5'-Cyclophosphonic acid modified nucleotides

Abstract

To provide 5'-cyclo-phosphonate modified nucleotides useful for incorporation into oligomeric compounds, such as RNAi agents.SOLUTION: Described are 5'-cyclo-phosphonate modified nucleotides and oligonucleotides such as oligonucleotide interference (RNAi) agents, containing 5'-cyclo-phosphonate modified nucleotides. The RNAi agents having either double-stranded or single-stranded oligonucleotides described herein comprising 5'cyclo-phosphonate modified nucleotides are useful in modulating gene expression and in therapeutic, diagnostic, target validation, and genomic discovery applications. The RNAi agents and single-stranded antisense oligonucleotides comprising 5'-cyclo-phosphonate modified nucleotides are useful in treatment of diseases or conditions that respond to inhibition of gene expression or activity in a cell, tissue or organism.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications

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

[0003] This specification discloses 5'-cyclophosphonic acid-modified nucleotides that are useful for incorporation into oligomeric compounds such as RNAi agents. [Background technology]

[0004] Oligomer compounds contain nucleotide sequences that are at least partially complementary to the target nucleic acid and have been shown to alter the function and activity of the target both in vitro and in vivo. When delivered to cells containing the target nucleic acid (such as messenger RNA (mRNA)), oligomer compounds have been shown to modulate the expression of the target and alter the transcription or translation of the target nucleic acid. In certain examples, oligomer compounds can reduce gene expression by repressing the nucleic acid target and / or causing degradation of the target nucleic acid.

[0005] When the target nucleic acid is mRNA, one mechanism by which expression repression oligomer compounds can regulate the expression of mRNA targets is through RNA interference (RNAi). RNAi is a biological process in which RNA or RNA-like molecules (such as chemically modified RNA molecules) can stop gene expression through degradation. Post-transcriptional gene silencing is considered an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes.

[0006] RNAi response mechanisms typically involve an endonuclease complex, commonly known as the RNA-induced silencing complex (RISC), which is thought to incorporate a single strand of RNA or RNA-like molecule complementary to its target into its structure. RISC is understood to mediate the cleavage of single-stranded RNA (i.e., mRNA) through the complementarity of the single-stranded RNA or RNA-like molecule incorporated into RISC via 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) describe RNAi induced by introducing double-stranded synthetic 21-nucleotide RNA molecules into cultured mammalian cells. The types of synthetic RNA or RNA-like molecules that can interact with RISC and induce RNAi response mechanisms can consist of modified nucleotides and / or one or more non-phosphorylated diester bonds.

[0008] Furthermore, single-stranded RNA and RNA-like molecules may contain modified nucleotides and have one or more non-phosphorylated diester bonds, which can alter the expression of target mRNA.

[0009] We have shown that certain known modified nucleotides, when incorporated into oligomeric compounds and administered in vivo, enhance the duration and / or activity of the expression-suppressing oligomeric compounds. [Overview of the project]

[0010] Novel modified nucleotides are needed that can provide improved or enhanced stability and / or capabilities to oligomeric compounds such as RNAi agents. For example, novel modified nucleotides are needed that can improve the stability of the phosphate group at the 5' end of an RNAi agent.

[0011] The present specification describes 5'-cyclo-phosphonic acid modified nucleotides, and oligomeric compounds such as RNAi containing the 5'-cyclo-phosphonic acid modified nucleotides. The described 5'-cyclo-phosphonic acid modified nucleotides can be incorporated into double-stranded oligonucleotides (such as short interfering RNA) or single-stranded oligonucleotides (such as antisense oligonucleotides). Oligomeric compounds such as RNAi agents comprising one or more 5'-cyclo-phosphonic acid modified nucleotides can also have a targeting ligand such as an N-acetyl-galactosamine cluster or a peptide that binds to the RNAi agent. Oligomeric compounds such as RNAi agents comprising one or more 5'-cyclo-phosphonic acid modified nucleotides can also have a pharmacokinetic modulator such as a polyethylene glycol (PEG) moiety or a lipid that binds to the RNAi agent.

