5'-Cyclic phosphonate-modified nucleotides

Abstract

Described herein are oligonucleotides such as 5'-cyclic phosphonate modified nucleotides and RNA interference (RNAi) agents comprising such nucleotides. The RNAi agents described herein, comprising double-stranded or single-stranded oligonucleotides comprising 5'-cyclic phosphonate modified nucleotides, can be used to modulate gene expression and for therapeutic, diagnostic, target validation, and genomic development applications. RNAi agents and single-stranded antisense oligonucleotides comprising 5'-cyclic phosphonate modified nucleotides can be used to treat diseases or conditions that respond to inhibition of gene expression or activity in cells, tissues, or organisms.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 346,304, filed June 6, 2016, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] Disclosed herein is a 5'-cyclic phosphonate modified nucleotide useful for incorporation into oligomeric compounds such as RNAi agents. Background Art

[0004] Oligomeric compounds comprising a nucleotide sequence that is at least partially complementary to a target nucleic acid have been shown to alter the function and activity of the target both in vitro and in vivo. When delivered to cells containing a target nucleic acid (e.g., messenger RNA (mRNA)), oligomeric compounds have been shown to modulate the expression of the target, resulting in altered transcription or translation of the target nucleic acid. In some cases, oligomeric compounds can reduce gene expression by inhibiting the nucleic acid target and / or initiating degradation of the target nucleic acid.

[0005] If the target nucleotide is mRNA, one mechanism by which expression of an inhibitory oligomeric compound can modulate the expression of the mRNA target is through RNA interference (RNAi). RNAi is a biological process by which RNA or RNA-like molecules (e.g., chemically modified RNA molecules) are able to silence gene expression through degradation. The process of post-transcriptional gene silencing is believed to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of exogenous genes.

[0006] The RNAi response mechanism is believed to be characterized by a nuclease complex, often referred to as an RNA-induced silencing complex (RISC), in which a single strand of RNA or RNA-like molecule complementary to its target is incorporated. RISC is believed to mediate the cleavage of single-stranded RNA (e.g., mRNA) by virtue of the complementarity between the single-stranded RNA or RNA-like molecule bound to RISC and the mRNA.

[0007] Synthetic RNA and RNA-like molecules have been shown to induce RNA interference in vitro and in vivo. For example, Elbashir et al. (Nature 2000, 411, 494-98) described the induction of RNAi by introducing a duplex of a synthetic 21-nucleotide RNA molecule into cultured mammalian cells. The type of synthetic RNA or RNA-like molecule that can interact with RISC and trigger the RNAi response mechanism can be composed of modified nucleotides and / or one or more non-phosphodiester linkages.

[0008] Additionally, single-stranded RNA and RNA-like molecules (which may also contain modified nucleotides and have one or more non-phosphodiester linkages) can also alter the expression of target mRNAs.

[0009] Certain known modified nucleotides have been shown to increase the duration and / or activity of expression inhibitory oligomeric compounds when incorporated into oligomeric compounds upon in vivo administration. Summary of the Invention

[0010] There is a need for novel modified nucleotides that can provide improved or increased stability and / or potency to oligomeric compounds such as RNAi agents. For example, there is a need for novel modified nucleotides that can provide increased stability to the phosphate moiety on the terminal nucleotide at the 5' end of an RNAi agent.

[0011] Described herein are novel 5'-cyclic phosphonate modified nucleotides and oligomeric compounds, such as RNAi agents, comprising the 5'-cyclic phosphonate modified nucleotides. The 5'-cyclic phosphonate modified nucleotides can be incorporated into double-stranded oligonucleotides (e.g., short interfering RNAs) or single-stranded oligonucleotides (e.g., antisense oligonucleotides). Oligomeric compounds, such as RNAi agents, comprising one or more 5'-cyclic phosphonate modified nucleotides can also have a targeting ligand, such as an n-acetylgalactosamine cluster or a peptide, attached to the RNAi agent. Oligomeric compounds, such as RNAi agents, comprising one or more 5'-cyclic phosphonate modified nucleotides can also have a pharmacokinetic modulator, such as a polyethylene glycol (PEG) moiety or a lipid, attached to the RNAi agent.

[0012] The 5'-cyclic phosphonate modified nucleotides described herein can provide RNAi agents with improved stability and / or efficacy. In some embodiments, the 5'-cyclic phosphonate modified nucleotides described herein provide greater stability and resistance to endonucleases and exonucleases that can cleave the phosphodiester bonds of the oligonucleotide strand in vivo. Additionally, without wishing to be bound by theory, it is believed that the 5'-phosphorylation state of the termini of the RNAi agent is a factor in the strand's binding to RISC. Thus, a 5'-cyclic phosphonate modified nucleotide at the 5' end of the antisense strand can increase the likelihood that the 5' end of the oligonucleotide will become and remain phosphorylated. This can increase the likelihood of loading a specific strand into RISC, thereby allowing the RNAi agent to enter the RNAi pathway, leading to improved and enhanced gene knockdown and gene silencing activity.

[0013] The 5'-cyclic phosphonate modified nucleotides described herein have a cyclic group or cyclic portion located at the 5' carbon of the sugar of the nucleotide (or at the corresponding position of a sugar alternative substitution moiety). The 5'-cyclic phosphonate modified nucleotides described herein form a phosphonate group at the 5' end of the oligonucleotide (or, as described herein, a phosphonate mimetic group by introducing, for example, a 5'-C-malonyl group).

[0014] In some embodiments, the 5'-cyclic phosphonate modified nucleotide compound has a structure represented by Formula A:

[0015]

[0016] in:

[0017] "Ring" is an optionally substituted divalent cyclic moiety having 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, for example, cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl), cycloalkenyl (e.g., cyclopentenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl or cycloheptenyl), aryl (e.g., phenyl), heteroaryl (e.g., pyridyl, pyrimidinyl, pyridazinyl, pyrrole, pyrazole, imidazole, thiophene, benzothiophene, thiazole, benzothiazole, furan, Azoles, isocyanates Azoles, benzofurans, indoles, indazoles, benzimidazoles, oxadiazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, quinolyl, isoquinolyl or quinoxalinyl) or a heterocyclic group (such as tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, diazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, quinolyl, isoquinolyl or quinoxalinyl) or a heterocyclic group (such alkane or dioxolane);

[0018] X′ is

[0019]

[0020] or contain sugar-substituting substitution moieties;

[0021] X″ is (i) an internucleoside linkage linking the 5′-cyclic phosphonate modified nucleotide to the rest of the RNAi agent, or (ii) a phosphoramidite group;

[0022] D is O, S, CH2-CH2, CH=CH, OCH2, N(R 1 )、C(R 2 )(R 3 )、C(R 2 )(R 3 )C(R 4 )(R 2 )、C(R 2)=C(R 4 )、OC(R 2 )(R 3 )、OC(H)(X 3 ) or OC(R 2 )(X 3 );

[0023] R 1 is H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl;

[0024] R 2 、R 3 and R 4 Each is independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0025] When D is O, S, CH2-CH2, CH=CH, OCH2, N(R 1 )、C(R 2 )(R 3 )、C(R 2 )(R 3 )C(R 4 )(R 2 )、C(R 2 )=C(R 4 )、OC(R 2 )(R 3 ), X is a heterocyclic base moiety;

[0026] When D is OC(H)(X 3 ) or OC(R 2 )(X 3 ), X is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, and X 3 is a heterocyclic base moiety;

[0027] Z is H, -OH, F, OCH3, -O-(CH2)2-OCH3; halogen; -OCH2F, -OCHF2, -OCF3, -OCH2CH3, -O(CH2)2F, -OCH2CHF2, -OCH2CF3, -OCH2-CH=CH2, -O(CH2)2-OCH3, -O(CH2)2-SC H3, -O(CH2)2-OCF3, -O(CH2)2-O(CH2)2-N(CH3)2, -OCH2C(=O)-N(H)CH3, -OCH2C(=O)-N(H)-(CH2)2-N(CH3)2, -O(CH2)2-N(H)-C(=NH)(NH2), -O(CH2)3-N(R 5 )(R 6 )、-O(CH2)2-ON(R 5 )(R 6 )、-O(CH2)2-O(CH2)2-N(R 5 )(R 6 )、-OCH2C(=O)-N(R 5 )(R 6 )、-OCH2C(=O)-N(R 7 )-(CH2)2-N(R 5 )(R 6 )、-O(CH2)2-N(R 7 )-C(=R 8 )[N(R 5 )(R 6 )], optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, wherein R 5 、R 6 、R 7 and R 8 Each independently is H or C1-C6 alkyl;

[0028] Y 1 、Y 2 、Y 3 and Y 4 are each independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; or, Y 4 With Y 1 or Y 2 One of the connections, wherein the connection comprises a member selected from the group consisting of O, S, NR 9 、C(R 10 )(R 11)、C(R 10 )=C(R 11 ), C[=C(R 10 )(R 11 )] and a divalent group of C(=O), and Y 1 、Y 2 and Y 3 The other two are each independently H, halogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, wherein R 9 、R 10 and R 11 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl;

[0029] J is O, S, NR 12 ,NN(R 13 )2 or N-OR 13 ,in:

[0030] R 12 It is H, OH, halogen, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0031]

[0032] where R 14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or NH-C=(NH)NH2, wherein R 15 Selected from H, C1-C 18 alkyl or aryl;

[0033] where R 13 It is H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0034]

[0035] where R 14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or NH-C=(NH)NH2, and wherein R15 Selected from H, C1-C 18 alkyl or aryl; and

[0036] K and L are each independently selected from OH, OR 16 SR 16 or NR 16 , where R 16 Selected from H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0037]

[0038] where R 14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or -NH-C=(NH)NH2, and R 15 Selected from H, C1-C 18 Alkyl or aryl.

[0039] In some embodiments, the "ring" in the structure of Formula A is selected from the group consisting of:

[0040]

[0041]

[0042] In some embodiments, the "ring" in the structure of Formula A is a cyclic functional group, wherein the cyclic functional group is connected to the phosphonate moiety of Formula A and X' at the following positions of the cyclic functional group represented by standard IUPAC nomenclature: 1,2; 1,3; 1,4; 1,5; 1,6; 1,7; 2,3; 2,4; 2,5; 2,6; 2,7; 3,4; 3,5; 3,6; 3,7; 4,5; 4,6; 4,7; 5,6; 5,7 or 6,7.

[0043] In some embodiments, the "Ring" in the structure of Formula A is substituted. In some embodiments, the "Ring" in the structure of Formula A is a substituted cyclic moiety that is attached to the phosphonate moiety of Formula A and X' at the following positions of the substituted cyclic functionality represented by standard IUPAC nomenclature: 1,2; 1,3; 1,4; 1,5; 1,6; 1,7; 2,3; 2,4; 2,5; 2,6; 2,7; 3,4; 3,5; 3,6; 3,7; 4,5; 4,6; 4,7; 5,6; 5,7 or 6,7.

[0044] In some embodiments, X' of Formula A is or comprises a sugar alternative substitution moiety. In some embodiments, X' of Formula A is or comprises a sugar alternative substitution moiety, wherein the sugar alternative substitution moiety is morpholinyl. In some embodiments, X' of Formula A is or comprises a sugar alternative substitution moiety, wherein the sugar alternative substitution moiety is cyclohexenyl. In some embodiments, X' of Formula A is or comprises a sugar alternative substitution moiety, wherein the sugar alternative substitution moiety is cyclohexenyl.

[0045] In some embodiments, X' of Formula A is or comprises a sugar alternative substitution moiety, wherein the sugar alternative substitution moiety is acyclic. In some embodiments, X' of Formula A is or comprises an unlocked nucleobase analog (UNA) as a sugar alternative substitution moiety (see, e.g., U.S. Patent No. 8,314,227). In some embodiments, X' of Formula A is or comprises a glycerol nucleic acid structure as a sugar alternative substitution moiety (see, e.g., WO 2016 / 028649).

[0046] In some embodiments, X' of Formula A is or comprises a locked nucleic acid.

[0047] In some embodiments, the compounds described herein have a 5'-cyclopropylphosphonate group of Formula Ib or Formula II-b:

[0048]

[0049] in,

[0050] D is O, S, CH2-CH2, CH=CH, OCH2, N(R 1 )、C(R 2 )(R 3 )、C(R 2 )(R 3 )C(R 4 )(R 2 )、C(R 2 )=C(R 4 )、OC(R 2 )(R 3 )、OC(H)(X 3 ) or OC(R 2 )(X 3 );

[0051] R 1 is H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl;

[0052] R 2 、R3 and R 4 Each is independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0053] When D is O, S, CH2-CH2, CH=CH, OCH2, N(R 1 )、C(R 2 )(R 3 )、C(R 2 )(R 3 )C(R 4 )(R 2 )、C(R 2 )=C(R 4 )、OC(R 2 )(R 3 ), X is a heterocyclic base moiety;

[0054] When D is OC(H)(X 3 ) or OC(R 2 )(X 3 ), X is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, and X 3 is a heterocyclic base moiety;

[0055] Z is H, -OH, F, OCH3, -O-(CH2)2-OCH3; halogen; -OCH2F, -OCHF2, -OCF3, -OCH2CH3, -O(CH2)2F, -OCH2CHF2, -OCH2CF3, -OCH2-CH=CH2, -O(CH2)2-OCH3, -O(CH2)2-SC H3, -O(CH2)2-OCF3, -O(CH2)2-O(CH2)2-N(CH3)2, -OCH2C(=O)-N(H)CH3, -OCH2C(=O)-N(H)-(CH2)2-N(CH3)2, -O(CH2)2-N(H)-C(=NH)(NH2), -O(CH2)3-N(R 5 )(R 6 )、-O(CH2)2-ON(R 5 )(R 6 )、-O(CH2)2-O(CH2)2-N(R 5 )(R 6 )、-OCH2C(=O)-N(R 5)(R 6 )、-OCH2C(=O)-N(R 7 )-(CH2)2-N(R 5 )(R 6 )、-O(CH2)2-N(R 7 )-C(=R 8 )[N(R 5 )(R 6 )], optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, wherein R 5 、R 6 、R 7 and R 8 Each independently is H or C1-C6 alkyl;

[0056] Y 1 、Y 2 、Y 3 and Y 4 are each independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; or, Y 4 With Y 1 or Y 2 One of the connections, wherein the connection comprises a member selected from the group consisting of O, S, NR 9 、C(R 10 )(R 11 )、C(R 10 )=C(R 11 ), C[=C(R 10 )(R 11 )] and a divalent group of C(=O), and Y 1 、Y 2 and Y 3 The other two are each independently H, halogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, wherein R 9 、R 10 and R 11 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl;

[0057] J is O, S, NR 12 ,NN(R 13 )2 or N-OR13 ,in:

[0058] R 12 It is H, OH, halogen, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0059]

[0060] where R 14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or NH-C=(NH)NH2, wherein R 15 Selected from H, C1-C 18 alkyl or aryl;

[0061] where R 13 It is H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0062]

[0063] where R 14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or NH-C=(NH)NH2, and wherein R 15 Selected from H, C1-C 18 alkyl or aryl;

[0064] K and L are each independently selected from OH, OR 16 SR 16 or NR 16 , where R 16 Selected from H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0065]

[0066] where R 14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or -NH-C=(NH)NH2, and R 15 Selected from H, C1-C 18 alkyl or aryl;

[0067] Q is selected from O, S, N (R 30 ) or C(R 31 )(R 32 ) of the divalent part, wherein R 30 is H, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, and R 31 and R 32 are each independently H, halogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl; and

[0068] A is (i) an internucleoside linkage linking the 5'-cyclic phosphonate modified nucleotide of Formula I to the remainder of the RNAi agent, or (ii) a phosphoramidite group; and

[0069] G 1 , G 2 , G 3 and G 4 Each independently selected from the group consisting of H, F, halogen, C1-C6 alkyl, CN, CH2(R 33 )、CH2-O-(R 33 )、C(=O)(R 33 )、C(=S)(R 33 ) or (R 34 )(R 33 ), where R 33 It is O(R 35 )、S(R 35 )、N(R 35 )(R 36 ), where R 34 、R 35 and R 36 Each is independently selected from H, halogen or C1-C6 alkyl.

[0070] In some embodiments, the compounds described herein comprise a 5-cyclopropylphosphonate group and are linked to an RNAi agent and have Formula III or Formula IV as follows:

[0071]

[0072] in:

[0073] X is a heterocyclic base moiety;

[0074] Z is H, -OH, F, OCH3, -O-(CH2)2-OCH3; halogen; -OCH2F, -OCHF2, -OCF3, -OCH2CH3, -O(CH2)2F, -OCH2CHF2, -OCH2CF3, -OCH2-CH=CH2, -O(CH2)2-OCH3, -O(CH2)2-SC H3, -O(CH2)2-OCF3, -O(CH2)2-O(CH2)2-N(CH3)2, -OCH2C(=O)-N(H)CH3, -OCH2C(=O)-N(H)-(CH2)2-N(CH3)2, -O(CH2)2-N(H)-C(=NH)(NH2), -O(CH2)3-N(R 5 )(R 6 )、-O(CH2)2-ON(R 5 )(R 6 )、-O(CH2)2-O(CH2)2-N(R 5 )(R 6 )、-OCH2C(=O)-N(R 5 )(R 6 )、-OCH2C(=O)-N(R 7 )-(CH2)2-N(R 5 )(R 6 )、-O(CH2)2-N(R 7 )-C(=R 8 )[N(R 5 )(R 6 )], optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, wherein R 5 、R 6 、R 7 and R 8 Each independently is H or C1-C6 alkyl;

[0075] J and J' are each independently O or S;

[0076] L, L' and K are each independently selected from OH, OR 16 SR 16 or NR 16 , where R 16 Selected from H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0077]

[0078] where R 14is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or -NH-C=(NH)NH2, and R 15 Selected from H, C1-C 18 alkyl or aryl; and

[0079] The remainder of the RNAi agent is contained.

[0080] In some embodiments, the 5-cyclic phosphonate modified nucleotide compound is a phosphoramidite compound.

