising linkers of the invention can be administered in various different administration methods as described in this example. Triacetylene linkers can also be used with various targeting ligands. In this example, the targeting ligand conjugated to the triacetylene linker is an αvβ6 targeting ligand.

[0619] In this example, the triyne linker of compound 22 was added to the sense strand following solid support synthesis in the manner described in Example 22.

[0620] On study day 1, male Sprague Dawley rats were dosed by 200 μL oropharyngeal aspiration (OP) using a pipette according to the following dosing groups:

[0621] Table 13. Dosage groups of rats in Example 8

[0622] Group RNAi agents and dosage Dosage regimen 1 Isotonic saline (without RNAi agent) Single OP dose on day 1 2 0.5 mg / kg of AD05347 conjugated to a tridentate small molecule αvβ6 epithelial cell targeting ligand (compound 22-IX, Tri-avB6 SM2) via an amine (NH2-C6) bond on the 5' end of the sense strand, formulated in isotonic saline Single OP dose on day 1 3 0.5 mg / kg of AD05347 conjugated to a tridentate small molecule αvβ6 epithelial cell targeting ligand (Compound 2-IX, Tri-avB6 SM6.1) via an amine (NH2-C6) bond on the 5' end of the sense strand, formulated in isotonic saline Single OP dose on day 1 4 0.5 mg / kg of AD05453 conjugated to a tridentate small molecule αvβ6 epithelial cell targeting ligand (compound 22-IX, Tri-avB6 SM2) via an amine (NH2-C6) bond on the 5' end of the sense strand, formulated in isotonic saline Single OP dose on day 1 5 0.5 mg / kg of AD05453 conjugated to a tridentate small molecule αvβ6 epithelial cell targeting ligand (compound 22-IX, Tri-avB6 SM9) via an amine (NH2-C6) bond on the 5' end of the sense strand, formulated in isotonic saline Single OP dose on day 1 6 0.5 mg / kg of AD05453 conjugated to a tridentate small molecule αvβ6 epithelial cell targeting ligand (compound 22-IX, Tri-avB6 SM6) via an amine (NH2-C6) bond on the 5' end of the sense strand, formulated in isotonic saline Single OP dose on day 1 7 0.5 mg / kg of AD05453 conjugated to a tridentate small molecule αvβ6 epithelial cell targeting ligand (compound 22-IX, Tri-avB6 SM8) via an amine (NH2-C6) bond on the 5' end of the sense strand, formulated in isotonic saline Single OP dose on day 1 8 0.5 mg / kg of AD05453 conjugated to a tridentate small molecule αvβ6 epithelial cell targeting ligand (compound 22-IX, Tri-avB6 SM6.1) via an amine (NH2-C6) bond on the 5' end of the sense strand, formulated in isotonic saline Single OP dose on day 1 9 0.5 mg / kg of AD05453 conjugated to a tridentate small molecule αvβ6 epithelial cell targeting ligand (compound 22-IX, Tri-avB6 SM10) via an amine (NH2-C6) bond on the 5' end of the sense strand, formulated in isotonic saline Single OP dose on day 1 10 0.5 mg / kg of AD05453 conjugated to a tridentate small molecule αvβ6 epithelial cell targeting ligand (compound 22-IX, Tri-avB6 SM10) via an amine (NH2-C6) bond on the 5' end of the sense strand, formulated in isotonic saline Single OP dose on day 1 11 0.5 mg / kg of AD05453 conjugated to a tridentate-based αvβ6 epithelial cell targeting ligand using compound 22-IX via an amine (NH2-C6) bond on the 5' end of the sense strand, formulated in isotonic saline Single OP dose on day 1

[0623] In each group, compound 22 was reacted with the amine bond on the 5' end of the sense strand. The structure of the αvβ6 targeting ligand is shown below:

[0624]

[0625]

[0626] ,in Indicates the point of attachment to the linker.

[0627] Four (4) rats were dosed in each group (n=4). Rats were euthanized on study day 9 post-injection and total RNA was isolated from both lungs after collection and homogenization. Alpha-ENaC (SCNN1A) mRNA expression was quantified by probe-based quantitative PCR, normalized to GAPDH expression and expressed as a fraction of the vehicle control group (geometric mean, + / - 95% confidence interval).