[0012] The 5'-cyclo-phosphonic acid modified nucleotides described in the present specification can improve the stability and / or potency of RNAi agents. In some embodiments, the 5'-cyclo-phosphonic acid modified nucleotides disclosed in the present specification can increase the stability and resistance to endonucleases and exonucleases that are capable of cleaving phosphodiester bonds of oligonucleotide strands in vivo. Furthermore, while not wishing to be bound by theory, the terminal 5'-phosphorylation state of an RNAi agent is believed to be a factor for the incorporation of the strand into RISC. Therefore, the 5'-cyclo-phosphonic acid modified nucleotide located at the 5'-end of the antisense strand can phosphorylate the 5'-end of the oligonucleotide and increase the possibility of maintaining the phosphorylation. This can increase the possibility of loading a specific strand into RISC, thereby allowing the RNAi agent to enter the RNAi pathway, resulting in improved and enhanced knockdown and gene silencing activities.

[0013] The 5'-cyclo-phosphonic acid modified nucleotides disclosed in the present specification have a cyclic group or cyclic moiety located at the 5' carbon of the sugar of the nucleotide (or an equivalent position in a sugar surrogate substituent). The 5'-cyclo-phosphonic acid modified nucleotides disclosed in the present specification form a phosphonic acid group (or a phosphonic acid mimetic group, for example by incorporating 5'-C-malonyl as described in the present specification) at the 5' end of an oligonucleotide.

[0014] In some embodiments, the disclosed 5'-cyclo-phosphonic acid modified nucleotides have a structure represented by Formula A,

[0015]

化

[0016] Cyclo 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, oxazole, isoxazole, benzofuran, indole, indazole, benzimidazole, oxadiazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, quinolinyl, isoquinolinyl, or quinoxalinyl), or heterocyclyl (e.g., tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, dioxane, or dioxolane),

[0017] X' is

[0018]

化

[0019] or comprises a sugar replacement substituent, X'' is (i) an internucleoside linkage that binds the 5'-cyclo-phosphonic acid modified nucleotide to the rest of the RNAi agent, or (ii) a phosphoramidite group,

[0020] D is O, S, CH2-CH 2、 CH=CH, OCH 2、 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 ),

[0021] 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,

[0022] R 2 , R 3 and R 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,

[0023] D is O, S, CH2-CH 2、 CH=CH, OCH 2、 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 When ), X is a heterocyclic base part, D is OC(H)(X 3 ) or OC(R 2 )(X 3 When ), 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 This is the heterocyclic base part,

[0024] Z is H, -OH, F, OCH 3、 -O-(CH2)2-OCH3; halogen; -OCH2F, -OCHF2, -OCF 3、 -OCH2CH3, -O(CH2)2F, -OCH2CHF2, -OCH2CF3, -OCH2-CH=CH2, -O(CH2)2-OCH3, -O(CH2)2-SCH3, -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)], a substituted C1-C6 alkyl, a substituted C1-C6 alkoxy, a substituted C2-C6 alkenyl, or a substituted C2-C6 alkynyl, R 5 , R 6 , R 7 and R 8 Each of these is independently H or C1-C6 alkyl,

[0025] Y 1 , Y 2 , Y 3 , and Y 4 Each of these 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, or Y 4 is Y 1 Or Y 2 It is bonded to one of the following, and the bond is O, S, NR 9 , C(R 10 )(R 11 ), C(R 10 )=C(R 11 ), C[=C(R 10 )(R 11 The Y 1 , Y 2 and Y 3 The other two are 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 each R 9、 R 10 and R 11 These are 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.

[0026] J is O, S, NR 12 , NN(R 13 )2, or N-OR 13is,

[0027] R 12 is H, OH, halogen, C1-C 10 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or the following,

[0028]

Chemical formula

[0029] R 14 is selected from H or C1-C4 alkyl which may be substituted with 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted with hydroxyl, heteroaryl optionally substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, R 15 is selected from H, C1-C 18 alkyl or aryl,

[0030] R 13 is H, C1-C 10 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl or the following,

[0031]

Chemical formula

[0032] R 14 is selected from H or C1-C4 alkyl which may be substituted with 1 to 3 substituents independently selected from SH, S-(C1-C4 alkyl), aryl optionally substituted with hydroxyl, heteroaryl optionally substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, R 15 is selected from H, C1-C 18 alkyl or aryl,