[0081] In some embodiments, the 5-cyclic phosphonate modified nucleotide compound is a phosphoramidite compound of Formula Ib-5 or Formula II-b-5:

[0082]

[0083] in,

[0084] D is O, S, CH2-CH2, CH=CH, OCH2, N(R 1 )、C(R 2 )(R 3 )、C(R 2 )(R 3 )C(R 4 )(R 2 )、C(R 2 )=C(R 4 )、OC(R 2 )(R 3 )、OC(H)(X 3 ) or OC(R 2 )(X 3 );

[0085] R 1 is H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl;

[0086] R 2 、R 3 and R 4 Each is independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0087] When D is O, S, CH2-CH2, CH=CH, OCH2, N(R 1 )、C(R 2 )(R 3 )、C(R 2 )(R 3 )C(R 4 )(R 2 )、C(R 2 )=C(R 4 )、OC(R 2 )(R 3 ), X is a heterocyclic base moiety;

[0088] When D is OC(H)(X 3 ) or OC(R 2 )(X 3 ), X is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, and X 3 is a heterocyclic base moiety;

[0089] Z is H, -OH, F, OCH3, -O-(CH2)2-OCH3; halogen; -OCH2F, -OCHF2, -OCF3, -OCH2CH3, -O(CH2)2F, -OCH2CHF2, -OCH2CF3, -OCH2-CH=CH2, -O(CH2)2-OCH3, -O(CH2)2-SC H3, -O(CH2)2-OCF3, -O(CH2)2-O(CH2)2-N(CH3)2, -OCH2C(=O)-N(H)CH3, -OCH2C(=O)-N(H)-(CH2)2-N(CH3)2, -O(CH2)2-N(H)-C(=NH)(NH2), -O(CH2)3-N(R 5 )(R 6 )、-O(CH2)2-ON(R 5 )(R 6 )、-O(CH2)2-O(CH2)2-N(R 5 )(R 6 )、-OCH2C(=O)-N(R 5 )(R 6 )、-OCH2C(=O)-N(R 7 )-(CH2)2-N(R 5 )(R 6 )、-O(CH2)2-N(R 7 )-C(=R 8 )[N(R5 )(R 6 )], optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, wherein R 5 、R 6 、R 7 and R 8 Each independently is H or C1-C6 alkyl;

[0090] Y 1 、Y 2 、Y 3 and Y 4 are each independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; or, Y 4 With Y 1 or Y 2 One of the connections, wherein the connection comprises a member selected from the group consisting of O, S, NR 9 、C(R 10 )(R 11 )、C(R 10 )=C(R 11 ), C[=C(R 10 )(R 11 )] and a divalent group of C(=O), and Y 1 、Y 2 and Y 3 The other two are each independently H, halogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, wherein R 9 、R 10 and R 11 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl;

[0091] J is O, S, NR 12 ,NN(R 13 )2 or N-OR 13 ,in:

[0092] R 12 It is H, OH, halogen, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0093]

[0094] where R 14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or NH-C=(NH)NH2, wherein R 15 Selected from H, C1-C 18 alkyl or aryl;

[0095] where R 13 It is H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0096]

[0097] where R 14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or NH-C=(NH)NH2, and wherein R 15 Selected from H, C1-C 18 alkyl or aryl;

[0098] K and L are each independently selected from OH, OR 16 SR 16 or NR 16 , where R 16 Selected from H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0099]

[0100] where R 14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or -NH-C=(NH)NH2, and R 15 Selected from H, C1-C 18 alkyl or aryl; and

[0101] G 1 , G 2 , G 3 and G 4 Each independently selected from the group consisting of H, F, halogen, C1-C6 alkyl, CN, CH2(R 33 )、CH2-O-(R 33)、C(=O)(R 33 )、C(=S)(R 33 ) or (R 34 )(R 33 ), where R 33 It is O(R 35 )、S(R 35 )、N(R 35 )(R 36 ), where R 34 、R 35 and R 36 Each is independently selected from H, halogen or C1-C6 alkyl.

[0102] In some embodiments, the phosphoramidite-containing compound comprising a 5'-cyclopropylphosphonate modified nucleotide has the following structure:

[0103]

[0104]

[0105] In some embodiments, the 5'-cyclic phosphonate modified nucleotide compound has a structure represented by Formula B:

[0106]

[0107] in:

[0108] "Ring" and X' are each as defined above in Formula A; and

[0109] The M of formula B is or comprises a 5'-phosphonate mimetic group. In some embodiments, the M of formula B is a 5'-C-malonyl group. In some embodiments, the M of formula B is a carboxylic acid, a sulfonic acid or a boric acid. In some embodiments, the M of formula B is a dicarboxylic acid, a disulfonic acid or a diboric acid. In some embodiments, the M of formula B is a diprotic acid selected from a mixture of carboxylic acid, sulfonic acid, boric acid and phosphoric acid. In some embodiments, the phosphonate mimetic group (such as the M of formula B) is connected to the "ring" of formula B by a single bond. In some embodiments, the phosphonate mimetic group is connected to the "ring" of formula B by more than one bond.

[0110] In some embodiments, the compound of Formula B has a structure represented by the following structure:

[0111]

[0112] wherein "ring" and X' are each as defined above in formula A.

[0113] In some embodiments, the compound of Formula B has a structure represented by the following structure:

[0114]

[0115] wherein "ring", D, X and Z are each as defined above in Formula A.

[0116] As used herein, the term "linked" when referring to a connection between two molecules means that the two molecules are connected by a covalent bond, or that the two molecules are associated by intermolecular forces (e.g., hydrogen bonds, van der Waals forces, or ionic bonds). In some embodiments, the term "linked" refers to an association between two molecules by intermolecular forces, where the association between the two different molecules has a specific affinity of less than 1x10 in a physiologically acceptable buffer (e.g., phosphate buffered saline). -4 M (e.g. less than 1x10 -5 M, less than 1x10 -6 M or less than 1x10 -7 M)K D .

[0117] As used herein, the term "directly connected" refers to a first compound or group that is connected to a second compound or group without any intervening atoms or groups of atoms. As used herein, the term "indirectly connected" refers to a first compound that is connected to a second compound or group through an intermediate atom, group, compound, or molecule (e.g., a linking group). Unless otherwise indicated, the term "connected" as used herein includes "directly connected" and "indirectly connected" as defined herein.

[0118] As used herein, an "oligomeric compound" is a nucleotide sequence containing about 10 to 50 nucleotides or nucleotide base pairs. In some embodiments, the oligomeric compound has a nucleobase sequence that is at least partially complementary to a core sequence in a target nucleic acid or target gene expressed in a cell. In some embodiments, the oligomeric compound can inhibit the expression of the corresponding gene after being delivered to a cell expressing a gene, and is referred to herein as an "expression-inhibiting oligomeric compound." Gene expression can be inhibited in vitro or in vivo. "Oligomeric compounds" include, but are not limited to, oligonucleotides, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and dicer enzyme substrates.

[0119] As used herein, the terms "oligonucleotide" and "polynucleotide" refer to a polymer of linked nucleosides, wherein each nucleoside may independently be modified or unmodified.

[0120] As used herein, the term "single-stranded oligonucleotide" refers to a single-stranded oligomeric compound having a sequence that is at least partially complementary to a target mRNA and that is capable of hybridizing to the target mRNA via hydrogen bonds under mammalian physiological conditions (or similar in vitro conditions). In some embodiments, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide. The 5'-cyclic phosphonate modified nucleotides described herein can be incorporated into the single-stranded antisense oligonucleotide. In some embodiments, the position of the 5'-cyclic phosphonate modified nucleotide is the terminal nucleotide at the 5' end of the single-stranded oligonucleotide.

[0121] As used herein, "RNAi agent" refers to an agent containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that can reduce or inhibit the translation of mRNA transcripts of target messenger RNA (mRNA) in a sequence-specific manner. As used herein, RNAi agents can act through an RNA interference mechanism (e.g., by inducing RNA interference through interaction with the RNA interference pathway mechanism of mammalian cells (RNA-induced silencing complex or RISC)) or other arbitrary mechanisms or pathways. Although it is believed that the term RNAi agent used herein acts primarily through an RNA interference mechanism, the RNAi agent is not limited to or confined to any particular pathway or mechanism of action. RNAi agents include, but are not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer enzyme substrates. The RNAi agents described herein are composed of oligonucleotides having a chain that is at least partially complementary to the target mRNA. In some embodiments, the RNAi agents described herein are double-stranded and consist of an antisense strand and a sense strand that is at least partially complementary to the antisense strand. The RNAi agent can be composed of modified nucleotides and / or one or more non-phosphodiester linkages.In some embodiments, the RNAi agent described herein is single-stranded.

[0122] As used herein, the terms "silencing," "reducing," "inhibiting," "downregulating," or "knockdown" when referring to the expression of a given gene mean that when a cell, cell cluster, tissue, organ, or subject is treated with an oligomeric compound such as an RNAi agent described herein, the expression of the gene is reduced as compared to a second cell, cell cluster, tissue, organ, or subject that has not been treated as such, as measured by the level of RNA transcribed from the gene or the level of a polypeptide, protein, or protein subunit translated from mRNA in the cell, cell cluster, tissue, organ, or subject in which the gene is transcribed.

[0123] As used herein, the term "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides, expressed in alphabetical order using standard nucleotide nomenclature.

[0124] As used herein, "nucleotide bases" or "nucleobases" are heterocyclic pyrimidine or purine compounds that are standard components of all nucleic acids and include bases that form adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U) nucleotides. Nucleobases can be further modified to include, but are not limited to, universal bases, hydrophobic bases, promiscuous bases, size-extended bases, and fluorinated bases.

[0125] As used herein, the term "heterocyclic base moiety" is a nucleobase or modified nucleobase as defined herein. In some embodiments, the heterocyclic base moiety is a pyrimidine, a substituted pyrimidine, a purine, or a substituted purine. In some embodiments, the heterocyclic base moiety is a naturally occurring purine or a substituted purine. In some embodiments, the heterocyclic base moiety is a non-naturally occurring purine or a substituted purine. In some embodiments, the heterocyclic base moiety is a naturally occurring pyrimidine or a substituted pyrimidine. In some embodiments, the heterocyclic base moiety is a non-naturally occurring pyrimidine or a substituted pyrimidine. In some embodiments, particularly when the 5'-cyclic phosphonate modified nucleotides described herein are phosphoramidite compounds, the heterocyclic base moiety comprises one or more protecting groups.

[0126] As used herein, "sugar surrogate substitution moiety" refers to a structure that is capable of replacing the 5-membered furanose ring of a naturally occurring ribonucleotide.

[0127] As used herein, and unless otherwise indicated, the term "complementary" when used to describe the relationship between a first nucleotide sequence (e.g., an RNAi agent sense strand or a target mRNA) and a second nucleotide sequence (e.g., a single-stranded antisense oligonucleotide or a double-stranded RNAi agent antisense strand) refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize (form base pairing hydrogen bonds under mammalian physiological conditions (or similar in vitro conditions)) and form a duplex or double helical structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions. Complementary sequences comprise Watson-Crick base pairing or non-Watson-Crick base pairing and comprise natural or modified nucleotides or nucleotide mimetics to at least the extent that they meet the above hybridization requirements. Sequence identity or complementarity is independent of modification. For example, for the purpose of determining identity or complementarity, a and Af are complementary to U (or T) and are equivalent to A.

[0128] As used herein, "perfect complementarity" or "complete complementarity" means that all (100%) bases in a contiguous sequence of a first polynucleotide hybridize to the same number of bases in a contiguous sequence of a second polynucleotide. The contiguous sequence may comprise all or part of the first or second nucleotide sequence.

[0129] As used herein, "partially complementary" refers to a hybridization pair of nucleobase sequences in which at least 70%, but not all, of the bases in a contiguous sequence of a first polynucleotide hybridize to the same number of bases in a contiguous sequence of a second polynucleotide.

[0130] As used herein, "substantially complementary" means that in a pair of hybridized nucleobase sequences, at least 85%, but not all, of the bases in the contiguous sequence of a first polynucleotide hybridize to the same number of bases in the contiguous sequence of a second polynucleotide. The terms "complementary," "fully complementary," and "substantially complementary" herein can be used in relation to base matching between the sense and antisense strands of a double-stranded RNAi agent, between the antisense strand of a double-stranded RNAi agent and the sequence of a target mRNA, or between a single-stranded antisense oligonucleotide and the sequence of a target mRNA.

[0131] As used herein, the terms "treat," "treat," "treat," and like terms refer to methods or steps employed to relieve or lessen the amount, severity, and / or frequency of one or more symptoms of a disease or condition in a subject.

[0132] As used herein, the phrase "introduced into a cell" when referring to an RNAi agent comprising a 5'-cyclic phosphonate modified nucleotide refers to the functional delivery of the RNAi agent into a cell. The phrase "functional delivery" means that the RNAi agent is delivered to the cell in a manner that enables the RNAi agent to have the intended biological activity, such as sequence-specific inhibition of gene expression.

[0133] Unless otherwise specified, the symbols used in this paper are The use of means that any group may be attached thereto, according to the scope of the invention described herein.

[0134] As used herein, the term "isomer" refers to compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers." Stereoisomers that are not mirror images of one another are termed "diastereomers," and stereoisomers that are non-superimposable mirror images are termed "enantiomers," or sometimes optical isomers. A carbon atom bonded to four different substituents is termed a "chiral center."

[0135] As used herein, unless a specific conformation is specifically indicated on the structure, for each structure in which asymmetric centers are present, resulting in enantiomers, diastereomers, or other stereoisomeric configurations, each structure described herein is intended to represent all such possible isomers, including their optically pure and racemic forms. For example, the structures described herein are intended to encompass mixtures of diastereomers as well as individual stereoisomers. The 5'-cyclic phosphonate modified nucleotides described herein contain one or more asymmetric centers, resulting in the production of enantiomers, diastereomers, and other stereoisomeric configurations, which can be defined in absolute stereochemical terms as (R) or (S), α or β (e.g., for sugar anomers), or (D) or (L) (e.g., for amino acids). The 5'-cyclic phosphonate modified nucleotides described herein include all such possible isomers, including their racemic and optically pure forms. Unless otherwise indicated, when a compound described herein (e.g., in an olefin or imine) contains a double bond, it is intended to represent that the compound contains both E and Z geometric isomers or cis and trans isomers. Likewise, all tautomeric forms are also intended to be included.The configuration of any bond appearing herein is chosen for convenience only and is not intended to be limited to a specific configuration unless otherwise indicated by the context.

[0136] As used herein, the term "substituted" means that any one or more hydrogens on a designated atom (typically a carbon, oxygen, or nitrogen atom) are replaced by any group as defined herein, provided that the conventional valence of the designated atom is not exceeded and that the substitution results in a stable compound. When the substituent is a ketone or an oxygen (e.g., =O), two (2) hydrogens on the atom are replaced. As used herein, a cyclic double bond is a double bond formed between two adjacent ring atoms (e.g., C=C, C=N, N=N, etc.). In some embodiments, the substituent of a cyclic functional group is another cyclic group or an aromatic group. As used herein, a bicyclic group is considered a substituted cyclic functional group. Examples of organic functional groups include, but are not limited to: hydrogen; halogens (e.g., F, Cl, Br, I); cyano; -CO2R a ;-CONR a R a ; R selected by 1 or 2 independently a Optionally substituted C 1-6 Alkyl; C 1-4 Haloalkyl; C 1-4 Alkoxy; C 1-4 Haloalkoxy; cycloalkyl, wherein the cycloalkyl is substituted by 1 to 4 independently selected R a An optionally substituted heterocyclic group containing 5 to 8 ring atoms, wherein 1 to 3 ring atoms are independently selected from N(R a ), O and S, wherein the heterocyclic group is replaced by 1 to 4 independently selected R a Optionally substituted; 1 to 4 Ra Optionally substituted C 6-10 Aryl; heteroaryl containing 5 to 10 ring atoms, wherein 1 to 4 ring atoms are independently selected from N, N (R a ), O and S, and wherein the heteroaryl is surrounded by 1 to 3 R a Optionally substituted; –N3; –CO2H; –OH; –SO 1-2 (R a );–NR a R a ;–SO 1-2 (NR a R a ); and thioalkoxy; wherein each R a Independently selected from C 1-6 Alkyl, –OH, –halogen, –NH2, –N(C 1-4 Alkyl)2, C 1-4 Alkoxy, C 1-4 Haloalkoxy, –C(=O)O(C 1-4 alkyl), –C(=O)(C 1-4 alkyl), –C(=O)OH, –CON(C 1-4 Alkyl)2, –S(O) 1-2 (C 1-4 alkyl)2 and cyano.

[0137] Some compounds of the present invention may exist in tautomeric forms, which are also intended to fall within the scope of the present invention. "Tautomers" are compounds whose structures differ significantly in the arrangement of atoms, but which readily and rapidly form equilibrium. It should be understood that the compounds of the present invention may be described as different tautomers. It should also be understood that when a compound has tautomeric forms, all tautomeric forms are intended to fall within the scope of the present invention, and the nomenclature of the compound does not exclude any tautomeric form.

[0138] The compounds and pharmaceutically acceptable salts of the present invention may exist in one or more tautomeric forms, including keto-enol, amide-nitrile, lactam-lactim, amide-imidic acid (e.g., in the nucleobases guanine, thymine, and cytosine), amine-enamine, and enamine-enamine, as well as geometric isomers and mixtures thereof. Ring-chain tautomerism, which occurs in glucose and other sugars, results from the reaction of an aldehyde group (-CHO) in a sugar chain molecule with a hydroxyl group (-OH) in the same molecule, resulting in a cyclic (annular) form. All such tautomeric forms are encompassed by the present invention. Tautomers exist as a mixture of tautomer groups in solution. In solid form, a single tautomer typically predominates. While a single tautomer may be described, the present invention encompasses all tautomers of the compounds described herein. The concept of tautomers that can interconvert through tautomerism is called tautomerism. In tautomerism, simultaneous migration of electrons and hydrogen atoms occurs.

[0139] Tautomerization is catalyzed by bases produced by: 1) deprotonation; 2) formation of a delocalized anion (e.g., an enolate); and 3) protonation at a different position of the anion. Tautomerization is catalyzed by acids produced by: 1) protonation; 2) formation of a delocalized cation; and 3) deprotonation at a different position adjacent to the cation.

[0140] As used herein, "protecting group" refers to an unstable chemical moiety known in the art for preventing reactive groups (e.g., hydroxyl, amino, carboxyl, and sulfhydryl) from undergoing undesirable reactions during synthesis. Protecting groups are typically used selectively and / or orthogonally to protect sites at other reactive sites during a reaction and are subsequently removed to release the unprotected group, making it available for further reactions. In some embodiments, a "substituted" group or substituent comprises a protecting group.

[0141] As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon group having 1 to 10 carbon atoms, which is straight or branched, unless otherwise specified. For example, "C1-C6 alkyl" includes alkyl groups having 1, 2, 3, 4, 5, or 6 carbon atoms in a straight or branched arrangement. As used herein, the term "aminoalkyl" refers to an alkyl group as defined above substituted with one or more amino groups at any position as permitted by conventional valence conditions. The amino group may be unsubstituted, monosubstituted, or disubstituted.

[0142] As used herein, the term "cyclic functional group" is intended to refer to a functional group that forms a ring structure. Cyclic functional groups include, but are not limited to, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, and aryl.