[0628] Table 14. Mean relative rENaC mRNA expression at sacrifice (day 9) in Example 8

[0629] Group ID Mean relative rENaC mRNA expression Low(error) High (error) Group 1 (isotonic saline) 1.000 0.162 0.193 Group 2 (0.5 mg / kg AD05347-Compound 22-IX, Tri-SM2) 0.469 0.101 0.129 Group 3 (0.5 mg / kg AD05347-Compound 22-IX, Tri-SM6.1) 0.358 0.078 0.100 Group 4 (0.5 mg / kg AD05453-Compound 22-IX, Tri-SM2) 0.562 0.086 0.102 Group 5 (0.5 mg / kg AD05453-Compound 22-IX, Tri-SM9) 0.620 0.168 0.230 Group 6 (0.5 mg / kg AD05453-Compound 22-IX, Tri-SM6) 0.559 0.099 0.120 Group 7 (0.5 mg / kg AD05453-Compound 22-IX, Tri-SM8) 0.691 0.072 0.081 Group 8 (0.5 mg / kg AD05453-Compound 22-IX, Tri-SM6.1) 0.454 0.055 0.063 Group 9 (0.5 mg / kg AD05453-Compound 22-IX, Tri-SM10) 0.454 0.080 0.097 Group 10 (0.5 mg / kg AD05453-Compound 22-IX, Tri-SM11) 0.577 0.113 0.140 Group 11 (0.5 mg / kg AD05453-Compound 22-IX, tridentate peptide ligand) 0.558 0.057 0.064

[0630] As shown in Table 14 above, various different targeting ligand structures linked to respective RNAi agents using the triyne linker compounds disclosed herein exhibited inhibition of gene expression compared to controls.

[0631] Other Implementations

[0632] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims. Sequence Listing <110> ARROWHEAD PHARMACEUTICALS, INC. <120> Triacetylene linkers and methods of use <130> 30665-WO1 <150> 62 / 790,300 <151> 2019-01-09 <150> 62 / 663,763 <151> 2018-04-27 <150> 62 / 646,739 <151> 2018-03-22 <150> 62 / 631,683 <151> 2018-02-17 <160> 13 <210> 1 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi Antisense Strand Modification Sequence <400> 1 uuucaugaaa ucguuacguu g 21 <210> 2 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi Antisense Strand Modification Sequence <400> 2 uauuuguucu gguugcacag g 21 <210> 3 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi Antisense Strand Modification Sequence <400> 3 uauuuguucu gguugcacag g 21 <210> 4 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent sense strand modification sequence <400> 4 caacguaacg auuucaugaa a 21 <210> 5 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent sense strand modification sequence <400> 5 caacguaacg auuucaugaa a 21 <210> 6 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent sense strand modification sequence <400> 6 caacguaacg auuucaugaa a 21 <210> 7 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent sense strand modification sequence <400> 7 caacguaacg auuucaugaa a 21 <210> 8 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent sense strand modification sequence <400> 8 caacguaacg auuucaugaa a 21 <210> 9 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent sense strand modification sequence <400> 9 caacguaacg auuucaugaa a 21 <210> 10 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent sense strand modification sequence <400> 10 caacguaacg auuucaugaa a 21 <210> 11 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent sense strand modification sequence <400> 11 caacguaacg auuucaugaa a 21 <210> 12 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent sense strand modification sequence <400> 12 ccugugcaac cagaacaaau a 21 <210> 13 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> RNAi agent sense strand modification sequence <400> 13 ccugugcaac cagaacaaau a 21

Claims

1. A compound of formula V, or a pharmaceutically acceptable salt thereof, in, L 1 , L 2 and L 3 All have the formula: L 4 Selected from: in Indicates the connection point; R 4 is H or optionally substituted alkyl; TL is the targeting ligand; Y is O or S; and The RNA comprises or consists of a RNAi agent.

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

3. Compounds of Formula IX or a pharmaceutically acceptable salt thereof, in, L 1 , L 2 and L 3 All have the formula: L 4 Selected from: in Indicates the connection point; TL is the targeting ligand; R 4 Each instance of is H or optionally substituted alkyl; and The RNA comprises or consists of a RNAi agent.

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

5. A compound of formula III a method of reacting with a targeting ligand comprising an azide to form a compound of formula V, in, L 1 , L 2 and L 3 All have the formula: L 4 Selected from: in Indicates the connection point; R 4 is H or optionally substituted alkyl; TL is the targeting ligand; Y is O or S; and The RNA comprises or consists of a RNAi agent.

6. A compound of formula VII a method of reacting with a targeting ligand comprising an azide to form a compound of formula IX, in, L 1 , L 2 and L 3 All have the formula: L 4 Selected from: in Indicates the connection point; R 4 Each instance of is H or optionally substituted alkyl; TL is a targeting ligand; and The RNA comprises or consists of a RNAi agent.

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