[0033] K and L are each independently OH, OR 16 , SR 16 , or NR16 is selected from, R 16 is H, C1-C 10 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, or selected from the following,

[0034]

Chemical Formula

[0035] R 14 is selected from H or SH, S-(C1-C4 alkyl), aryl optionally substituted with hydroxyl, heteroaryl optionally substituted with hydroxyl, amino, hydroxyl, oxo, or C1-C4 alkyl optionally substituted with 1 to 3 substituents independently selected from -NH-C=(NH)NH2, R 15 is H, C1-C 18 selected from alkyl or aryl,

[0036] In some embodiments, the cyclo of the structure of Formula A is selected from the group consisting of the following.

[0037]

Chemical Formula

[0038] In some embodiments, the cyclo of the structure of Formula A is a cyclic functional group, and the cyclic functional group is bonded to the phosphate moiety and X' of Formula A at the following positions of the substituted cyclic functional group, which are indicated using 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.

[0039] In some embodiments, the cyclo in the structure of formula A is substituted. In some embodiments, the cyclo in the structure of formula A is a substituted 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, at the following positions: the phosphate moiety and the substituted cyclic moiety bonded to X' of formula A.

[0040] In some embodiments, X' in formula A is a sugar substitution moiety or contains one. In some embodiments, X' in formula A is a sugar substitution moiety or contains one, and the sugar substitution moiety is morpholino. In some embodiments, X' in formula A is a sugar substitution moiety or contains one, and the sugar substitution moiety is cyclohexenyl. In some embodiments, X' in formula A is a sugar substitution moiety or contains one, and the sugar substitution moiety is cyclohexitol.

[0041] In some embodiments, X' in formula A is or includes a sugar substitution moiety, and the sugar substitution moiety is acyclic. In some embodiments, X' in formula A is or includes an unlocked nucleonucleotide analog (UNA) as the sugar substitution moiety (see, for example, U.S. Patent No. 8,314,227). In some embodiments, X' in formula A is or includes a glycerol nucleic acid structure as the sugar substitution moiety (see, for example, International Publication No. 2016 / 028649).

[0042] In some embodiments, X' in formula A is or contains a locked nucleic acid.

[0043] In some embodiments, the compounds disclosed herein have a 5'-cyclopropylphosphonic acid group having formula Ib or formula II-b,

[0044] [ka]

[0045] D is O, S, CH2-CH 2、 CH=CH, OCH 2、 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 ) and

[0046] R 1 These are 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.

[0047] R 2、 R 3 and R 4 Each of these 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.

[0048] 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 When ), X is a heterocyclic base part,

[0049] D is OC(H)(X 3 ) or OC(R 2 )(X 3 When ), 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 This is the heterocyclic base part,

[0050] Z is H, -OH, F, OCH3, -O-(CH2)2-OCH3; halogen; -OCH2F, -OCHF2, -OCF 3、 -OCH2CH3, -O(CH2)2F, -OCH2CHF2, -OCH2CF3, -OCH2-CH=CH2, -O(CH2)2-OCH3, -O(CH2)2-SCH3, -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 )], a substituted C1-C6 alkyl, a substituted C1-C6 alkoxy, a substituted C2-C6 alkenyl, or a substituted C2-C6 alkynyl, R 5 , R 6 , R 7 and R 8Each of these is independently H or C1-C6 alkyl,

[0051] Y 1 , Y 2 , Y 3 , and Y 4 Each of these 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, or Y 4 is Y 1 Or Y 2 It is bonded to one of the following, and the bond is O, S, NR 9 , C(R 10 )(R 11 ), C(R 10 )=C(R 11 ), C[=C(R 10 )(R 11 The Y 1 , Y 2 and Y 3 The other two are 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 each R 9 , R 10 and R 11 These are 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.