[0143] As used herein, the term "cycloalkyl" refers to a saturated or unsaturated non-aromatic hydrocarbon ring group having 3 to 14 carbon atoms, unless otherwise specified. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, methylcyclopropyl, 2,2-dimethylcyclobutyl, 2-ethylcyclopentyl, or cyclohexyl. Cycloalkyl groups may contain multiple spiro or fused rings. Cycloalkyl groups may be optionally mono-, di-, tri-, tetra-, or penta-substituted at any position, as permitted by conventional valence conditions.

[0144] As used herein, the term "alkenyl" refers to a linear or branched, non-aromatic hydrocarbon group having from 2 to 10 carbon atoms, unless otherwise specified. Up to 5 carbon-carbon double bonds may be present in such a group. For example, a "C2-C6" alkenyl group is defined as an alkenyl group having from 2 to 6 carbon atoms. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, and cyclohexenyl. The linear, branched, or cyclic portion of the alkenyl group may contain double bonds and may be optionally mono-, di-, tri-, tetra-, or penta-substituted at any position as permitted by conventional valence conditions. The term "cycloalkenyl" refers to a monocyclic hydrocarbon group having a specified number of carbon atoms and at least one carbon-carbon double bond.

[0145] As used herein, the term "alkynyl" refers to a straight or branched hydrocarbon group having 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond, unless otherwise specifically stated. Up to 5 carbon-carbon triple bonds may be present. 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 contain a triple bond, where conventional valence permits, and may be optionally mono-, di-, or tri-substituted at any position, where conventional valence permits.

[0146] As used herein, "alkoxy" refers to an alkyl group as defined above having the specified number of carbon atoms attached through an oxygen bridge. For example, C 1–6 Alkoxy is intended to include C1, C2, C3, C4, C5 and C6 alkoxy. For example, C 1–8 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-hexoxy and n-octoxy.

[0147] As used herein, "ketone" refers to any alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, or aryl group as defined herein, linked by a carbonyl bridge. Examples of ketone groups include, but are not limited to, alkanoyl (e.g., acetyl, propionyl, butyryl, pentanoyl, hexanoyl), alkenoyl (e.g., acryloyl), alkynoyl (e.g., ethynyl, propioyl, butynoyl, pentynyl, hexynyl), aroyl (e.g., benzoyl), heteroaroyl (e.g., pyrroloyl, imidazolyl, quinolinyl, pyridinoyl).

[0148] As used herein, "alkoxycarbonyl" refers to any alkoxy group as defined above attached through a carbonyl bridge (e.g., -C(O)O-alkyl). Examples of alkoxycarbonyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, or n-pentoxycarbonyl.

[0149] As used herein, "aryloxycarbonyl" refers to any aryl group as defined above attached through an oxycarbonyl bridge (eg, -C(O)O-aryl). Examples of aryloxycarbonyl groups include, but are not limited to, phenoxycarbonyl and naphthoxycarbonyl.

[0150] As used herein, "heteroaryloxycarbonyl" refers to any heteroaryl group as defined above (e.g., -C(O)O-heteroaryl) attached through an oxycarbonyl bridge. Examples of heteroaryloxycarbonyl groups include, but are not limited to, 2-pyridyloxycarbonyl, 2- oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl or pyrimidinyloxycarbonyl.

[0151] As used herein, the term "aryl" refers to any stable monocyclic or polycyclic carbocyclic ring having up to 7 atoms per 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. Where an 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 penta-substituted at any position as permitted by conventional valence conditions.

[0152] As used herein, the term "heteroaryl" refers to a stable monocyclic or polycyclic ring system having up to 7 atoms per ring, wherein at least one ring is aromatic and contains 1 to 4 heteroatoms selected from O, N and S. Examples of heteroaryl groups include, but are not limited to, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, benzimidazolonyl, benzotriazolyl, benzothiophene, benzofuranyl, benzoimidazolonyl, benzothiophene ... oxazolidinyl, quinolinyl, isoquinolinyl, dihydroisoindolinyl, imidazopyridinyl, isoindolinyl, indazolyl, Azolyl, Oxazolyl, iso oxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline. "Heteroaryl" is also understood to include N-oxide derivatives of any nitrogen-containing heteroaryl. In the case where the heteroaryl substituent is bicyclic and one ring is non-aromatic or does not contain heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom-containing ring. Heteroaryl is optionally mono- or di-substituted at any position under conventional valence conditions.

[0153] As used herein, the term "heterocycle", "heterocyclic" or "heterocyclyl" refers to a 3- to 14-membered aromatic or non-aromatic heterocyclic ring containing 1 to 4 heteroatoms selected from O, N and S, including polycyclic groups. As used herein, the term "heterocyclic" is also considered synonymous with the terms "heterocycle" and "heterocyclyl" and is understood to have the same definition as described herein. "Heterocyclyl" includes the above-mentioned heteroaryl groups as well as their dihydro and tetrahydro analogs. Examples of heterocyclyl groups include, but are not limited to, azetidinyl, benzimidazolyl, benzofuranyl, benzofurazyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzo oxazolyl, carbazolyl, carbolyl, cinnolinyl, furyl, imidazolyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolinyl, isothiazolyl, isothiazolyl, Azolyl, naphthopyridinyl, Oxadiazole, oxo Oxazolidinyl, Azolyl, Oxazoline, oxopiperazinyl, oxopyrrolidinyl, oxomorpholinyl, iso oxazolinyl, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridinyl, pyridonyl, pyrimidinyl, pyrimidonyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl, tetrazolyl, tetrazolyl, tetrazolopyridinyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, 1,4-diazoles, Alkyl, hexahydroazepine yl, piperazinyl, piperidinyl, pyridin-2-one, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzo oxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindole, dihydroisoquinoline Azolyl, dihydroisothiazolyl, dihydro Oxazolyl, dihydrogen The heterocyclic radical substituents can be connected by carbon atoms or heteroatoms. Heterocyclic radicals can be optionally substituted by single, double, three, four or five substituents at any position under the conditions that conventional valence allows.

[0154] 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 significantly affect the basic and novel characteristics of the claimed invention.

[0155] Those skilled in the art will readily appreciate and understand that the compounds and compositions described 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. Thus, as used herein, the structures described herein take into account that certain functional groups, such as OH, SH, or NH, may be protonated or deprotonated. The disclosure herein is intended to encompass the above-described compounds and compositions, regardless of their protonation state based on the pH of the environment, as will be readily understood by those skilled in the art.

[0156] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Although methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of conflict, the present specification (including definitions) shall prevail. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0157] Other features and advantages of the present invention will become apparent from the following detailed description and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0158] Figure 1 This graph shows relative cF12 expression in cynomolgus monkeys following administration of RNAi agents targeting factor 12. Only AD04443 contains a 5'-cyclopropylphosphonate modified nucleotide located at the 5' end of the antisense strand.

[0159] Figure 2Graph showing mean HBsAg in pHBV model mice after administration of RNAi agents targeting HBV, normalized to pre-treatment and saline controls. Only AD04580 contains a 5'-cyclopropylphosphonate modified nucleotide at the 5' end of the antisense strand. DETAILED DESCRIPTION

[0160] Described herein are 5'-cyclic phosphonate modified nucleotides and RNAi agents (also referred to as RNAi initiators) comprising 5'-cyclic phosphonate modified nucleotides. In some embodiments, one or more 5'-cyclic phosphonate modified nucleotides are attached to the end of the RNAi agent and form the terminal nucleotide of the RNAi agent. In some embodiments, the 5'-cyclic phosphonate modified nucleotide is attached to the 5' end or 5' end of the RNAi agent and forms the terminal nucleotide on the 5' end of the RNAi agent. In some embodiments, the 5'-cyclic phosphonate modified nucleotide is attached to the 5' end of the antisense strand of a double-stranded RNAi agent and forms the terminal nucleotide on the 5' end of the antisense strand of the double-stranded RNAi agent.

[0161] The 5'-cyclic phosphonate modified nucleotides described herein have a cyclic group or cyclic moiety located at the 5' carbon of the sugar of the nucleotide (or at the corresponding position of a sugar alternative substitution moiety).

[0162] In some embodiments, the 5'-terminal nucleotide of an RNAi agent comprises or is a 5'-cyclic phosphonate-modified nucleotide. In some embodiments, the 5'-terminal nucleotide of the antisense strand of a double-stranded RNAi agent comprises or is a 5'-cyclic phosphonate-modified nucleotide. In some embodiments, a 5'-cyclic phosphonate-modified nucleotide is located at the 5' end of an oligomeric compound, such as a single-stranded RNAi agent or the antisense strand of a double-stranded RNAi agent, to facilitate loading of the oligomeric compound into RISC and thereby contribute to the RNAi mechanism.

[0163] In some embodiments, one or more 5'-cyclic phosphonate modified nucleotides are attached to the terminus of a single-stranded antisense oligonucleotide and form the terminal nucleotide of the single-stranded antisense oligonucleotide. In some embodiments, a 5'-cyclic phosphonate modified nucleotide is attached to the 5' terminus of a single-stranded antisense oligonucleotide and forms the terminal nucleotide on the 5' end of the single-stranded antisense oligonucleotide.

[0164] In some embodiments, the compound has a structure represented by Formula A described above in the Summary of the Invention section.

[0165] In some embodiments, the compound of Formula A has a 5'-cyclic phosphonate structure represented by Formula I:

[0166]

[0167] in:

[0168] "Ring" is an optionally substituted divalent cyclic moiety having 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, for example, cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl), cycloalkenyl (e.g., cyclopentenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl or cycloheptenyl), aryl (e.g., phenyl), heteroaryl (e.g., pyridyl, pyrimidinyl, pyridazinyl, pyrrole, pyrazole, imidazole, thiophene, benzothiophene, thiazole, benzothiazole, furan, Azoles, isocyanates Azoles, benzofurans, indoles, indazoles, benzimidazoles, oxadiazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, quinolyl, isoquinolyl or quinoxalinyl) or a heterocyclic group (such as tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, diazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, quinolyl, isoquinolyl or quinoxalinyl) or a heterocyclic group (such alkane or dioxolane);

[0169] D is O, S, CH2-CH2, CH=CH, OCH2, N(R 1 )、C(R 2 )(R 3 )、C(R 2 )(R 3 )C(R 4 )(R 2 )、C(R 2 )=C(R 4 )、OC(R 2 )(R 3 )、OC(H)(X 3 ) or OC(R 2 )(X 3 );

[0170] R 1 is H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl;

[0171] R 2 、R 3 and R 4 Each is independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0172] When D is O, S, CH2-CH2, CH=CH, OCH2, N(R 1 )、C(R 2 )(R 3)、C(R 2 )(R 3 )C(R 4 )(R 2 )、C(R 2 )=C(R 4 )、OC(R 2 )(R 3 ), X is a heterocyclic base moiety;

[0173] When D is OC(H)(X 3 ) or OC(R 2 )(X 3 ), X is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, and X 3 is a heterocyclic base moiety;

[0174] Z is H, -OH, F, OCH3, -O-(CH2)2-OCH3; halogen; -OCH2F, -OCHF2, -OCF3, -OCH2CH3, -O(CH2)2F, -OCH2CHF2, -OCH2CF3, -OCH2-CH=CH2, -O(CH2)2-OCH3, -O(CH2)2-SC H3, -O(CH2)2-OCF3, -O(CH2)2-O(CH2)2-N(CH3)2, -OCH2C(=O)-N(H)CH3, -OCH2C(=O)-N(H)-(CH2)2-N(CH3)2, -O(CH2)2-N(H)-C(=NH)(NH2), -O(CH2)3-N(R 5 )(R 6 )、-O(CH2)2-ON(R 5 )(R 6 )、-O(CH2)2-O(CH2)2-N(R 5 )(R 6 )、-OCH2C(=O)-N(R 5 )(R 6 )、-OCH2C(=O)-N(R 7 )-(CH2)2-N(R 5 )(R 6 )、-O(CH2)2-N(R 7 )-C(=R 8 )[N(R 5 )(R 6)], optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, wherein R 5 、R 6 、R 7 and R 8 Each independently is H or C1-C6 alkyl;

[0175] Y 1 、Y 2 、Y 3 and Y 4 are each independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; or, Y 4 With Y 1 or Y 2 One of the connections, wherein the connection comprises a member selected from the group consisting of O, S, NR 9 、C(R 10 )(R 11 )、C(R 10 )=C(R 11 ), C[=C(R 10 )(R 11 )] and a divalent group of C(=O), and Y 1 、Y 2 and Y 3 The other two are each independently H, halogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, wherein R 9 、R 10 and R 11 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl;

[0176] J is O, S, NR 12 ,NN(R 13 )2 or N-OR 13 ,in:

[0177] R 12 It is H, OH, halogen, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0178]

[0179] where R14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or NH-C=(NH)NH2, wherein R 15 Selected from H, C1-C 18 alkyl or aryl;

[0180] where R 13 It is H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0181]

[0182] where R 14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or NH-C=(NH)NH2, and wherein R 15 Selected from H, C1-C 18 alkyl or aryl; and

[0183] K and L are each independently selected from OH, OR 16 SR 16 or NR 16 , where R 16 Selected from H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0184]

[0185] where R 14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or -NH-C=(NH)NH2, and R 15 Selected from H, C1-C 18 alkyl or aryl;

[0186] Q is selected from O, S, N (R 30 ) or C(R 31 )(R 32 ) of the divalent part, wherein R 30 is H, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, and R 31 and R32 are each independently H, halogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl; and

[0187] A is (i) an internucleoside linkage linking the 5'-cyclic phosphonate modified nucleotide of Formula I to the remainder of the RNAi agent, or (ii) a phosphoramidite group.

[0188] In some embodiments, when A is a phosphoramidite group, A is coupled to Q in Formula I via a phosphoramidite-forming reagent to form a phosphoramidite compound.

[0189] In some embodiments, Q in Formula I is O.

[0190] In some embodiments, the compound has a 5'-cyclic phosphonate structure represented by Formula II:

[0191]

[0192] Among them, "ring", D, X, Z, Y 1 、Y 2 、Y 3 、Y 4 , J, K, L, Q and A are each as defined in Formula I above.

[0193] In some embodiments, Y in the structures of Formula I and Formula II 1 、Y 2 、Y 3 and Y 4 Both are H.

[0194] In some embodiments, the "ring" in the structures of Formula I and Formula II is a cycloalkyl group containing 3, 4, 5, 6, or 7 carbon atoms.

[0195] In some embodiments, the "ring" in the structures of Formula I and Formula II is a cycloalkenyl group containing 4, 5, 6, or 7 carbon atoms.

[0196] In some embodiments, the "ring" in the structures of Formula I and Formula II is a cycloalkynyl group containing 5, 6, or 7 carbon atoms.

[0197] In some embodiments, the "ring" in the structures of Formula I and Formula II is an aryl group containing 3, 4, 5, 6, or 7 carbon atoms.

[0198] In some embodiments, the "ring" in the structures of Formula I and Formula II is a heterocyclic group containing 2, 3, 4, 5, or 6 carbon atoms and one or more non-carbon atoms.

[0199] In some embodiments, the "ring" in the structures of Formula I and Formula II is an aryl or heterocyclic group containing 2, 3, 4, 5, or 6 carbon atoms and one or more non-carbon atoms.

[0200] In some embodiments, the "ring" in the structures of Formula I and Formula II is a bicyclic group.

[0201] In some embodiments, the "ring" in the structures of Formula I and Formula II is selected from the group consisting of:

[0202]

[0203] In some embodiments, the "ring" in the structures of Formula I and Formula II is a cyclic functional group, wherein the cyclic functional group is attached to the phosphonate moiety and sugar ring of Formula I and Formula II at the following positions of the cyclic functional group represented by standard IUPAC nomenclature: 1,2; 1,3; 1,4; 1,5; 1,6; 1,7; 2,3; 2,4; 2,5; 2,6; 2,7; 3,4; 3,5; 3,6; 3,7; 4,5; 4,6; 4,7; 5,6; 5,7 or 6,7.

[0204] In some embodiments, the "ring" in the structures of Formula I and Formula II is selected from the group consisting of:

[0205]

[0206]

[0207] In some embodiments, the "ring" in the structures of Formula I and Formula II is substituted. In some embodiments, the "ring" in the structures of Formula I and Formula II is a substituted cyclic moiety that is attached to the phosphonate moiety and sugar ring of Formula I and Formula II at the following positions of the substituted cyclic functionality represented by standard IUPAC nomenclature: 1,2; 1,3; 1,4; 1,5; 1,6; 1,7; 2,3; 2,4; 2,5; 2,6; 2,7; 3,4; 3,5; 3,6; 3,7; 4,5; 4,6; 4,7; 5,6; 5,7 or 6,7.

[0208] In some embodiments, the "ring" in the structures of Formula I and Formula II is a cyclopropyl group having a specific stereochemistry, for example:

[0209]

[0210] In some embodiments, the "ring" in the structures of Formula I and Formula II is a substituted cyclopropyl functional group selected from the group consisting of:

[0211]

[0212] In some embodiments, the compound has a structure represented by Formula Ia or Formula II-a:

[0213]

[0214] Among them, "ring", D, X, Z, Y 1 、Y 2 、Y 3 、Y 4 , J, K, L and A are each as defined above in Formula I and Formula II.

[0215] As used herein, unless otherwise indicated, references to Formula I include Formula Ia and Formula Ib, and references to Formula II include Formula II-a and Formula II-b, as long as such references should be understood by a person of ordinary skill in the art to be applicable in light of the disclosure herein.

[0216] In some embodiments, the heterocyclic base moiety (e.g., X in the structure of any of Formulas I-VIII (including all subgroups or species of formulas (e.g., Formula Ib-5)) is a pyrimidine, a substituted pyrimidine, a purine, or a substituted purine. In some embodiments, the heterocyclic base moiety is a naturally occurring purine or a substituted purine. In some embodiments, the heterocyclic base moiety is a non-naturally occurring purine or a substituted purine. In some embodiments, the heterocyclic base moiety is a naturally occurring pyrimidine or a substituted pyrimidine. In some embodiments, the heterocyclic base moiety is a non-naturally occurring pyrimidine or a substituted pyrimidine.

[0217] In some embodiments, the heterocyclic base moiety (e.g., X in the structure of any of Formulas I to VIII (including all subgroups or species of formulas (e.g., Formula Ib-5)) is uracil, thymine, cytosine, 5-methylcytosine, adenine, guanine, or inosine.

[0218] In some embodiments, the heterocyclic base moiety (e.g., X in the structure of any of Formulas I to VIII (including all subgroups or species of formulas (e.g., Formula Ib-5)) is a substituted uracil, a substituted thymine, a substituted cytosine, a substituted 5-methylcytosine, a substituted adenine, a substituted guanine, or a substituted inosine. In some embodiments, the substituted group is a protecting group.

[0219] In some embodiments, hydrogen isotopes such as deuterium and tritium may be incorporated at one or more positions where hydrogen is present. In some embodiments, isotopes of other atoms (e.g., C, N, O, or F) are present.

[0220] In some embodiments, the compound has a 5'-cyclopropylphosphonate structure represented by Formula Ib or Formula II-b:

[0221]

[0222] in:

[0223] D is O, S, CH2-CH2, CH=CH, OCH2, N(R 1 )、C(R 2 )(R 3 )、C(R 2 )(R 3 )C(R 4 )(R 2 )、C(R 2 )=C(R 4 )、OC(R 2 )(R 3 )、OC(H)(X 3 ) or OC(R 2 )(X 3 );

[0224] R 1 is H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl;

[0225] R 2 、R 3 and R 4 Each is independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0226] When D is O, S, CH2-CH2, CH=CH, OCH2, N(R 1 )、C(R 2 )(R 3 )、C(R 2 )(R 3 )C(R 4 )(R 2 )、C(R 2 )=C(R 4 )、OC(R 2 )(R 3 ), X is a heterocyclic base moiety;

[0227] When D is OC(H)(X 3 ) or OC(R 2 )(X 3), X is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, and X 3 is a heterocyclic base moiety;

[0228] Z is H, -OH, F, OCH3, -O-(CH2)2-OCH3; halogen; -OCH2F, -OCHF2, -OCF3, -OCH2CH3, -O(CH2)2F, -OCH2CHF2, -OCH2CF3, -OCH2-CH=CH2, -O(CH2)2-OCH3, -O(CH2)2-SC H3, -O(CH2)2-OCF3, -O(CH2)2-O(CH2)2-N(CH3)2, -OCH2C(=O)-N(H)CH3, -OCH2C(=O)-N(H)-(CH2)2-N(CH3)2, -O(CH2)2-N(H)-C(=NH)(NH2), -O(CH2)3-N(R 5 )(R 6 )、-O(CH2)2-ON(R 5 )(R 6 )、-O(CH2)2-O(CH2)2-N(R 5 )(R 6 )、-OCH2C(=O)-N(R 5 )(R 6 )、-OCH2C(=O)-N(R 7 )-(CH2)2-N(R 5 )(R 6 )、-O(CH2)2-N(R 7 )-C(=R 8 )[N(R 5 )(R 6 )], optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, wherein R 5 、R 6 、R 7 and R 8 Each independently is H or C1-C6 alkyl;

[0229] Y 1 、Y 2 、Y 3 and Y 4are each independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; or, Y 4 With Y 1 or Y 2 One of the connections, wherein the connection comprises a member selected from the group consisting of O, S, NR 9 、C(R 10 )(R 11 )、C(R 10 )=C(R 11 ), C[=C(R 10 )(R 11 )] and a divalent group of C(=O), and Y 1 、Y 2 and Y 3 The other two are each independently H, halogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, wherein R 9 、R 10 and R 11 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl;

[0230] J is O, S, NR 12 ,NN(R 13 )2 or N-OR 13 ,in:

[0231] R 12 It is H, OH, halogen, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0232]

[0233] where R 14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or NH-C=(NH)NH2, wherein R 15 Selected from H, C1-C 18 alkyl or aryl;

[0234] where R 13 It is H, C1-C 10Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0235]

[0236] where R 14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or NH-C=(NH)NH2, and wherein R 15 Selected from H, C1-C 18 alkyl or aryl;

[0237] K and L are each independently selected from OH, OR 16 SR 16 or NR 16 , where R 16 Selected from H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0238]

[0239] where R 14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or -NH-C=(NH)NH2, and R 15 Selected from H, C1-C 18 alkyl or aryl;

[0240] Q is selected from O, S, N (R 30 ) or C(R 31 )(R 32 ) of the divalent part, wherein R 30 is H, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, and R 31 and R 32 are each independently H, halogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl;

[0241] G 1 , G 2 , G 3 and G 4 Each independently selected from the group consisting of H, F, halogen, C1-C6 alkyl, CN, CH2(R 33)、CH2-O-(R 33 )、C(=O)(R 33 )、C(=S)(R 33 ) or (R 34 )(R 33 ), where R 33 It is O(R 35 )、S(R 35 )、N(R 35 )(R 36 ), where R 34 、R 35 and R 36 are each independently selected from H, halogen or C1-C6 alkyl; and

[0242] A is (i) an internucleoside linkage linking the 5'-cyclic phosphonate modified nucleotide of Formula Ib or Formula II-b to the remainder of the RNAi agent, or (ii) a phosphoramidite group.

[0243] As used herein, unless otherwise indicated, references to Formula Ib include Formula Ib-1, Formula Ib-2, Formula Ib-3, Formula Ib-4, Formula Ib-5, and references to Formula II-b include Formula II-b-1, Formula II-b-2, Formula II-b-3, Formula II-b-4, and Formula II-b-5, as long as such references are understood by a person of ordinary skill in the art to be applicable in light of the disclosure herein.

[0244] In some embodiments, the compound has a 5'-cyclopropylphosphonate structure represented by Formula Ib-1 or Formula II-b-1:

[0245]

[0246] Among them, D, X, Z, Y 1 、Y 2 、Y 3 、Y 4 , J, K, L, A and Q are each as defined above in Formula Ib and Formula II-b.

[0247] In some embodiments, the compound has a structure represented by Formula Ib-2 or Formula II-b-2:

[0248]

[0249] Among them, D, X, Z, Y 1 、Y 2 、Y 3 、Y 4 , J, K, L, G 1 , G 2 , G 3 , G4 and A are each as defined above in Formula Ib and Formula II-b.

[0250] In some embodiments, the compound has a structure represented by Formula Ib-3 or Formula II-b-3:

[0251]

[0252] Among them, D, X, Z, Y 1 、Y 2 、Y 3 、Y 4 , J, K, L and A are each as defined above in Formula Ib and Formula II-b.

[0253] In some embodiments, the compound has a structure represented by Formula Ib-4 or Formula II-b-4:

[0254]

[0255] Among them, D, X, Z, Y 1 、Y 2 、Y 3 、Y 4 , J, K, L, Q, A, G 1 , G 2 , G 3 and G 4 Each is as defined above in Formula Ib and Formula II-b.

[0256] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonate modified nucleotide represented by the following Formula III or Formula IV:

[0257]

[0258] in:

[0259] X is a heterocyclic base moiety;

[0260] Z is H, -OH, F, OCH3, -O-(CH2)2-OCH3; halogen; -OCH2F, -OCHF2, -OCF3, -OCH2CH3, -O(CH2)2F, -OCH2CHF2, -OCH2CF3, -OCH2-CH=CH2, -O(CH2)2-OCH3, -O(CH2)2-SC H3, -O(CH2)2-OCF3, -O(CH2)2-O(CH2)2-N(CH3)2, -OCH2C(=O)-N(H)CH3, -OCH2C(=O)-N(H)-(CH2)2-N(CH3)2, -O(CH2)2-N(H)-C(=NH)(NH2), -O(CH2)3-N(R 5 )(R 6 )、-O(CH2)2-ON(R 5 )(R 6 )、-O(CH2)2-O(CH2)2-N(R 5 )(R 6 )、-OCH2C(=O)-N(R 5 )(R 6 )、-OCH2C(=O)-N(R 7 )-(CH2)2-N(R 5 )(R 6 )、-O(CH2)2-N(R 7 )-C(=R 8 )[N(R 5 )(R 6 )], optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, wherein R 5 、R 6 、R 7 and R 8 Each independently is H or C1-C6 alkyl;

[0261] J and J' are each independently O or S;

[0262] L, L' and K are each independently selected from OH, OR 16 SR 16 or NR 16 , where R 16 Selected from H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0263]

[0264] where R 14is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or -NH-C=(NH)NH2, and R 15 Selected from H, C1-C 18 alkyl or aryl; and

[0265] The remainder of the RNAi agent is contained.

[0266] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonate modified nucleotide represented by the following formula III-a or formula IV-a:

[0267]

[0268] Among them, X, Z, J, K, L, J′, L′ and Each is as defined above in Formula III and Formula IV.

[0269] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonate modified nucleotide represented by the following formula III-b and formula IV-b:

[0270]

[0271] Among them, X, Z, J, J′ and Each is as defined above in Formula III and Formula IV.

[0272] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of a double-stranded RNAi agent is a 5'-cyclopropylphosphonate modified nucleotide comprising a thymine heterocyclic base moiety and a 2'-methoxyethyl (2'-O-2-methoxyethyl or "2'-MOE") modification (cPrpTMs), as shown in Structure i below:

[0273]

[0274] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonate modified nucleotide, wherein the modified nucleotide comprises a thymine heterocyclic base moiety and a 2'-methoxyethyl (2'-O-2-methoxyethyl or "2-MOE") modification (cPrp™), as shown in structure ii below:

[0275]

[0276] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonate modified nucleotide, wherein the modified nucleotide comprises a thymine heterocyclic base moiety and a 2'-H modification (cPrpdT), as shown in the following structure iii:

[0277]

[0278] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of a double-stranded RNAi agent is a 5'-cyclopropylphosphonate modified nucleotide comprising a uracil heterocyclic base moiety and a 2'-methoxyethyl (2'-O-2-methoxyethyl or "2'-MOE") modification (cPrpUMs), as shown in Structure iv below:

[0279]

[0280] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonate modified nucleotide, wherein the modified nucleotide comprises a uracil heterocyclic base moiety and a 2'-O-methyl modification (cPrpu), as shown in the following structure v:

[0281]

[0282] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonate modified nucleotide, wherein the modified nucleotide comprises a uracil heterocyclic base moiety and a 2'-O-methyl modification (cPrpus), as shown in the following structure vi:

[0283]

[0284] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonate modified nucleotide, wherein the modified nucleotide comprises a uracil heterocyclic base moiety and a 2'-deoxy modification (cPrpdU), as shown in the following structure vii:

[0285]

[0286] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonate modified nucleotide, wherein the modified nucleotide comprises a uracil heterocyclic base moiety and a 2'-O-methyl modification (cPrpdUs), as shown in the following structure viii:

[0287]

[0288] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonate modified nucleotide, wherein the modified nucleotide comprises an adenine heterocyclic base moiety and a 2'-O-methyl modification (cPrpa), as shown in the following structure ix:

[0289]

[0290] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonate modified nucleotide, wherein the modified nucleotide comprises an adenine heterocyclic base moiety and a 2'-O-methyl modification (cPrpas), as shown in the following structure x:

[0291]

[0292] In some embodiments, structure i, ii, iii, iv, v, vi, vii, viii, ix, or x can be located at the 5' end (terminal nucleotide) of the antisense strand of a double-stranded RNAi agent.

[0293] In some embodiments, structure i, ii, iii, iv, v, vi, vii, viii, ix, or x can be located at the 5' end (terminal nucleotide) of the single-stranded antisense oligonucleotide.

[0294] Structures i-x are merely exemplary in nature. As discussed elsewhere herein, for example, sugar alternative substituents can be used in conjunction with the above structures to modify the 5-membered furanose ring into different structures that can replace the 5-membered furanose ring, such as morpholinyl, cyclohexenyl, cyclohexanehexol, or acyclic structures. Such modifications are contemplated and fall within the scope of the invention described herein.

[0295] In some embodiments, a 5'-phosphonate mimetic, such as a 5'-C-malonyl group, is linked to a cyclopropyl group, and the terminus (or terminal nucleotide) of the antisense strand of the RNAi agent is a 5'-cyclopropyl-C-malonyl modified nucleotide represented by the following formulas V and VI:

[0296]

[0297] in:

[0298] X is a heterocyclic base moiety;

[0299] Z is H, -OH, F, OCH3, -O-(CH2)2-OCH3; halogen; -OCH2F, -OCHF2, -OCF3, -OCH2CH3, -O(CH2)2F, -OCH2CHF2, -OCH2CF3, -OCH2-CH=CH2, -O(CH2)2-OCH3, -O(CH2)2-SC H3, -O(CH2)2-OCF3, -O(CH2)2-O(CH2)2-N(CH3)2, -OCH2C(=O)-N(H)CH3, -OCH2C(=O)-N(H)-(CH2)2-N(CH3)2, -O(CH2)2-N(H)-C(=NH)(NH2), -O(CH2)3-N(R 5 )(R 6 )、-O(CH2)2-ON(R 5 )(R 6 )、-O(CH2)2-O(CH2)2-N(R 5 )(R 6 )、-OCH2C(=O)-N(R 5 )(R 6 )、-OCH2C(=O)-N(R 7 )-(CH2)2-N(R 5 )(R 6 )、-O(CH2)2-N(R 7 )-C(=R 8 )[N(R 5 )(R 6 )], optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, wherein R 5 、R 6 、R 7 and R 8 Each independently is H or C1-C6 alkyl;

[0300] J and J' are each independently O or S; and

[0301] L' is selected from OH, OR 16 SR 16 or NR 16 , where R 16 Selected from H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0302]

[0303] where R 14is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or -NH-C=(NH)NH2, and R 15 Selected from H, C1-C 18 alkyl or aryl; and

[0304] The remainder of the RNAi agent is contained.

[0305] It will be readily understood and appreciated by those of ordinary skill in the art that, under sufficiently alkaline conditions, the protic groups of Formula I, II, III, IV, V, VI, VII and / or VIII (including subsets or species of formulae (e.g., Formula Ib-5)) and / or Structures i, ii, iii, iv, v, vi, vii, viii, ix, x, xi, xii and / or xiii exist in a partially or completely deprotonated state. Similarly, under sufficiently acidic conditions, the groups or atoms comprising the basic sites are protonated. All of these protonated or deprotonated forms of the groups described herein fall within the scope of the present invention. For example, where a carboxyl group falls within the scope of the present invention or claims, the corresponding carboxylate / salt also falls within the scope of the present invention or claims. For example, where an amino group falls within the scope of the present invention or claims, the corresponding ammonium group also falls within the scope of the present invention or claims.

[0306] In some embodiments, the compound is a phosphoramidite compound having a structure represented by Formula Ib-5 or Formula II-b-5:

[0307]

[0308] in:

[0309] D is O, S, CH2-CH2, CH=CH, OCH2, N(R 1 )、C(R 2 )(R 3 )、C(R 2 )(R 3 )C(R 4 )(R 2 )、C(R 2 )=C(R 4 )、OC(R 2 )(R 3 )、OC(H)(X 3 ) or OC(R 2 )(X 3 );

[0310] R 1is H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl;

[0311] R 2 、R 3 and R 4 Each is independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;

[0312] When D is O, S, CH2-CH2, CH=CH, OCH2, N(R 1 )、C(R 2 )(R 3 )、C(R 2 )(R 3 )C(R 4 )(R 2 )、C(R 2 )=C(R 4 )、OC(R 2 )(R 3 ), X is a heterocyclic base moiety;

[0313] When D is OC(H)(X 3 ) or OC(R 2 )(X 3 ), X is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, and X 3 is a heterocyclic base moiety;

[0314] Z is H, -OH, F, OCH3, -O-(CH2)2-OCH3; halogen; -OCH2F, -OCHF2, -OCF3, -OCH2CH3, -O(CH2)2F, -OCH2CHF2, -OCH2CF3, -OCH2-CH=CH2, -O(CH2)2-OCH3, -O(CH2)2-SC H3, -O(CH2)2-OCF3, -O(CH2)2-O(CH2)2-N(CH3)2, -OCH2C(=O)-N(H)CH3, -OCH2C(=O)-N(H)-(CH2)2-N(CH3)2, -O(CH2)2-N(H)-C(=NH)(NH2), -O(CH2)3-N(R 5 )(R 6)、-O(CH2)2-ON(R 5 )(R 6 )、-O(CH2)2-O(CH2)2-N(R 5 )(R 6 )、-OCH2C(=O)-N(R 5 )(R 6 )、-OCH2C(=O)-N(R 7 )-(CH2)2-N(R 5 )(R 6 )、-O(CH2)2-N(R 7 )-C(=R 8 )[N(R 5 )(R 6 )], optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, wherein R 5 、R 6 、R 7 and R 8 Each independently is H or C1-C6 alkyl;

[0315] Y 1 、Y 2 、Y 3 and Y 4 are each independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; or, Y 4 With Y 1 or Y 2 One of the connections, wherein the connection comprises a member selected from the group consisting of O, S, NR 9 、C(R 10 )(R 11 )、C(R 10 )=C(R 11 ), C[=C(R 10 )(R 11 )] and a divalent group of C(=O), and Y 1 、Y 2 and Y 3 The other two are each independently H, halogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl, wherein R 9 、R 10 and R 11are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl;

[0316] J is O, S, NR 12 ,NN(R 13 )2 or N-OR 13 ,in:

[0317] R 12 It is H, OH, halogen, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0318]

[0319] where R 14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or NH-C=(NH)NH2, wherein R 15 Selected from H, C1-C 18 alkyl or aryl;

[0320] where R 13 It is H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0321]

[0322] where R 14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or NH-C=(NH)NH2, and wherein R 15 Selected from H, C1-C 18 alkyl or aryl;

[0323] K and L are each independently selected from OH, OR 16 SR 16 or NR 16 , where R 16 Selected from H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or

[0324]

[0325] where R14 is selected from H or C1-C4 alkyl optionally substituted by 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted by hydroxy, heteroaryl optionally substituted by hydroxy, amino, hydroxy, oxygen or -NH-C=(NH)NH2, and R 15 Selected from H, C1-C 18 alkyl or aryl; and

[0326] G 1 , G 2 , G 3 and G 4 Each independently selected from the group consisting of H, F, halogen, C1-C6 alkyl, CN, CH2(R 33 )、CH2-O-(R 33 )、C(=O)(R 33 )、C(=S)(R 33 ) or (R 34 )(R 33 ), where R 33 It is O(R 35 )、S(R 35 )、N(R 35 )(R 36 ), where R 34 、R 35 and R 36 Each is independently selected from H, halogen or C1-C6 alkyl.

[0327] In some embodiments, X in Formula Ib-5 and Formula II-b-5 comprises one or more protecting groups.

[0328] In some embodiments, the compound represented by Formula Ib-5 or Formula II-b-5 has the following structure:

[0329]

[0330] (2-cyanoethyl((2R,3R,4R,5R)-2-(2-(diethoxyphosphoryl)cyclopropyl)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl)diisopropylphosphoramidite);

[0331]

[0332] (dimethyl [2-[(2R,3R,4R,5R)-3-([[bis(propan-2-yl)amino](2-cyanoethoxy)phosphino]oxy)-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)oxolan-2-yl]cyclopropyl]phosphonate); or

[0333]

[0334] (2R,3R,4R,5R)-5-(6-Benzamido-9H-purin-9-yl)-2-(2-(diethoxyphosphoryl)cyclopropyl)-4-methoxytetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite).