[0052] J is O, S, NR 12 , NN(R 13 )2, or N-OR 13 And,

[0053] R 12 H, OH, halogen, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, or the following:

[0054] [ka]

[0055] R 14 R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, 15 H, C1-C 18 Selected from alkyl or aryl,

[0056] R 13 H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, or the following:

[0057] [ka]

[0058] R 14 R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, 15 H, C1-C 18 Selected from alkyl or aryl,

[0059] K and L are OH and OR, respectively, independently. 16 , SR 16 , or NR 16 Selected from, R 16 H, C1-C 10 Selected from alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl, or the following:

[0060] [ka]

[0061] R 14 R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, 15 H, C1-C 18 Selected from alkyl or aryl,

[0062] Q is O, S, N(R 30 ), or C(R 31 )(R 32 The divalent part is selected from ) and R 30 R is H, an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 alkoxy, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl, 31 and R 32 Each of these is independently H, a halogen, an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 alkoxy, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl.

[0063] A is (i) a nucleoside bond that connects the 5'-cyclophosphonic acid modified nucleotide of formula I to the rest of the RNAi agent, or (ii) a phosphoramidite group.

[0064] G 1 , G 2 , G 3 and G 4 These are independently 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 33Selected from the group consisting of ), R 33 is O(R 35 ), S(R 35 ), N(R 35 )(R 36 ) and R 34 , R 35 , and R 36 Each of these is independently selected from H, halogen, or C1-C6 alkyl.

[0065] In some embodiments, the compounds disclosed herein contain a 5-cyclopropylphosphonic acid group, are bound to an RNAi agent, and have the following formula III or formula IV:

[0066] [ka]

[0067] X is the heterocyclic base part,

[0068] 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-S CH3, -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(R7 )-C(=R 8 )[N(R 5 )(R 6 )], a substituted C1-C6 alkyl, a substituted C1-C6 alkoxy, a substituted C2-C6 alkenyl, or a substituted C2-C6 alkynyl, R 5 , R 6 , R 7 and R 8 Each of these is independently H or C1-C6 alkyl,

[0069] J and J' are independently either O or S.

[0070] L, L', and K are OH and OR respectively, independently of each other. 16 , SR 16 , or NR 16 Selected from, R 16 H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, or selected from the following:

[0071] [ka]

[0072] R 14 R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, 15 H, C1-C 18 Selected from alkyl or aryl,

[0073] [ka]

[0074] The above formula includes the remaining RNAi agent.

[0075] In some embodiments, the 5'-cyclophosphonic acid-modified nucleotide compound disclosed is a phosphoramidite compound.

[0076] In some embodiments, the disclosed 5'-cyclophosphonic acid-modified nucleotide compounds are phosphoramidite compounds having formula Ib-5 or formula II-b-5.

[0077] [ka]

[0078] 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 ) and

[0079] R 1 These are 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.

[0080] R 2 , R 3 and R 4 Each of these 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.

[0081] D is O, S, CH2-CH2, CH=CH, OCH 2、 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 When ), X is a heterocyclic base part, D is OC(H)(X 3 ) or OC(R 2 )(X 3 When ), 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 This is the heterocyclic base part,

[0082] 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-S CH3, -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 )], a substituted C1-C6 alkyl, a substituted C1-C6 alkoxy, a substituted C2-C6 alkenyl, or a substituted C2-C6 alkynyl, R 5 , R 6 , R 7 and R 8 Each of these is independently H or C1-C6 alkyl,

[0083] Y 1 , Y 2 , Y 3 , and Y 4 Each of these 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, or Y 4 is Y 1 Or Y 2 It is bonded to one of the following, and the bond is O, S, NR 9 , C(R 10 )(R 11 ), C(R 10 )=C(R 11 ), C[=C(R 10 )(R 11 The Y 1 , Y 2 and Y 3 The other two are 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 each R 9 , R 10 and R 11 These are 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.

[0084] J is O, S, NR 12 , NN(R 13 )2, or N-OR 13 And,

[0085] R 12 H, OH, halogen, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, or the following:

[0086] [ka]

[0087] R 14 R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, 15 H, C1-C 18 Selected from alkyl or aryl,

[0088] R 13 H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, or the following formulas:

[0089] [ka]

[0090] R 14 R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, 15 H, C1-C 18 Selected from alkyl or aryl,

[0091] K and L are OH and OR, respectively, independently. 16 , SR 16 , or NR 16 Selected from, R 16 H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, or selected from the following formulas:

[0092] [ka]

[0093] R 14 R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, 15 H, C1-C 18 Selected from alkyl or aryl,

[0094] G 1 , G 2 , G 3 and G 4 These are independently 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 Selected from the group consisting of ), R 33 is O(R 35 ), S(R 35 ), N(R 35 )(R 36 ) and R 34 , R 35 , and R 36 Each of these is independently selected from H, halogen, or C1-C6 alkyl.