[0335] In some embodiments, structure xi, xii, or xiii can be added to the 5' end (terminal nucleotide) of the antisense strand of a double-stranded RNAi agent.

[0336] In some embodiments, structure xi, xii, or xiii can be added to the 5' end (terminal nucleotide) of a single-stranded antisense oligonucleotide.

[0337] Structures xi, xii, and xiii are merely exemplary in nature. As discussed elsewhere herein, for example, sugar-substituent moieties can be used in conjunction with the above structures to modify the 5-membered furanose ring into different structures that can replace the 5-membered furanose ring, such as morpholinyl, cyclohexenyl, cyclohexanehexol, or an acyclic structure. In addition, as discussed elsewhere herein, modifications at the 2' or 3' position of the modified nucleotide can vary from various modifications known in the art, and / or the heterocyclic base moiety can be modified from the specific structures described herein. Such variations are contemplated and fall within the scope of the invention described herein.

[0338] When the 5'-cyclic phosphonate modified nucleotide is in the form of a phosphoramidite compound as described herein, it can be used to link the 5'-cyclic phosphonate modified nucleotide using phosphoramidite synthesis methods for nucleotides known in the art. The 5'-cyclic phosphonate modified nucleotide can be prepared as a phosphoramidite compound by linking the phosphorus atoms of the phosphoramidite forming reagent through a coupling reaction (e.g., phosphorylation) to form the phosphoramidite compound.

[0339] In some embodiments, a 5'-cyclic phosphonate modified nucleotide-phosphoramidite compound is used to attach a 5'-cyclic phosphonate modified nucleotide to the 5' end of the antisense strand of a double-stranded RNAi agent. In some embodiments, a 5'-cyclic phosphonate modified nucleotide-phosphoramidite compound is used to attach a 5'-cyclic phosphonate modified nucleotide to the 5' end of a single-stranded RNAi agent.

[0340] As used herein, RNAi agents and single-stranded antisense oligonucleotides comprising 5'-cyclopropylphosphonate modified nucleotides described herein contain one or more asymmetric centers, thereby giving rise to enantiomers, diastereomers, and other stereoisomeric configurations. The configuration of any bond presented herein is selected for convenience only and is not intended to limit a particular configuration unless otherwise indicated herein.

[0341] In some embodiments, the compound has a structure of Formula B represented by Formula IX or Formula X:

[0342]

[0343] Among them, "ring", D, X, Z, Y 1 、Y 2 、Y 3 、Y 4 , J, K, L, Q and A are each as defined above in Formula I and Formula II.

[0344] RNAi agents, targeting ligands, and delivery polymers

[0345] Oligomeric compounds (eg, RNAi agents) having a sequence at least partially complementary to a target nucleic acid have been shown to alter the function and activity of the target nucleic acid both in vitro and in vivo. As used herein, an RNAi agent may comprise one or more 5'-cyclic phosphonate modified nucleotides.

[0346] In some embodiments, the RNAi agent comprising a 5'-cyclic phosphonate modified nucleotide described herein is double-stranded. In the case of a double-stranded RNAi agent, the sense and antisense strands of the RNAi agent are independently 16 to 30 nucleotides in length. In some embodiments, the double-stranded RNAi agent comprises a sense strand and an antisense strand that are at least partially complementary to each other (at least 70% complementary). The antisense strand comprises a region having a sequence that is perfectly complementary (100% complementary) or at least partially complementary (at least 85% complementary) to a sequence in the target mRNA. The sense and antisense strands of the double-stranded RNAi agent can each be 16 to 30 nucleotides in length. The sense and antisense strands can be the same length or different lengths. In some embodiments, the sense and antisense strands are each independently 17 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are each independently 17 to 21 nucleotides in length. In some embodiments, the sense and antisense strands are both 21 to 26 nucleotides in length. In some embodiments, the sense strand is about 19 nucleotides in length and the antisense strand is about 21 nucleotides in length. In some embodiments, the sense strand is about 21 nucleotides in length and the antisense strand is about 23 nucleotides in length. In some embodiments, the sense and antisense strands are both 26 nucleotides in length. In some embodiments, the sense and antisense strands of an RNAi agent are each independently 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. In some embodiments, a double-stranded RNAi agent has a duplex length of about 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides. This region of perfect or partial complementarity between the sense and antisense strands is typically 15 to 25 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides in length and is located at or near the 5' end of the antisense strand (e.g., this region is 1, 2, 3, or 4 nucleotides from the 5' end of the antisense strand that is not perfectly or partially complementary). In some embodiments, the 5'-cyclic phosphonate modified nucleotide is the terminal nucleotide on the 5' end of the antisense strand of the double-stranded RNAi agent.

[0347] In some embodiments, the RNAi agent comprising a 5'-cyclic phosphonate modified nucleotide is a single-stranded antisense oligonucleotide. In some embodiments, the single-stranded antisense oligonucleotide is each independently about 8 to about 40 nucleotides in length.

[0348] In some embodiments, the RNAi agent comprising a 5'-cyclic phosphonate modified nucleotide is a double-stranded molecule having a sense strand and an antisense strand. In some embodiments, the RNAi agent comprising a 5'-cyclic phosphonate modified nucleotide is a single-stranded antisense oligonucleotide.

[0349] In some embodiments, a 5'-cyclic phosphonate modified nucleotide is attached to the end of the RNAi agent and improves the nuclease stability of the RNAi agent. In some embodiments, the RNAi agent described herein comprising a 5'-cyclic phosphonate modified nucleotide at the end is delivered to cells and can inhibit or knock down the expression of the target gene in vitro or in vivo through the biological process of RNA interference (RNAi).

[0350] In the case of double-stranded RNAi agents, the sense strand and / or antisense strand may optionally and independently contain additional 1, 2, 3, 4, 5, or 6 nucleotides (extension sequences) at the 3' end, 5' end, or both 3' and 5' ends of the core sequence. If additional sense strand nucleotides are present, they may be identical or different from the corresponding sequence in the target mRNA. If additional antisense strand nucleotides are present, they may be complementary or non-complementary to the other nucleotides (if any) of the corresponding sense strand. In the case of single-stranded RNAi agents, if additional nucleotides are present, they may be complementary or non-complementary to the corresponding sequence in the target mRNA.

[0351] In some embodiments, the sense strand and antisense strand of a double-stranded RNAi agent comprising a terminal 5'-cyclic phosphonate modified nucleotide as described herein contain the same number of nucleotides. In some embodiments, the sense strand and antisense strand of an RNAi agent as described herein contain different numbers of nucleotides. In some embodiments, the sense strand 5' end and the antisense strand 3' end of the RNAi agent form a blunt end. In some embodiments, the sense strand 3' end and the antisense strand 5' end of the RNAi agent form a blunt end. In some embodiments, both ends of the RNAi agent form a blunt end. In some embodiments, both ends of the RNAi agent are not blunt ends. As used herein, the term "blunt end" refers to the end of a double-stranded RNAi agent in which the terminal nucleotides of the two annealed chains are complementary (forming complementary base pairing).

[0352] In some embodiments, the 5' end of the sense strand and the 3' end of the antisense strand of the RNAi agent form a defective end. In some embodiments, the 3' end of the sense strand and the 5' end of the antisense strand of the RNAi agent form a defective end. In some embodiments, both ends of the RNAi agent form defective ends. In some embodiments, both ends of the RNAi agent are not defective ends. As used herein, a defective end refers to the end of a double-stranded initiator molecule where the terminal nucleotides of the two annealed chains form a pair (i.e., do not form an overhang) but are not complementary (i.e., form a non-complementary pair). As used herein, an overhang refers to an extension of one or more unpaired nucleotides at the end of one chain of a double-stranded RNAi agent. The unpaired nucleotides can be located on the sense strand or the antisense strand to generate a 3' or 5' overhang. In some embodiments, the RNAi agent contains: one blunt end and one missing end, one blunt end and one 5' overhang, one blunt end and one 3' overhang, one missing end and one 5' overhang, one missing end and one 3' overhang, two 5' overhangs, two 3' overhangs, one 5' overhang and one 3' overhang, two missing ends, or two blunt ends.

[0353] In some embodiments, the RNAi agent contains at least one nucleotide with a modified backbone (also referred to herein as an internucleoside linkage). In some embodiments, the modified backbone or internucleoside linkage is one or more phosphorothioate linkages.

[0354] In some embodiments, the sense strand of the RNAi agent contains 1 to 4 phosphorothioate linkages. In other embodiments, the antisense strand of the RNAi agent contains 1 to 4 phosphorothioate linkages. In some embodiments, both the sense strand and the antisense strand contain 1 to 4 phosphorothioate linkages.

[0355] In some embodiments where the RNAi agent is single-stranded, all or substantially all linkages of nucleotides or modified nucleotides in the molecule of the RNAi agent contain phosphorothioate linkages.

[0356] In some embodiments, the RNAi agent comprising a 5'-cyclic phosphonate modified nucleotide having a structure of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII and / or Formula VIII (including all subgroups or species of formula (e.g., Formula Ib-5)) and / or Structure i, Structure ii, Structure iii, Structure iv, Structure v, Structure vi, Structure vii, Structure viii, Structure ix, Structure x, Structure xi, Structure xii and / or Structure xiii is double-stranded. A double-stranded RNAi agent can be formed by annealing the antisense and sense strands together. In some embodiments, the RNAi agent comprising a 5'-cyclic phosphonate modified nucleotide having a structure of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII and / or Formula VIII (including all subgroups or species of formula (e.g., Formula Ib-5)) and / or Structure I, Structure II, Structure III, Structure IV, Structure V, Structure Vi, Structure Vii, Structure VIII, Structure IX, Structure X, Structure Xi, Structure Xii and / or Structure XIII is a single-stranded oligonucleotide. The RNAi agents described herein are synthesized using methods commonly used in the art.

[0357] In some embodiments, the RNAi agent comprises one or more modified nucleotides. As used herein, "modified nucleotides" are nucleotides other than ribonucleotides (2'-hydroxy nucleotides). As used herein, deoxyribonucleotides are considered a class of modified nucleotides. In some embodiments, at least 50%, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% of the nucleotides of the RNAi agent are modified. As used herein, modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides (denoted herein as X, Ab), 2'-modified nucleotides, 3'-3' linked (inverted) nucleotides (denoted herein as invdN, invN, invn, invX, invAb), nucleotides containing non-natural bases, bridged nucleotides, peptide nucleic acids (PNAs), 2', 3'-open ring nucleotide mimics (non-locked nucleobase analogs, denoted herein as N UNA or NUNA), locked nucleotides (represented herein as N LNA or NLNA), 3'-O-methoxy (2' internucleoside linked) nucleotides (denoted herein as 3'-OMen), 2'-F-arabinosyl nucleotides (denoted herein as NfANA or Nf ANA), 5'-Me, 2'-fluoro nucleotides (denoted herein as 5Me-Nf), morpholino nucleotides, vinylphosphonate deoxyribonucleotides (denoted herein as vpdN), vinylphosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleotides (cPrpN). 2'-modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides (denoted herein as lowercase "n" in the nucleotide sequence), 2'-deoxy-2'-fluoro nucleotides (denoted herein as Nf, also denoted as 2'-fluoro nucleotides), 2'-deoxy nucleotides (denoted herein as dN), 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides (denoted herein as NM or 2'-MOE), 2'-amino nucleotides, and 2'-alkyl nucleotides. It is not necessary to uniformly modify all positions in a given compound. Rather, more than one modification can be introduced into a single RNAi agent or even a single nucleotide thereof. RNAi agents can be synthesized and / or modified by methods known in the art. Modifications on one nucleotide are independent of modifications on another nucleotide.

[0358] Modified nucleobases include synthetic and natural nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, (e.g., 2-aminopropyladenine, 5-propynyluracil or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothreonine ... Adenine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

[0359] In some embodiments, all or substantially all of the nucleotides of the RNAi agent are modified nucleotides.

[0360] In some embodiments, one or more nucleotides of the RNAi agent are ribonucleotides. As used herein, ribonucleotides are represented herein as "N" (capital letters with no other symbols).

[0361] The nucleotides of the RNAi agents described herein can be linked by phosphate-containing or phosphate-free covalent internucleoside linkages. Modified internucleoside linkages or backbones include, but are not limited to, 5'-phosphorothioate groups (represented herein by a lowercase "s" before the nucleotide, such as sN, sn, sNf, or sdN), chiral phosphorothioates, phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkyl phosphonates (e.g., methyl phosphonate or 3'-alkylenephosphonate), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidate, aminoalkylphosphoramidate, or thiocarbonylphosphoramidate), thiocarbonylalkylphosphonates, thiocarbonylalkylphosphotriesters, morpholino linkages, boranophosphates with a conventional 3'-5' linkage, 2'-5' linkage analogs of boranophosphates, or boranophosphates with opposite polarity in which a pair of adjacent nucleoside units is shifted from 3'-5' to 5'-5' or from 2'-5' to 5'-2'. In some embodiments, the modified internucleoside linkage or backbone does not have a phosphorus atom. Modified internucleoside linkages without phosphorus atoms include, but are not limited to, linkages between short-chain alkyl or cycloalkyl sugars, linkages between mixed heteroatoms and alkyl or cycloalkyl sugars, or linkages between one or more short-chain heteroatoms or heterocyclic sugars. In some embodiments, the modified internucleoside backbone includes, but is not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formyl and thioformyl backbones, methyleneformyl and thioformyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazinyl backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH2 components.

[0362] In some embodiments, the RNAi agent comprising a 5'-cyclic phosphonate modified nucleotide inhibits the expression of a target mRNA in a cell, cell mass, tissue, or subject. In some embodiments, a therapeutically effective amount of an RNAi agent comprising a 5'-cyclic phosphonate modified nucleotide as described herein is administered to a subject, thereby inhibiting the expression of a target mRNA in the subject.

[0363] In some embodiments, the RNAi agent is used to treat, prevent or control clinical manifestations related to the expression of the target mRNA. In some embodiments, a therapeutically or prophylactically effective amount of one or more RNAi agents is administered to a subject in need of such treatment, prevention or control.

[0364] The RNAi agents and methods comprising 5'-cyclic phosphonate modified nucleotides can be used to treat or prevent at least one symptom in a subject suffering from a disease or disorder that would benefit from reduction or inhibition of target mRNA expression. In some embodiments, a therapeutically effective amount of one or more RNAi agents is administered to a subject to treat the at least one symptom. In other embodiments, a prophylactically effective amount of one or more RNAi agents is administered to a subject to prevent the at least one symptom.

[0365] In some embodiments, the gene expression level and / or mRNA level of the target in a subject administered the targeting ligand conjugated to an expression-inhibiting oligomeric compound is decreased by at least about 5%, such as at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, relative to the subject before administration of the targeting ligand conjugate or a subject that did not receive the targeting ligand conjugate. The gene expression level and / or mRNA level in the subject can be decreased in cells, cell populations, and / or tissues of the subject. In some embodiments, the protein level in a subject administered with the targeting ligand conjugated to an expression-inhibiting oligomeric compound is decreased by at least about 5%, such as at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, relative to the subject before administration of the targeting ligand conjugate or the subject not receiving the targeting ligand conjugate. The protein level in the subject can be decreased in the subject's cells, cell populations, tissues, blood, and / or other fluids. A decrease in gene expression, mRNA, or protein levels can be assessed by any method known in the art. A decrease or reduction in mRNA levels and / or protein levels is collectively referred to herein as inhibition, reduction, or decrease in target gene expression.

[0366] The RNAi medicament comprising 5'-cyclic phosphonate modified nucleotides as described herein and the composition comprising RNAi medicament as described herein can be delivered to cells, cell masses, tumors, tissues or objects using oligonucleotide delivery technology known in the art. In general, any method suitable for delivering nucleic acid molecules (in vitro or in vivo) known in the art can be applied to the RNAi medicament comprising one or more 5'-cyclic phosphonate modified nucleotides as described herein. For example, delivery can be by topical administration (such as direct injection, implantation or external administration), systemic administration or by subcutaneous, intravenous, oral, intraperitoneal or parenteral route, including intracranial (such as intraventricular, soft tissue and intrathecal), intramuscular, transdermal, airway (aerosol), intranasal, rectal or external (including buccal and sublingual) administration. In some embodiments, the composition is administered by subcutaneous or intravenous infusion or injection.

[0367] In some embodiments where RNAi agents are double-stranded, the RNAi agent may contain a non-nucleotide group connected to the 3' or 5' end of one of the sense strand or antisense strand. In some embodiments, a targeting ligand or a targeting group, a linking group, or a delivery vehicle is covalently linked to the sense strand. In some embodiments, the targeting ligand, a linking group, and / or a delivery vehicle are connected to the 3' and / or 5' end of the sense strand. In some embodiments, the targeting ligand, a linking group, and / or a delivery vehicle are connected to the 5' end of the sense strand. In some embodiments, the targeting ligand, a linking group, and / or a delivery vehicle are directly or indirectly connected to the 3' or 5' end of the sense strand through a joint. In some embodiments, the targeting ligand is connected to the RNAi agent through an unstable, cleavable, or reversible bond or a joint / spacer.

[0368] In some embodiments where the RNAi agent is single-stranded, the RNAi agent may contain a targeting ligand or targeting group, linker, or delivery vehicle attached to the end where the terminal 5'-cyclic phosphonate modified nucleotide is absent. In some embodiments where the RNAi agent is single-stranded, the 5'-cyclic phosphonate modified nucleotide is attached to the 5' end of the RNAi agent, and the targeting ligand, linker, or delivery vehicle is attached to the 3' end of the RNAi agent.

[0369] In some embodiments, a delivery vector can be used to deliver the RNAi agent to a cell or tissue. A delivery vector is a compound that facilitates the delivery of the RNAi agent to a cell or tissue. The delivery vector can include or be composed of, but is not limited to, a polymer such as an amphiphilic polymer, a membrane active polymer, a peptide, melittin, a melittin analog, a lipid, a reversibly modified polymer or peptide, or a reversibly modified membrane active polyamine.

[0370] The RNAi medicament comprising 5'-cyclic phosphonate modified nucleotides can be combined with lipid, nanoparticle, polymer, liposome, micelle, dynamic multiconjugate (DPC) or other delivery system available in this area. RNAi medicament can also be chemically coupled with targeting group or targeting moiety, lipid (including but not limited to cholesterol and cholesterol base derivatives), nanoparticle, polymer, liposome, micelle, DPC (see, for example, WO 2000 / 053722, WO 2008 / 0022309, WO 2011 / 104169 and WO 2012 / 083185, WO 2013 / 032829, WO 2013 / 158141, which are each incorporated herein by reference) or other delivery system available in this area.

[0371] In some embodiments, one or more 5'-cyclic phosphonate-modified nucleotides linked to an RNAi agent are included in a pharmaceutical composition for in vivo delivery to cells. Such pharmaceutical compositions may include, but are not limited to, an RNAi agent comprising one or more 5'-cyclic phosphonate-modified nucleotides conjugated to a delivery polymer to form an RNAi initiator-delivery polymer conjugate. In some embodiments, the delivery polymer is a membrane-active polyamine. In some embodiments, the delivery polymer is a reversibly modified membrane-active polyamine.

[0372] In some embodiments, the targeting ligand or targeting group is a galactose cluster that is connected to an RNAi agent comprising one or more 5'-cyclic phosphonate modified nucleotides. In some embodiments, the RNAi agent described herein is connected to a galactose cluster. As used herein, a galactose cluster includes molecules having two to four terminal galactose derivatives. As used herein, the term galactose derivative includes galactose and galactose derivatives with an affinity equal to or greater than galactose for asialoglycoprotein receptors. The terminal galactose derivative is typically connected to the molecule via its C-1 carbon. In some embodiments, the galactose cluster has three terminal galactosamines or galactosamine derivatives (e.g., N-acetylgalactosamine) that each have an affinity for asialoglycoprotein receptors. In some embodiments, the galactose cluster has three terminal N-acetylgalactosamines. Other terms commonly used in the art include triantennary galactose, trivalent galactose, and galactose trimers. It is known that triantennary galactose derivative clusters bind to ASGPr with greater affinity than diantennary or monoantennary galactose derivative structures (Baenziger and Fiete, Cell, 1980, 22, 611-620; Connolly et al., J. Biol. Chem., 1982, 257, 939-945).

[0373] In some embodiments, the galactose cluster contains three galactose derivatives, each of which is connected to a central branch point. In some embodiments, the galactose cluster contains four galactose derivatives, each of which is connected to a central branch point. The galactose derivatives are connected to the central branch point via the C-1 carbon of the sugar. In some embodiments, the galactose derivatives are connected to the central branch point via a linker or a spacer.

[0374] In some embodiments, the galactose derivative comprises N-acetylgalactosamine (GalNAc or NAG). Other saccharides with affinity for asialoglycoprotein receptors are selected from the group consisting of galactose, galactosamine, N-formylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, and N-isobutyrylgalactosamine. The affinity of various galactose derivatives for asialoglycoprotein receptors has been studied (e.g., see Iobst, ST and Drickamer, KJBC 1996, 271, 6686) or has been determined using methods known and commonly used in the art.

[0375] Known targeting ligands suitable for use in targeted RNAi agents comprising 5'-cyclic phosphonate modified nucleotides are known in the art, for example, see US Patent Application Serial Nos. 14 / 452,626, 15 / 452,324, 15 / 452,423, and 62 / 415,752, the entire contents of which are incorporated herein by reference in their entirety.

[0376] Pharmaceutical compositions and preparations

[0377] Oligomeric compounds such as RNAi agents comprising 5'-cyclic phosphonate modified nucleotides described herein can be used to treat subjects (e.g., humans or animals, such as mammals, e.g., apes, monkeys, pigs, sheep, goats, cattle, horses, dogs, cats, rabbits, rats, or mice) suffering from a disease or disorder that would benefit from administration of the compound. In some embodiments, at least one RNAi agent comprising a 5'-cyclic phosphonate modified nucleotide is used to prepare a pharmaceutical composition (i.e., a drug) for treating a subject that would benefit from reduction or inhibition of gene expression. These pharmaceutical compositions are used to inhibit gene expression in a cell, tissue, or organism. In some embodiments, the pharmaceutical compositions are used to treat a subject that would benefit from reduction or inhibition of gene expression.

[0378] In some embodiments, RNAi agents comprising 5'-cyclic phosphonate modified nucleotides can be used to treat subjects (e.g., humans) suffering from diseases or disorders that would benefit from reduction or inhibition of target mRNA expression. A therapeutically effective amount of any one or more RNAi agents is administered to the subject. The subject can be a human, a patient, or a human patient. The subject can be an adult, a teenager, a child, or an infant. The pharmaceutical composition comprising a targeting ligand linked to an expression-inhibiting oligomeric compound can be used to provide a therapeutic treatment method for a disease. This method comprises administering the pharmaceutical composition described herein to a human or animal.

[0379] Thus, in some embodiments, the pharmaceutical compositions described herein can comprise one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions described herein can be formulated for administration to a subject.

[0380] As used herein, a pharmaceutical composition or drug comprises a pharmacologically effective amount of at least one RNAi agent and / or RNAi agent conjugate as described herein and one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance other than an active pharmaceutical ingredient (API, therapeutic product, such as an RNAi agent or RNAi trigger), which has been properly evaluated for safety and is intentionally included in a drug delivery system. The excipient does not produce or tends to produce a therapeutic effect at the planned dose. The excipient may play the following roles: a) assist in the processing of the drug delivery system during the manufacturing process; b) protect, support or improve the stability, bioavailability or patient acceptability of the API; c) help product identification; and / or d) improve one or more of the overall safety and effectiveness of API delivery during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0381] Excipients include, but are not limited to, absorption enhancers, antiadherents, antifoaming agents, antioxidants, binders, buffers, carriers, coatings, pigments, delivery enhancers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, isotonic agents, vehicles, water repellents, and wetting agents.

[0382] The pharmaceutical composition may contain other components commonly found in pharmaceutical compositions. Such other components include, but are not limited to, antipruritic agents, astringents, local anesthetics, anti-inflammatory drugs, or antihistamines (e.g., diphenhydramine, doxylamine, acrivastine, or cetirizine). It is also contemplated that cells, tissues, or isolated organs expressing or containing RNAi triggers comprising 5'-cyclic phosphonate modified nucleotides as defined herein can be used as "pharmaceutical compositions." As used herein, "pharmacologically effective amount," "therapeutically effective amount," or simply "effective amount" refers to the amount of the RNAi agent that produces the desired pharmacological, therapeutic, or preventive result.

[0383] In some embodiments, the RNAi elicitor is combined with one or more other therapies or treatments, including but not limited to: a second RNAi elicitor or other RNAi agents, small molecule drugs, antibodies, antibody fragments, and / or vaccines.

[0384] The RNAi initiators comprising 5'-cyclic phosphonate modified nucleotides and pharmaceutical compositions comprising the RNAi initiators described herein can be packaged or included in a kit, container, package or dispenser. The RNAi initiators and pharmaceutical compositions comprising the RNAi initiators can be packaged in pre-filled syringes or vials.

[0385] Cells, tissues, and non-human organisms comprising at least one RNAi initiator comprising a 5'-cyclic phosphonate modified nucleotide as described herein are contemplated. The cells, tissues, or non-human organisms are produced by delivering the RNAi initiator to the cells, tissues, or non-human organisms using any means available in the art. In some embodiments, the cells are mammalian cells, including but not limited to human cells. The cells, tissues, or non-human organisms can be used for research or as research tools (e.g., drug trials or diagnostics).

[0386] In some embodiments, the RNAi agents described herein comprising 5'-cyclic phosphonate modified nucleotides can be used to treat a subject suffering from a disease or disorder that would benefit from reduction or inhibition of target gene expression. In some embodiments, the RNAi agents are used to treat or prevent at least one symptom in a subject suffering from a disease or disorder that would benefit from reduction or inhibition of target gene expression. A therapeutically effective amount of any one or more RNAi agents described herein is administered to the subject, thereby treating the symptom.

[0387] In some embodiments, RNAi agents comprising 5'-cyclic phosphonate modified nucleotides are used to treat or manage clinical manifestations, wherein a therapeutically or prophylactically effective amount of one or more RNAi agents described herein is administered to a subject in need of such treatment, prevention, or management. In some embodiments, the method comprises administering to a mammal (e.g., a human) to be treated a composition comprising an RNAi initiator molecule described herein.

[0388] In some embodiments, RNAi initiators comprising 5'-cyclic phosphonate modified nucleotides can be used to inhibit the expression of a target gene in a cell, cell mass, or tissue in a subject. In some embodiments, RNAi initiators can be used to formulate compositions for inhibiting the expression of a target gene in, for example, a cell, cell mass, or tissue in a subject. In some embodiments, a therapeutically effective amount of one (or more different types) of RNAi agents described herein is administered to a subject, thereby inhibiting the expression of a target gene in the subject (e.g., an amount effective to inhibit the expression of a target gene in the subject).

[0389] In some embodiments, the gene expression level and / or mRNA level of the target in a subject administered the RNAi initiator comprising a 5'-cyclic phosphonate modified nucleotide is decreased by at least about 5%, for example, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, relative to the subject before administration of the RNAi initiator or a subject that did not receive the RNAi initiator. The gene expression level and / or mRNA level in the subject can be decreased in cells, cell populations, and / or tissues of the subject. In some embodiments, the protein level in a subject administered with the RNAi initiator comprising a 5'-cyclic phosphonate modified nucleotide is decreased by at least about 5%, for example, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%, relative to the subject before administration of the RNAi initiator or to a subject that has not received the RNAi initiator. The protein level in the subject can be decreased in the subject's cells, cell populations, tissues, blood, and / or other fluids. The decrease in gene expression, mRNA, or protein levels can be assessed by any method known in the art. A decrease or reduction in mRNA levels and / or protein levels is collectively referred to herein as inhibition, reduction, or decrease in target gene or target mRNA expression.

[0390] The route of administration is the path by which the RNAi initiator comprising a 5'-cyclic phosphonate modified nucleotide comes into contact with the body. In general, methods for administering drugs and nucleotides for treating a subject are well known in the art and can be applied to the administration of the compositions described herein. The compounds described herein can be administered by any suitable route in the form of a formulation suitable for that particular route. Thus, the compounds described herein can be administered by injection, for example, intravenously, intramuscularly, intradermally, subcutaneously, or intraperitoneally.

[0391] In some embodiments, RNAi initiator molecules or compositions as described herein can be delivered to cells, cell clusters, tissues or objects using oligonucleotide delivery technologies known in the art. In general, any method known in the art suitable for delivering nucleic acid molecules (in vitro or in vivo) can be applied to RNAi initiators comprising 5'-cyclic phosphonate modified nucleotides as described herein. For example, delivery can be by topical administration (such as direct injection, implantation or external administration), systemic administration, or by subcutaneous, intravenous, oral, intraperitoneal or parenteral routes, including intracranial (such as intraventricular, soft tissue and intrathecal), intramuscular, transdermal, airway (such as aerosol), intranasal, rectal or external (including buccal and sublingual) administration. In some embodiments, the composition is administered by subcutaneous or intravenous infusion or injection.

[0392] In general, the effective amount of the active compound is in the range of about 0.1 to about 100 mg / kg body weight / day, for example, about 1.0 to about 50 mg / kg body weight / day. In some embodiments, the effective amount of the active compound is in the range of about 0.25 to about 5 mg / kg body weight per dose. In some embodiments, the effective amount of the active ingredient is in the range of about 0.5 to about 3 mg / kg body weight per dose. The dosage may also depend on variables such as the patient's overall health, the relative biological efficacy of the compound delivered, the dosage form of the drug, the presence and type of excipients in the formulation, and the route of administration. It should also be understood that the initial dosage may be increased beyond the above upper limit in order to quickly reach the desired blood level or tissue level, or the initial dosage may be less than the optimal value.

[0393] In terms of the treatment of diseases or the formulation of drugs or compositions for treating diseases, the pharmaceutical compositions described herein comprising expression inhibitory oligomeric compounds such as RNAi agents containing one or more 5'-cyclic phosphonate modified nucleotides can be combined with an excipient or a second therapeutic agent or treatment, which includes but is not limited to: a second or other expression inhibitory oligomeric compound, a small molecule drug, an antibody, an antibody fragment and / or a vaccine.

[0394] The RNAi agent comprising one or more 5'-cyclic phosphonate modified nucleotides can be packaged in a kit, container, package or dispenser when added to a pharmaceutically acceptable excipient or adjuvant. The pharmaceutical composition described herein can be packaged in a pre-filled syringe or vial.

[0395] The embodiments and items provided above are now illustrated by the following non-limiting examples.

[0396] Example

[0397] The following examples are not limiting and are not intended to illustrate certain embodiments described herein.

[0398] Some abbreviations used in the following experimental details of the synthesis of the examples are defined as follows: H or hr = hour; min = minute; mol = mole; mmol = millimole; M = molar concentration; μM = micromolar concentration; g = gram; μg = microgram; rt or RT = room temperature; L = liter; mL = milliliter; wt = weight; Et2O = diethyl ether; THF = tetrahydrofuran; DMSO = dimethyl sulfoxide; EtOAc = ethyl acetate; Et3N or TEA = triethylamine; i-Pr2Net or DIPEA or DIEA = diisopropylethylamine; CH2Cl2 or DCM = dichloromethane; CHCl3 = chloroform; CDCl3 = deuterated chloroform; CCl4 = carbon tetrachloride; MeOH = methanol; EtOH = ethanol; DMF = dimethylformamide; BOC = tert-butyloxycarbonyl; CBZ = benzyloxycarbonyl; T BS = tert-butyldimethylsilyl; TBSCl or TBDMSCl = tert-butyldimethylchlorosilane; TFA = trifluoroacetic acid; DMAP = 4-dimethylaminopyridine; NaN3 = sodium azide; Na2SO4 = sodium sulfate; NaHCO3 = sodium bicarbonate; NaOH = sodium hydroxide; MgSO4 = magnesium sulfate; K2CO3 = potassium carbonate; KOH = potassium hydroxide; NH4OH = ammonium hydroxide; NH4Cl = ammonium chloride; SiO2 = silica; Pd–C = palladium on carbon; HCl = hydrogen chloride or hydrochloric acid; NMM = N-methylmorpholine; H2 = hydrogen; KF = potassium fluoride; EDC-HCl = N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; MTBE = tert-butyl methyl ether; MeOH = methanol; Ar = argon; N2 = nitrogen; SiO2 = silica; R T =Retention time.

[0399] Additionally, examples of RNAi agents suitable for use with the 5'-cyclic phosphonate modified nucleotides described herein are listed in the various tables in the Examples below.

[0400] The following symbols are used to represent modified nucleotides in the sequences listed in the tables described herein. As one of ordinary skill in the art will readily understand, when present in an oligonucleotide, the monomers are interconnected by 5'-3'-phosphodiester bonds, unless otherwise indicated:

[0401] N = 2'-OH (unmodified) ribonucleotide (no capital letter f or d)

[0402] n = 2'-OMe modified nucleotide

[0403] Nf = 2'-fluorinated nucleotide

[0404] dN = 2'-deoxynucleotide

[0405] N UNA = 2',3'-open ring nucleotide mimics (non-locked nucleobase analogs)

[0406] N LNA = Locked nucleotide

[0407] Nf ANA = 2'-F-arabinonucleotide

[0408] NM = 2'-methoxyethyl nucleotide

[0409] X or Ab = abasic ribose

[0410] R = Ribitol

[0411] (invdN) = inverted deoxyribonucleotide (3'-3' linker nucleotide)

[0412] (invAb) = inverted abasic nucleotide

[0413] (invX) = inverted abasic nucleotide

[0414] (invn) = inverted 2'-OMe nucleotide

[0415] s = phosphorothioate-linked nucleotide

[0416] vpdN = vinylphosphonate deoxyribonucleotide

[0417] (3'OMen) = 3'-OMe nucleotide

[0418] (5Me-Nf) = 5'-Me, 2'-fluoronucleotide

[0419] cPrp = cyclopropyl phosphonate

[0420] The compounds of the present invention can be prepared using synthetic chemistry techniques known to those skilled in the art and as described above.

[0421] Example 1. Synthesis of Compound 4 ([2-[(2R,3R,4R,5R)-3-([[bis(propan-2-yl)amino](2-cyanoethoxy)phosphino]oxy)-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)oxolan-2-yl]cyclopropyl]phosphonic acid dimethyl ester)

[0422] A. Synthesis of compound 2 (dimethyl [2-[(2R,3R,4R,5R)-3-[(tert-butyldiphenylsilyl)oxy]-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)oxolan-2-yl]cyclopropyl]phosphonate).

[0423]

[0424] Compound 1 (dimethyl [(E)-2-[(2R,3R,4R,5R)-3-[(tert-butyldiphenylsilyl)oxy]-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)oxolan-2-yl]vinyl]phosphonate) was synthesized according to a procedure similar to that of Whittaker, B. et al., Tetrahedron Lett. 49, 6984-6987 (2008) and Abbas, S. et al., Org. Lett., 3(21), 3365-3367 (2001).

[0425] A solution of sodium hydride (8.2 g, 341.67 mmol, 3.00 equiv) in dimethyl sulfoxide (500 mL) was added to a 1000-mL 3-necked round-bottom flask purged and maintained with a nitrogen inert atmosphere. A solution of trimethylsulfoxonium iodide (45 g, 204.48 mmol, 3.00 equiv) was then added. The resulting solution was stirred at 25°C for 0.5 h. Subsequently, a solution of compound 1 (45 g, 68.31 mmol, 1.00 equiv) in dimethyl sulfoxide (50 mL) was added dropwise with stirring at 25°C. The resulting solution was stirred at 25°C for 20 h. The reaction was then quenched by the addition of 50 mL of saturated aqueous ammonium chloride.

[0426] The resulting solution was extracted with 3 x 1000 mL of dichloromethane, and the organic layers were combined. The combined organic layers were washed with 1 x 2000 mL of saturated aqueous sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was flash chromatographed on silica gel, eluting with dichloromethane / methanol (100:1-10:1). This yielded 4.4 g (10%) of compound 2 (dimethyl [2-[(2R,3R,4R,5R)-3-[(tert-butyldiphenylsilyl)oxy]-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)oxolan-2-yl]cyclopropyl]phosphonate) as a white solid. (H-NMR: (CDCl3, 400MHz, ppm): δ8.55 (s, 1H), 7.71 (t, J = 6.4Hz, 2H), 7.66 (d, J = 6.8Hz ,2H),7.38-7.64(m,6H),7.03(s,1H),5.86(d,3.6Hz,1H),4.11(t,J=5.2Hz,1H),3.7 0-3.77(m,6H),3.55-3.61(m,2H),3.34-3.47(m,4H),3.30(s,3H),1.87(s,3H),1.40 -1.50(m,1H),1.10(s,9H),0.98-1.05(m,1H),0.80-0.90(m,1H),0.61-0.71(m,1H)).

[0427] B. Synthesis of compound 3 (dimethyl [2-[(2R,3R,4R,5R)-3-hydroxy-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)oxolan-2-yl]cyclopropyl]phosphonate).