[0095] In some embodiments, phosphoramidite-containing compounds comprising 5-cyclopropylphosphonic acid-modified nucleotides have the following structure.

[0096] [ka]

[0097] In some embodiments, the disclosed 5'-cyclophosphonic acid-modified nucleotide compounds have a structure represented by formula B,

[0098] [ka]

[0099] Cyclo and X' are defined by concatenating them with the aforementioned formula A,

[0100] In formula B, M is a 5'-phosphonic acid mock group, or contains one. In some embodiments, M in formula B is a 5'-C-malonyl group. In some embodiments, M in formula B is a carboxylic acid, sulfonic acid, or boronic acid. In some embodiments, M in formula B is a dicarboxylic acid, disulfonic acid, or diboronic acid. In some embodiments, M in formula B is a diacid selected from a mixture of carboxylic acids, sulfonic acids, boronic acids, and phosphoric acid. In some embodiments, the phosphonic acid mock group (i.e., M in formula B) is bonded to the cyclo of formula B by a single bond. In some embodiments, the phosphonic acid mock group (i.e., M in formula B) is bonded to the cyclo of formula B by more than one bond.

[0101] In some embodiments, the compound of formula B disclosed has the structure shown by the following structure,

[0102] [ka]

[0103] Cyclo and X' are defined by concatenating them with the aforementioned formula A,

[0104] In some embodiments, the compound of formula B disclosed has a structure represented by the following structure:

[0105] [ka]

[0106] Cyclo, D, X, and Z are defined by concatenating them with the aforementioned formula A.

[0107] When referring to a connection between two molecules, the term “bonded” as used herein means that two molecules are linked by a covalent bond or by intermolecular forces (e.g., hydrogen bonds, van der Waals forces, or ionic bonds). In some embodiments, when the term “bonded” refers to the bonding of two molecules by intermolecular forces, the bonding between two different molecules is 1 x 10⁻¹⁶ in a physiologically acceptable buffer (e.g., phosphate-buffered saline). -4 Less than M K D (For example, 1x10 -5 Less than M, 1x10 -6 Less than M, or 1x10 -7 It has (less than M)

[0108] The term "directly bonded," as used herein, refers to a first compound or group that is bonded to a second compound or group that does not have an intervening atom or atomic group. The term "indirectly bonded," as used herein, refers to a first compound that is bonded to a second compound or group by an intermediate atom, group, compound, or molecule, such as a bonding group. Unless otherwise specified, the term "bonded" as used herein includes both "directly bonded" and "indirectly bonded" as defined herein.

[0109] As used herein, an "oligomer compound" is a nucleotide sequence comprising approximately 10 to 50 nucleotides or nucleotide base pairs. In some embodiments, the oligomer compound has a nucleic acid base sequence that is at least partially complementary to the coding sequence of the expressed target nucleic acid or target gene in the cell. In some embodiments, when the oligomer compound is delivered to a gene-expressing cell, it can suppress the expression of the underlying gene, and is referred to herein as an "expression repression oligomer compound." It can suppress gene expression in vitro or in vivo. Examples of "oligomer 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 substrates.

[0110] As used herein, the terms “oligonucleotide” and “polynucleotide” mean polymers of bonded nucleosides that are independently modified or unmodified.

[0111] The term “single-stranded oligonucleotide,” as used herein, means a single-stranded oligomeric compound having a sequence at least partially complementary to the target mRNA and capable of hybridizing the target mRNA by hydrogen bonding under mammalian physiological conditions (or similar in vitro conditions). In some embodiments, the single-stranded oligonucleotide is a single-stranded antisense oligonucleotide. The 5'-cyclophosphonic acid-modified nucleotides disclosed herein can be incorporated into single-stranded antisense oligonucleotides. In some embodiments, the 5'-cyclophosphonic acid-modified nucleotide is positioned as the 5' terminal nucleotide of the single-stranded oligonucleotide.