[0428]

[0429] A solution of compound 2 (dimethyl [2-[(2R,3R,4R,5R)-3-[(tert-butyldiphenylsilyl)oxy]-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)oxolan-2-yl]cyclopropyl]phosphonate) (6.0 g, 8.92 mmol, 1.00 equiv) in tetrahydrofuran (60 mL) and triethylamine (4.18 g, 41.31 mmol, 3.00 equiv) were added to a 100-mL 3-necked round-bottom flask purged and maintained with a nitrogen inert atmosphere. Triethylamine trihydrochloride (13.34 g, 82.86 mmol, 6.00 equiv) was then added dropwise with stirring at 0°C. The resulting solution was stirred at 25°C for 18 h. The resulting mixture was concentrated in vacuo and diluted with 60 ml of dichloromethane.

[0430] The resulting solution was washed with 100 mL of saturated aqueous sodium bicarbonate solution and 100 mL of saturated aqueous sodium chloride solution. The organic solution was dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was flash chromatographed on silica gel and eluted with dichloromethane / methanol (100:1-10:1). This yielded 3 g (77%) of compound 3 (dimethyl [2-[(2R,3R,4R,5R)-3-hydroxy-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)oxolan-2-yl]cyclopropyl]phosphonate) as a white solid. (LC-MS: (ES, m / z): [M+H] + =435.H-NMR: (CDCl3, 300MHz, ppm): δ9.53(s,1H),8.28(s,1H),7.17(s,1H),5.75(s,1H),3.95-4.10(m,3H),3.61-3 .85(m,7H),3.51-3.58(m,2H),3.41(s,4H),1.93(s,3H),1.67-1.78(m,1H),1.19-1.28(m,1H),1.03-1.05(m,2H)).

[0431] C. Synthesis of compound 4 (dimethyl [2-[(2R,3R,4R,5R)-3-([[bis(propan-2-yl)amino](2-cyanoethoxy)phosphino]oxy)-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)oxolan-2-yl]cyclopropyl]phosphonate).

[0432]

[0433] A solution of compound 3 (dimethyl [2-[(2R,3R,4R,5R)-3-hydroxy-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)oxolan-2-yl]cyclopropyl]phosphonate) (2.5 g, 5.76 mmol, 1.00 equiv) in dichloromethane (50 mL) and 4,5-dicyanoimidazole (810 mg, 6.86 mmol, 1.20 equiv) were added to a 50-mL 3-necked round-bottom flask purged and maintained with a nitrogen inert atmosphere. 3-(bis(diisopropylamino)phosphinoyl)propionitrile (2.25 g, 7.46 mmol, 1.30 equiv) was then added dropwise with stirring at 0°C. The resulting solution was stirred at 25°C for 3 h. The resulting solution was diluted with 25 mL of dichloromethane.

[0434] The resulting solution was washed with 2 x 50 mL of saturated aqueous sodium bicarbonate and 50 mL of saturated aqueous sodium chloride. The organic solution was dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was flash chromatographed on silica gel and eluted with dichloromethane / ethyl acetate (5:1-1:5) (containing 0.5% triethylamine). This yielded 2.1 g (57%) of compound 4 (dimethyl [2-[(2R,3R,4R,5R)-3-([[bis(propan-2-yl)amino](2-cyanoethoxy)phosphino]oxy)-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)oxolan-2-yl]cyclopropyl]phosphonate) as a white solid. (LC-MS: (ES, m / z): [M+H] + =635.H-NMR: (CD3COCD3, 400MHz, ppm): δ10.11(s,1H),7.55-7.58(m,1H),5.94-5.99(m,1H), 4.42-4.48(m,2H),3.77-3.86(m,3H),3.69-3.76(m,9H),3.51-3.56(m,3H),3.30(d,J=2.8Hz ,3H),2.81-2.86(m,2H),1.86(s,3H),1.60-1.80(m,1H),1.21-1.27(m,12H),1.04-1.09(m,3 H).P-NMR: (CD3COCD3,161MHz,ppm): δ149.67,149.51,149.21,31.59,31.55,31.41,31.29.)

[0435] The above-mentioned compound 4 is a phosphoramidite compound that can be used to add 5'-cyclopropylphosphonate-2'-MOE modified nucleotides to form the ends of double-stranded RNAi agents and / or single-stranded antisense oligonucleotides. In general, a similar synthesis process can be used to prepare phosphoramidites that can be used to add 5'-cyclic phosphonate modified nucleotides as described herein to form the ends of the double-stranded RNAi agents and / or single-stranded antisense oligonucleotides. For example, those of ordinary skill in the art will understand and appreciate that the compound 1 of Example 1 can be synthesized with different groups, such as 2'-F, 2'-H or 2'-O-methyl at the 2' position. Similarly, as a non-limiting example, those of ordinary skill in the art will appreciate that different heterocyclic base moieties can be used to replace thymine (e.g., uracil, cytosine, guanine, 5-methylcytosine, etc.), as shown in Example 1.

[0436] Example 2. Synthesis of Compound 15 (2-cyanoethyl((2R,3R,4R,5R)-2-(2-(diethoxyphosphoryl)cyclopropyl)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl)diisopropylphosphoramidite).

[0437] A. Synthesis of compound 6 (1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione).

[0438]

[0439] Compound 5 was purchased from commercial channels. DMT-Cl (276 g, 813 mol) was added to a pyridine (1.5 L) solution of compound 5 (200 g, 775 mmol). The reaction mixture was stirred at 25 ° C for 8 h under N2 atmosphere. TLC (petroleum ether / ethyl acetate = 1 / 1, Rf = 0.2) showed that the reaction was complete. The solvent was removed under reduced pressure. The residue was diluted with EtOAc (4 L), washed with water (1 L x 2) and brine (1 L), dried over Na2SO4, filtered and concentrated under reduced pressure. The final residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 5: 1 to ethyl acetate) to give a light yellow gum compound 6 (448 g, contained in EtOAc). ( 1 H NMR:400MHz CDCl3.9.01(br s,1H),8.05(d,J=8.0Hz,1H),7.36-7.42(m,2H),7.27-7.34(m,6H),7.22-7.27(m,1H),6.81-6.8 9(m,4H),5.98(d,J=1.3Hz,1H),5.28(dd,J=1.9,8.2Hz,1H),4.48(dt,J=5.3,8.5Hz,1H),4.00(br d,J=8.0Hz,1H),3.81(d,J=0.8Hz,7H),3.65(s,3H),3.50-3.61(m,2H),2.64(br d,J=9.0Hz,1H)).

[0440] B. Synthesis of compound 7 (1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione).

[0441]

[0442] To a solution of compound 6 (448 g, 799 mmol) in DCM (2.8 L) were added imidazole (163 g, 2.4 mol) and TBSCl (241 g, 1.6 mol). The reaction mixture was stirred at 25°C for 8 h. TLC (petroleum ether / ethyl acetate = 1 / 1, Rf = 0.8) indicated that the reaction was complete. The resulting mixture was diluted with water (4.5 L) and extracted with DCM (5 L x 3). The combined organic layers were washed with brine (5 L), dried over Na2SO4, filtered, and concentrated under reduced pressure to afford compound 7 (620 g, crude) as a colorless gel. The crude product was used in the next step without further purification.

[0443] C. Synthesis of compound 8 (1-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione).

[0444]

[0445] A mixture of compound 7 (620 g, 919 mmol) and AcOH (4 L) in H2O (1 L) was degassed and purged with N2 three times, and then the mixture was stirred at 25 ° C under N2 atmosphere for 16 h. TLC (petroleum ether / ethyl acetate = 1 / 1, Rf = 0.2) showed that the reaction was complete. The reaction mixture was quenched by adding MeOH (50 mL) and Et3SiH (25 mL). NaHCO3 was added to the resulting mixture until pH = 7-8, followed by extraction with EtOAc (5 L x 3). The combined organic layers were washed with brine (5 L), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 5: 1 to 1: 1) to give compound 8 (296 g, yield 87%) as a white solid. ( 1 H NMR:400MHz CDCl3.9.27(s,1H),7.72(d,J=8.0Hz,1H),5.74(dd,J=1.3,8.0Hz,1H),5.69(d,J=4.0Hz,1H),4.36(t,J=5.3Hz,1H),4.04-4.0 9(m,1H),3.93-4.03(m,2H),3.75-3.76(m,1H),3.49(s,3H),2.85(dd,J=3.5,6.5Hz,1H),0.92(s,9H),0.11(d,J=5.3Hz,6H)).

[0446] D. Synthesis of compound 9 ((2S,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-carbaldehyde).

[0447]

[0448] To a solution of compound 8 (60 g, 161 mmol) in DCM (3 L) was added Dess-Martin periodinane (95.7 g, 226 mmol) and NaHCO₃ (1.35 g, 16.1 mmol) at 0°C. The reaction mixture was stirred at 0°C for 1 h, then at 25°C for 3 h. TLC (petroleum ether / ethyl acetate = 1 / 1, Rf = 0.16) indicated the reaction was complete. The resulting mixture was quenched with 4 L of a saturated Na₂S₂O₃ and saturated NaHCO₃ (1:1) solution and extracted with EtOAc (5 L x 3). The combined organic layers were washed with brine (5 L), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to yield compound 9 (48 g, crude) as an orange oil. This crude product was used in the next step without further purification.

[0449] E. Synthesis of compound 10.

[0450]

[0451] LiHMDS (1M in THF, 723 mL) was added to a slurry of compound 10-1 (231 g, 647 mmol) in THF (2.4 L). The yellow solution was stirred at 25°C for 15 min and then cooled to 5°C. The solution was treated with compound 10-2 (110 g, 583 mmol) and allowed to warm to 25°C, where it was maintained. The solution was then cooled to 5°C and treated with LiHMDS (1M in THF, 723 mL) and allowed to warm to 2°C. The solution was quenched by the addition of H2O (50 mL) at 5°C and diluted with EtOAc (1.5 L). The organic layer was separated, dried over Na2SO4, and concentrated under reduced pressure to give compound 10-3 as an oil. The crude oil was dissolved in THF (2.4 L) and cooled to 5°C. The solution was then treated with NaH (57 g, 1.43 mol, 60% dispersion in mineral oil) and allowed to warm to 25°C. The slurry was stirred at 25°C for 18 h. TLC (petroleum ether / ethyl acetate = 1 / 1, Rf = 0.3) indicated the reaction was complete. The NaH was removed by passing through a pad of Celite, and the filter cake was rinsed with THF (600 mL x 2). The filtrate was concentrated to afford compound 10 (250 g, crude) as a yellow oil.

[0452] F. Synthesis of compound 11 (((E)-2-((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl)vinyl)phosphorothioate O,O-diethyl ester).

[0453]

[0454] To a solution of compound 9 (165 g, 445 mmol) in THF (1.6 L) was added dropwise a solution of compound 10 (250 g, 583 mmol) in THF (0.8 L) at 5 ° C. The reaction was then warmed to 25 ° C and stirred for 1 h. TLC (petroleum ether / ethyl acetate = 1 / 1, Rf = 0.6) showed that the reaction was complete. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10:1 to 1:1) to give a clear oily compound 11 (89 g, purity 85%; 72 g, purity 65%; 59 g, purity 48%, yield 65%) (the impurity was identified as triphenylphosphine oxide (Ph3P=O)). ( 1 H NMR:400MHzCDCl3.8.46(br s,1H),7.30(d,J=8.3Hz,1H),6.76-6.89(m,1H),6.18-6.30(m,1H),5.85(d,J=2.0Hz,1H),5.79(d,J=8.3Hz,1H),4.51-4.59(m,1H),4.07- 4.18(m,4H),3.98(dd,J=5.0,7.5Hz,1H),3.73(dd,J=2.4,5.1Hz,1H),3.54(s,3H),1.33(dt,J=2.3,7.0Hz,6H),0.92(s,9H),0.11(s,6H)).

[0455] G. Synthesis of compound 12 ((2-((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl)cyclopropyl)phosphorothioate O,O-diethyl ester).

[0456]

[0457] To a solution of MeSOI (95.9 g, 436 mmol) in DMSO (800 mL) was added a THF slurry of sodium hydride (17.4 g, 436 mmol, 60% dispersion in mineral oil). The resulting white, frothy slurry was stirred at 25°C for 15 minutes, and the resulting white solution was added to a flask containing compound 11 (89 g, 85% purity, 145 mmol). The temperature was raised from 22°C to 26°C, and the yellow solution was stirred for 2 hours, then heated to 50°C for 2 hours. LCMS indicated the reaction was complete. The reaction mixture was cooled to 5°C and quenched with ice, maintaining the internal temperature at 30°C. The product was extracted with ethyl acetate (3 L), and the organic layer was washed with water (5 x 1 L). The organic layer was dried over NaSO, filtered, and concentrated under reduced pressure to afford compound 12 (206 g, crude) as a white solid (impurity: PhP=O; the crude product was used in the next step without further purification).

[0458] H. Synthesis of compound 13 ((2-((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)cyclopropyl)phosphorothioate O,O-diethyl ester).

[0459]

[0460] 12M HCl (340 mL, 4.1 mol) was added dropwise to a stirred solution of compound 12 (206 g, 385 mmol) in THF (1.3 L). The resulting mixture was stirred at 25 ° C for 1.5 h. TLC (DCM / ethyl acetate = 1 / 1, Rf = 0.2) showed that the reaction was complete. The reaction mixture was cooled with an ice bath and quenched with a saturated solution of sodium bicarbonate until pH = 8. The product was extracted with EtOAc (3.9 L x 2), and the combined organic layers were washed with brine (1.3 L). The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, DCM / ethyl acetate = DCM to 1:1) to give compound 13 (76 g, yield 47%) as a white solid. ( 1 H NMR:400MHz CDCl3.8.37(br.s.,1H),7.41(d,J=8.0Hz,1H),5.75-5.86(m,2H),4.00-4.18(m,5H),3.79-3.85(m,1H),3.56 -3.64(m,3H),3.41-3.53(m,1H),2.65-2.74(m,1H),1.49-1.62(m,1H),1.24-1.34(m,8H),0.97-1.09(m,1H)).

[0461] I. Synthesis of Compound 14 (diethyl (2-((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)cyclopropyl)phosphonate).

[0462]

[0463] Add the following to a solution of compound 13 (76 g, 181 mmol) in 1.4 L THF:H2O (1:1) cooled to 5°C: (194g, 316mmol). The reaction mixture was stirred at 5°C for 2h. LCMS showed that the reaction was complete. The reaction mixture was diluted with 200mL H2O and extracted with 2.5L DCM. The organic layer was collected and the aqueous layer was extracted again with DCM (2.5L x 4). The organic layers were combined, then dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, DCM / MeOH=DCM to 10:1) to give compound 14 (Cy3P-U-OMe-3-OH) (40g, crude) as a white solid. Finally, the crude product was purified by preparative HPLC (Gemini150*4.6mm (Luna200*25mm (C18, 10um, )+Gemini150*30(c18,5um, ), 0.1% TFA / CH3CN / H2O, 20 mL / Min) to obtain compound 14 (20.02 g, purity 99%, yield 27%) as a clear solid. ( 1 H NMR:400MHz CDC13.9.54(br.s.,1H),7.40-7.48(m,1H),5.75-5.89(m,2H),3.98-4.23(m,5H),3.84(dd,J=2.5,5.3Hz,1H),3.56-3 .64(m,3H),3.40(td,J=7.9,16.5Hz,1H),1.58-1.70(m,1H),1.30-1.40(m,6H),1.20-1.29(m,1H),0.92-1.09(m,2H)).

[0464] J. Synthesis of compound 15 (2-cyanoethyl((2R,3R,4R,5R)-2-(2-(diethoxyphosphoryl)cyclopropyl)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl)diisopropylphosphoramidite).

[0465]

[0466] To a solution of compound 14 (463 mg, 1.1 mmol) in DCM (6 mL) was added 4,5-dicyanoimidazole (54 mg, 0.46 mmol), followed by a solution of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphoramidite (518 mg, 1.7 mmol, 1.5 eq) in dichloromethane (3 mL). The reaction mixture was stirred overnight. After confirming by HPLC that all the starting materials had been consumed, the reaction mixture was concentrated under reduced pressure to a volume of ~2 mL. The crude solution was applied to a silica gel column and purified using an isocratic gradient (DCM: EtOAc: triethylamine: methanol (6:4:0.1:0.05), Rf = 0.25). Yield as a mixture of cyclopropyl and phosphoramidite diastereomers (4 in total): 421 mg (61%). ( 1 H NMR:400MHz DMSO-d6.11.41(s,1H),7.82–7.72(m,1H),5.73–5.64(m,1H),5.82–5.76(m,1H),4.49–4.18(m,2H),4.11–3.91(m, 4H),3.88–3.50(m,4H),3.42–3.32(m,4H),2.79(m,2H),1.73–1.53(m,1H),1.30–1.11(m,18H),1.10–0.79(m,3H)).

[0467] The above-mentioned compound 15 is a phosphoramidite compound that can be used to add 5'-cyclopropylphosphonate-2'-O-Me modified nucleotides to form the ends of double-stranded RNAi agents and / or single-stranded antisense oligonucleotides. In general, a similar synthesis process can be used to prepare phosphoramidites that can be used to add 5'-cyclic phosphonate modified nucleotides as described herein to form the ends of the double-stranded RNAi agents and / or single-stranded antisense oligonucleotides. For example, those of ordinary skill in the art will understand and appreciate that the compound 5 of Example 2 can be synthesized with different groups, such as 2'-F or 2' deoxy groups at the 2' position. Similarly, as a non-limiting example, those of ordinary skill in the art will appreciate that different heterocyclic base moieties can be used to replace uracil (e.g., thymine, cytosine, guanine, 5-methylcytosine, etc.), as shown in Example 2.

[0468] Example 3. Synthesis of RNAi Agents.

[0469] The synthesis of RNAi agents begins with the separate synthesis of sense and antisense strands. The two complementary strands are first synthesized on a solid support resin, followed by cleavage and deprotection, followed by purification using reverse phase or ion exchange chromatography, and finally annealing to form the RNAi agent.

[0470] A) Solid Phase Synthesis: RNAi agents are synthesized using the solid phase phosphoramidite technique employed in oligonucleotide synthesis. Depending on the scale, commercially available automated oligonucleotide synthesizers, such as (Bioautomation), (BioAutomation) or AKTA Oligopilot (GE). Solid-phase synthesis starts from the 3' end of the sequence and phosphoramidite building blocks are added sequentially in the desired order in each synthesis cycle to grow the oligomer. Each synthesis cycle consists of four chemical steps: 1) unblocking or detritylation; 2) coupling; 3) oxidation; 4) end-capping. Phosphoramidites are derived from naturally occurring or chemically modified nucleotides, or small molecules such as N-acetylgalactosamine targeting ligands. In controlled pore glass (CPG, or The synthesis was performed on a solid support made from Prime Synthesis (Aston, PA, USA). All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the following 2'-O-methyl phosphoramidites were used: (5'-O-dimethoxytrityl-N 6 -(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, 5'-O-dimethoxytrityl-N 4 -(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, (5'-O-dimethoxytrityl-N 2 -(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and 5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite. 2'-Deoxy-2'-fluorophosphoramidites carry the same protecting groups as 2'-O-methyl RNA amidites. Targeting ligands 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 were added. 5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used as the activator solution. Coupling times were 10 min (RNA), 15 min (targeting ligand), 90 sec (2'OMe), and 60 sec (2'F). To introduce the phosphorothioate linkage, a 100 mM solution of 3-phenyl-1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Leominster, MA, USA) in anhydrous acetonitrile was used.