[0112] As used herein, “RNAi agent” means an agent comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of sequence-specifically degrading or repressing the translation of a messenger RNA (mRNA) transcript of a target mRNA. As used herein, RNAi agents may act by an RNA interference mechanism (i.e., by inducing RNA interference through interaction with the RNA interference pathway mechanism (RNA-induced silencing complex or RISC) in mammalian cells) or by an alternative mechanism or pathway. As used herein, RNAi agents are considered to act primarily by an RNA interference mechanism, but the RNAi agents disclosed are not bound or limited to a specific pathway or mechanism of activity. Examples of 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 substrates. The RNAi agents described herein consist of oligonucleotides having a strand at least partially complementary to the target mRNA. In some embodiments, the RNAi agent described herein is double-stranded and consists of an antisense strand and a sense strand at least partially complementary to the antisense strand. The RNAi agent may consist of a modified nucleotide and / or one or more non-phosphorylated diester bonds. In some embodiments, the RNAi agent described herein is single-stranded.

[0113] When used herein to refer to the expression of a given gene, the terms “silence,” “reduce,” “suppress,” “downregulate,” or “knockdown” mean that the gene expression, as measured by the level of RNA or polypeptide transcribed from the gene, or by the level of protein or protein subunit translated from mRNA in cells, cell populations, tissues, organs, or the target on which the gene is transcribed, is reduced in cells, cell populations, tissues, or organs of a target treated with an RNAi agent or other oligomeric compound described herein, compared to a second cell population, cell group, tissue, or organ of a target that has not been treated or has never been treated.

[0114] The terms “sequence” or “nucleotide sequence” as used herein mean a sequence of nucleic acid bases or nucleotides written in consecutive letters using standard nucleotide nomenclature.

[0115] When used herein, “nucleotide base” or “nucleic acid base” refers to a heterocyclic pyrimidine or purine compound, the standard constituent of all nucleic acids, and includes the bases that form the nucleotides adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). Nucleic acid bases can be further modified to include, but are not limited to, universal bases, hydrophobic bases, random bases, expanded-size bases, and fluorinated bases.

[0116] The term “heterocyclic base moiety” as used herein refers to a nucleic acid base or a modified nucleic acid base 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 natural purine or a substituted purine. In some embodiments, the heterocyclic base moiety is a non-natural purine or a substituted purine. In some embodiments, the heterocyclic base moiety is a natural pyrimidine or a substituted pyrimidine. In some embodiments, the heterocyclic base moiety is a non-natural pyrimidine or a substituted pyrimidine. In some embodiments, in particular, when the 5'-cyclophosphonate-modified nucleotide disclosed herein is a phosphoramidite compound, the heterocyclic base moiety includes one or more protecting groups.

[0117] The term "sugar substitution moiety" refers to a structure that, when used herein, can substitute a five-membered furanose ring of a natural ribonucleotide.

[0118] The term “complementary,” as used herein, unless otherwise specified, means the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize (form base-pair hydrogen bonds under mammalian physiological conditions (or similar in vitro conditions)) to form a double-stranded or double-helical structure under specific conditions having the oligonucleotide or polynucleotide containing the second nucleotide sequence, when used to describe a first nucleotide sequence (e.g., an RNAi agent sense strand or target mRNA) in relation to a second nucleotide sequence (e.g., a single-stranded antisense oligonucleotide or a double-stranded RNAi agent antisense strand). Examples of complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and natural or modified nucleotides or nucleotide mimeographs, to the extent that they satisfy at least the aforementioned hybridization conditions. Sequence identity or complementarity is independent of modification. For example, a and Af are complementary to U (or T) and identical to A in order to determine identity or complementarity.

[0119] When used herein, “fully complementary” or “sufficiently complementary” means that all (100%) of the bases in the contiguous sequence of the first polynucleotide hybridize with the same number of bases in the contiguous sequence of the second polynucleotide. The contiguous sequence may include all or part of the first or second nucleotide sequence.

[0120] When used herein, "partially complementary" means that in a hybridized pair of nucleic acid base sequences, not all of the bases in the sequence of the first polynucleotide hybridize to the same number of bases as the sequence of the second polynucleotide, but at least 70% hybridize.