[0471] B) Hydrolysis of 5'-cyclopropylphosphonate modified nucleotides.

[0472] The hydrolysis of the 5'-cyclopropylphosphonate oligonucleotide bound to the support was performed in an AKTA Oligopilot (GE) synthesizer. A mixture of trimethylsilyl iodide / pyridine / acetonitrile (1:13:40 v / v) was passed through the reaction column at a flow rate of 2 mL / min for 30 to 90 minutes, depending on the scale of the reaction. The column was then washed with acetonitrile and removed from the synthesizer, and the oligonucleotide bound to the support was transferred to a funnel and washed with acetonitrile / water (1:1 v / v).

[0473] C) Cleavage and Deprotection of Support-Bound Oligonucleotides. After completion of the solid-phase synthesis and deprotection of the support-bound 5'-cyclopropylphosphonate oligonucleotides, the dried solid support was treated with a 1:1 volume solution of 40 wt% aqueous methylamine and 28 wt% aqueous ammonium hydroxide (Aldrich) at 30°C for two hours. The solution was evaporated, and the solid residue was redissolved in water (see below).

[0474] D) Purification. Purify the crude oligonucleotide by reversed-phase HPLC or ion exchange chromatography. Collect the components containing the product and confirm the purity and homogeneity of each single strand by LCMS. The concentration and productive rate of the oligonucleotide are evaluated by theoretical extinction coefficient by UV (260nm).

[0475] E) Annealing. The complementary strands were mixed by combining equimolar solutions of the sense and antisense strands. These solutions were kept at room temperature or placed in a 70°C thermomixer, heated to 95°C, held at 95°C for 5 minutes, and slowly cooled to room temperature. Some RNAi agents were lyophilized and stored at -15 to -25°C. The duplex concentration was determined by measuring the absorbance of the solution at 260 nm in a UV-Vis spectrometer. Unless otherwise stated, all conversion factors are 0.037 mg / (mL·cm). In some experiments, conversion factors were calculated from experimentally determined extinction coefficients.

[0476] Example 4. Exemplary RNAi agent sequences targeting LP(a) (human Apo(a) gene) comprising 5'-cyclic phosphonate modified nucleotides.

[0477] The following sequences listed in Table 1 below are exemplary only. The 5'-cyclic phosphonate modified nucleotides described herein can be conjugated to any RNAi agent or single-stranded antisense oligonucleotide targeting any gene.

[0478] Table 1. Modified RNAi agent sequences targeting LP(a) (human Apo(a) gene)

[0479]

[0480] The RNAi agents in Table 1 above were prepared according to the synthesis described in Example 3. As used in the tables herein, in the nucleotide sequences, 2'-O-methyl nucleotides are represented by lowercase "n"; 2'-deoxy-2'-fluoro nucleotides are represented by Nf; 2'-deoxy nucleotides are represented herein by dN; 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides are represented herein by NM; 3'-3' linked (inverted) nucleotides are represented herein by invdN, invN, invn, invX or invAb; 5'-cyclopropylphosphonic acid is represented herein by cPrp before the nucleotide, such as cPrpN, cPrpn, cPrpNf, cPrpdN or cPrpNM; abasic nucleotides (represented herein by Ab); n-acetylgalactosamine cluster targeting ligands are represented herein by NAG; 5'-phosphorothioate groups are represented herein by lowercase "s" before the nucleotide, such as sN, sn, sNf or sdN).

[0481] Example 5. In vivo analysis of RNAi agents targeting LP(a) (human Apo(a) gene) in transgenic mice.

[0482] A) To evaluate the efficacy of LP(a) RNAi agents in vivo, apo(a) transgenic mice were used (Frazer KA et al., Nature Genetics 9:424-431 (1995)). These mice express human apo(a) from a YAC containing the full-length LPA gene (encoding apo(a) protein) and additional sequences at the 5' and 3' ends. On day 1, mice were administered an RNAi agent conjugated to an N-acetylgalactosamine targeting ligand attached to the 5' end of the sense strand. Each mouse received a single subcutaneous (SC) injection of saline (n=4) or various treatment groups (n=3 for all treatment groups). Control serum (pre-treatment) samples were obtained from mice before injection on day -1. Post-injection serum samples were obtained from mice on days 8, 15, 22, 29, and 36.

[0483] B) Apo(a) protein levels. Human apo(a) protein levels were monitored in serum by testing serum from mice using an ELISA for apo(a) (Abcam). For normalization, the apo(a) levels of each animal at a time point were grouped according to the pre-treatment expression level in that animal (in this case, Day -1) to determine a "normalized to Day -1" expression level ratio. Next, the expression at a particular time point was normalized to the saline control group by dividing the "normalized to Day -1" ratio for one animal by the average "normalized to Day -1" ratio for all mice in the saline control group. This allowed the expression at each time point to be normalized to the expression in the control group, as shown in Table 2.

[0484] Table 2. Apo(a) knockdown (KD) in transgenic mice by a single 0.5 mg / kg dose of RNAi agents administered on day 1.

[0485]

[0486] As shown in Table 2 above, the lowest value for AD03541 was reached on day 8, while the lowest value for AD03158 was not reached until day 22. In addition, AD03541 achieved a greater degree of knockdown at all time points.

[0487] Example 6. Factor 12 knockdown (KD) in cynomolgus monkeys.

[0488] RNAi agents having sequences against factor 12 (F12) linked to an N-acetylgalactosamine targeting ligand at the 5' end of the sense strand were synthesized and combined with a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection.

[0489] On day 1, cynomolgus monkeys (Macaca fascicularis) were injected subcutaneously with 3 mg / kg of the F12 RNAi agent AD04254 or AD04443. Two (2) monkeys were dosed with treatment group AD04443, and three (3) monkeys were dosed with treatment group AD04254.

[0490] Table 3. F12 RNAi agents of Example 6.

[0491]

[0492] As shown in Table 3 above, AD04254 and AD04443 are composed of the same sense strand, and the difference between the RNAi agents is that the 5' end of the antisense strand contains a 2'-O-methyl u-modified nucleotide (AD04254) or a 5'-cyclopropylphosphonate modified nucleotide (which also contains a 2'-O-methyl u modification) (AD04443).

[0493] Serum samples were obtained from treated cynomolgus monkeys on days -29, -7, and 1 (pre-dose), as well as on days 8, 15, 22, and 29 to monitor knockdown. Knockdown was determined by quantifying the levels of monkey F12 protein (cF12) circulating in serum using a human F12 ELISA kit (Molecular Innovations). The cF12 levels of each animal at each time point were grouped according to the pre-treatment expression level in that animal (average of days -29, -7, and 1) to determine the "normalized to pre-dose" expression level ratio.

[0494] The average normalized relative expression of cF12 is shown in Figure 1 .like Figure 1 As shown, based on a single dose administration, the F12 RNAi agent comprising a 5'-cyclopropylphosphonate modified nucleotide of the present invention at the 5' end of the antisense strand of the sequence provided numerically increased efficacy and slightly faster knockdown compared to the same F12 RNAi agent without the 5'-cyclopropylphosphonate modified nucleotide.

[0495] Example 7. Factor 12 knockdown (KD) in wild-type mice.

[0496] F12 double-stranded RNAi agents were prepared, with N-acetylgalactosamine targeting ligands coupled to the 5' end of the sense strand. Each double-stranded RNAi agent was directed against F12 and combined with a pharmaceutically acceptable buffer for subcutaneous (SC) injection known in the art.

[0497] The F12 RNAi agents (AD04162 and AD04649) were delivered to wild-type mice by SC injection. On day 1, 200 μl of a solution containing saline, 0.5 mg / kg (mpk) of one RNAi agent in buffered saline, or 1.0 mg / kg (mpk) of one RNAi agent in buffered saline was injected SC into the loose skin of the back between the shoulders. There were four (4) wild-type mice in each of the five treatment groups.

[0498] Table 4. F12 RNAi agent of Example 7.

[0499]

[0500] As shown in Table 4 above, AD04162 and AD04649 are composed of the same sense strand, and the difference between the RNAi agents is that the 5' end of the antisense strand contains a 2'-O-methyluracil modified nucleotide (AD04162) or a 5'-cyclopropylphosphonate uracil modified nucleotide (which contains a 2'-deoxy modification) (AD04649).

[0501] Serum samples were obtained from treated mice on days -1 (pre-dose), 8, 15, 22, and 29 to monitor knockdown. Knockdown was determined by quantifying the levels of mouse F12 protein (mF12) circulating in serum using a Perkin Elmer assay. The mF12 levels of each animal at each time point were grouped according to the pre-treatment expression level in that animal to determine a "normalized to pre-dose" expression level ratio. Expression at a particular time point was then normalized to the saline control group by dividing the "normalized to pre-dose" ratio for one animal by the average "normalized to pre-dose" ratio for all mice in the saline control group. This allowed expression at each time point to be normalized to that in the control group. Experimental error was expressed as standard deviation, as shown in Table 5:

[0502] Table 5. Percent knockdown (KD) of mF12 in wild-type mice by a single dose of RNAi agents administered on day 1.

[0503]

[0504] As shown in Table 5 above, these data support that the addition of the 5'-cyclic phosphonate modified nucleotides of the present invention can provide higher potency for these F12 RNAi agents in wild-type mice, especially at a dose of 1.0 mg / kg.

[0505] Example 8. HBsAg reduction in pHBV model mice.

[0506] pHBV model mice were used to evaluate HBV surface antigen (HBsAg) reduction. Six- to eight-week-old female NOD.CB17-Prkdscid / NcrCrl (NOD-SCID) mice were transiently transfected with MC-HBV1.3 via tail vein injection 30 to 45 days prior to administration of an HBV RNAi agent or control (Yang PL et al., "Hydrodynamic injection of viral DNA: a common model of acute hepatitis B virus infection." PNAS USA 2002, Vol. 99: pp. 13825-13830). MC-HBV1.3 is a plasmid-derived minicircle containing the same terminal redundant human hepatitis B virus sequence, HBV1.3 (GenBank accession number #V01460), as found in HBV1.3.32 transgenic mice (Guidotti LG et al., "High-level hepatitis B virus replication in transgenic mice." J Virol 1995, Vol. 69, pp. 6158-6169). 5 μg of MC-HBV1.3 dissolved in Ringer's solution was injected into mice via the tail vein at a total dose of 10% of the animal's body weight to establish the pHBV model of chronic HBV infection. The solution was injected over 5-7 seconds using a 27-gauge needle as described above (Zhang G et al., "High levels of foreign gene expression in hepatocytes after tailvein injection of naked plasmid DNA." Human Gene Therapy 1999, Vol. 10, pp. 1735-1737). On day -1, the hepatitis B surface antigen (HBsAg) expression level in the serum was determined by ELISA, and the mice were divided into groups according to the average HBsAg expression level.

[0507] i) Serum Collection: Mice were anesthetized with 2-3% isoflurane, and blood samples were collected from the submandibular area into serum separator tubes (Sarstedt AG & Co., Neubrecht, Germany). The blood was allowed to clot at room temperature for 20 minutes. The tubes were centrifuged at 8,000 × g for 3 minutes to separate the serum, which was then stored at 4°C.

[0508] ii) Serum Hepatitis B Surface Antigen (HBsAg) Levels: Serum was collected and diluted 10-2000-fold with PBS containing 5% skim milk powder. A secondary HBsAg standard diluted in skim milk powder was prepared from serum from ICR mice (Harlan Sprague Dawley) transfected with 10 μg of the HBsAg expression plasmid pRc / CMV-HBs (Aldevron, Fargo, North Dakota). HBsAg levels were determined using the GS HBsAg EIA 3.0 kit (Bio-Rad Laboratories, Inc., Redmond, Washington) according to the manufacturer's instructions. Recombinant HBsAg protein, ayw subtype, was used as the primary standard (Aldevron), also diluted in skim milk powder in PBS.

[0509] To account for decreased expression of MC-HBV1.3 due to non-treatment reasons, HBsAg expression in each animal was normalized to that of a saline-injected control group of mice. First, to determine the "normalized to pre-treatment" expression level ratio, the HBsAg levels of each animal at a time point were grouped according to the pre-treatment expression level in that animal (day -1). Then, the "normalized to pre-treatment" ratio of an animal was divided by the average "normalized to pre-treatment" ratio of all mice in the saline control group, thereby normalizing the expression at a specific time point to that of the control group.

[0510] On day 1, each mouse received a single subcutaneous administration of 200 μl of 2 mg / kg (mpk) of a double-stranded HBV RNAi agent linked to an N-acetylgalactosamine targeting ligand at the 5' end of the sense strand, or 200 μl of phosphate-buffered saline (PBS) containing no HBV RNAi agent as a control. The administered HBV RNAi agents included AD04580 (containing a 5'-cyclopropylphosphonate uracil-modified nucleotide with a 2'-O-methyl modification at the 5' end of the antisense strand) and AD04178 (containing a 2'-O-methyl uracil-modified nucleotide at the 5' end of the antisense strand), formulated in phosphate-buffered saline. RNAi agents AD04580 and AD04178 are identical; however, the only difference between the two RNAi agents is that AD04580 contains a 5'-cyclopropylphosphonate moiety at the 5' end of the antisense strand, while AD04178 has a phosphate group at the 5' end of the antisense strand like a standard nucleotide.

[0511] Injections were administered subcutaneously into the loose skin over the entire décolletage area between the skin and muscle. Three (3) mice were tested per group (n=3). Serum was collected on days 8, 15, 22, and 29, and serum hepatitis B surface antigen (HBsAg) levels were determined.

[0512] The data from the experiments are shown in Figure 2 The average HBsAg value is used to represent the standardized average value of HBsAg. Figure 2 As shown, in pHBV model mice, an RNAi agent containing 5'-cyclopropylphosphonate modified nucleotides (AD04580) significantly outperformed an unmodified RNAi agent (AD04178).

[0513] Other implementations

[0514] It should be understood that although the invention has been described in conjunction with the detailed description, the above description is intended to illustrate rather than limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages and improvements are also within the scope of the following claims. Sequence Listing <110> Arrowhead Pharmaceuticals, Inc. Zhen Li Tao Pei Michael Lawler <120> 5'-Cyclic phosphonate-modified nucleotides <130> 30644-WO1 <150> US 62 / 346,304 <151> 2016-06-06 <160> 12 <210> 1 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent sense strand <400> 1 gccccuuauu guuauacgau u 21 <210> 2 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent antisense strand <400> 2 ucguauaaca auaaggggcu u 21 <210> 3 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent sense strand <400> 3 gccccuuauu guuauacgau u 21 <210> 4 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Antisense strand of RNAi agent containing 5'-cyclopropylphosphonate modified nucleotides <220> <221> Modified bases <222> 1 <223> / modified base = "2'-MOE-5'-cyclopropylphosphonate nucleoside" <400> 4 tcguauaaca auaaggggcu u 21 <210> 5 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent sense strand <400> 5 acucaauaaa gugcuuugaa a 21 <210> 6 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent antisense strand <400> 6 uuucaaagca cuuuauugag u 21 <210> 7 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent sense strand <400> 7 acucaauaaa gugcuuugaa a 21 <210> 8 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Antisense strand of RNAi agent containing 5'-cyclopropylphosphonate modified nucleotides <220> <221> Modified bases <222> 1 <223> / modified base = "2'-O-methyl-5'-cyclopropylphosphonate nucleoside" <400> 8 uuucaaagca cuuuauugag u 21 <210> 9 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent sense strand <400> 9 aacucaauaa agugcuuuga a 21 <210> 10 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent antisense strand <400> 10 uucaaagcac uuuauugagu u 21 <210> 11 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> RNAi agent sense strand <400> 11 aacucaauaa agugcuuuga a 21 <210> 12 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Antisense strand of RNAi agent containing 5'-cyclopropylphosphonate modified nucleotides <220> <221> Modified bases <222> 1 <223> / modified base = "2'-deoxy-5'-cyclopropylphosphonate nucleoside" <400> 12 uucaaagcac uuuauugagu u 21

Claims

1. A compound of formula Ib: in, D is O; X is a 1-pyrimidine base; Y 1 、Y 2 、Y 3 and Y 4 Each is H; Z is H, -OH, OCH3, or -O-(CH2)2-OCH3; G 1 , G 2 , G 3 and G 4 Each is H; J is O; K and L are each OR 16 , where R 16 Each independently selected from H or C1-C3 alkyl; Q is O; and A is (i) an internucleoside linkage linking the 5'-cyclic phosphonate modified nucleotide of Formula Ib to the remainder of the RNAi agent, or (ii) a phosphoramidite group.

2. The compound according to claim 1, wherein The RNAi agent is double-stranded.

3. The compound according to claim 1, wherein X is 1-uracil or 1-thymine.

4. The compound according to claim 1, wherein Z is -O-(CH2)2-OCH3 or OCH3.

5. The compound according to claim 1, wherein K is OH and L is OH.

6. A compound of formula III-b: in: X is a 1-pyrimidine base; Z is H, -OH, OCH3, or -O-(CH2)2-OCH3; J and J' are each O; and Contains substituents derived from RNAi agents.

7. The compound according to claim 6, characterized in that X is 1-uracil or 1-thymine.

8. The compound according to claim 6, wherein Z is -O-(CH2)2-OCH3 or OCH3.

9. A compound having a formula selected from the group consisting of: in: X" is (i) an internucleoside linkage that links the compound to the remainder of the RNAi agent; J is O; and Z is H, -OH, OCH3, or -O-(CH2)2-OCH3.

10. The compound according to claim 9, wherein The compound has the following formula:

11. The compound according to claim 9, wherein The compound has the following formula:

12. The compound according to claim 9, wherein The compound has the following formula:

13. The compound according to claim 9, wherein Z is -O-(CH2)2-OCH3 or OCH3. 14 . A pharmaceutical composition comprising the compound according to claim 1 and a pharmaceutically acceptable excipient.

15. The pharmaceutical composition according to claim 14, wherein The pharmaceutically acceptable excipient is saline.

16. The pharmaceutical composition according to claim 14, wherein The pharmaceutically acceptable excipient is water.

17. Use of the compound according to any one of claims 1 to 13 for preparing a preparation for inhibiting the expression of a target nucleic acid in a subject.

18. Use of a compound according to any one of claims 1 to 13 in the preparation of a formulation for treating a disease or disorder in a subject.

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