[0121] When used herein, "substantially complementary" means that in a hybridized pair of nucleic acid base sequences, not all bases of the first polynucleotide sequence hybridize to the same number of bases as the second polynucleotide sequence, but at least 85% hybridize. The terms "complementary," "fully complementary," and "substantially complementary" can be used herein to describe base matching between the sense and antisense strands of a double-stranded RNAi agent, the antisense strand of a double-stranded RNAi agent and the target mRNA sequence, or between a single-stranded antisense oligonucleotide and the target mRNA sequence.

[0122] The terms “to treat” and “treatment” as used herein mean methods or steps taken to provide relief or mitigation of one or more symptoms, severity, and / or frequency of the disease or condition in question.

[0123] The phrase "introducing into cells," as used herein, means functionally delivering an RNAi agent to cells, when it refers to an RNAi agent containing a 5'-cyclophosphonic acid-modified nucleotide. The phrase "functional delivery" means delivering an RNAi agent to cells in such a way that it can have its expected biological activity, such as sequence-specific repression of gene expression.

[0124] Unless otherwise stated, symbols

[0125] [ka]

[0126] This means that, when used herein, any group or combination of groups can be combined in accordance with the scope of the invention described herein.

[0127] As used herein, the term "isomer" refers to molecules that have the same molecular formula but differ in their atomic bonding properties, arrangement, or spatial configuration. Isomers that differ in spatial configuration are called "stereoisomers." Stereoisomers that are not mirror images of each other are called "diastereomers," while mirror images that cannot be superimposed are also called "enantiomers" or optical isomers. A carbon atom bonded to four non-identical substituents is called a "chiral center."

[0128] Unless otherwise specifically identified in a structure having a particular structure, each structure disclosed herein is intended to represent all possible isomers, including optically pure and racemic forms, for each structure in which an asymmetric center is present, resulting in an enantiomer, diastereomer, or other stereoisomeric structure. For example, the structures disclosed herein are intended to cover diastereomers and mixtures of one stereoisomer. The 5'-cyclophosphonic acid-modified nucleotides described herein contain one or more asymmetric centers, resulting in an enantiomer, diastereomer, or other stereoisomeric structure that can be defined in absolute stereochemistry as (R) or (S), as α or β (such as a sugar anomer), or as (D) or (L) (such as an amino acid). The 5'-cyclophosphonic acid-modified nucleotides described herein include all possible isomers, including racemic and optically pure forms. Unless otherwise specified, when a compound described herein contains a double bond (for example, in an alkene or imine), it is intended that the compound includes both E and Z geometric isomers and cis- and trans-isomers. Similarly, it is intended to include all tautomers. The bond structures described herein are selected independently for convenience and are not intended to limit the compound to any particular structure unless otherwise specified.

[0129] The term "substituted," as used herein, means that one or more hydrogen atoms on a specified atom, usually a carbon, oxygen, or nitrogen atom, are substituted with a group as defined herein, provided that the substitution results in a stable compound without exceeding the normal valence of the specified atom. When the substituent is keto or oxo (i.e., =0), two hydrogen atoms on the atom are substituted. A cyclic double bond, as used herein, is a double bond formed between two adjacent cyclic atoms (e.g., C=C, C=N, N=N). In some embodiments, the substituent of a cyclic functional group is an additional cyclic group or an aryl group. A bicyclic group, as used herein, is considered a substituted cyclic functional group. Examples of organic functional groups, but not limited to, include hydrogen; halo (e.g., F, CI, Br, I); cyano; -CO2R a;-CONR a R a ; 1-2 independently selected R a C may be replaced by 1-6 Alkyl; C 1-4 Haloalkyl; C 1-4 Alkoxy, C 1-4 Examples include haloalkoxys and cycloalkyls, where cycloalkyls consist of 1 to 4 independently selected R a They may be substituted with, and the heterocyclyl contains 5 to 8 cyclic atoms, with 1 to 3 cyclic atoms each independently being N(R) a ), selected from the group consisting of O and S, and the heterocyclyl is selected from 1 to 4 independently R a It may be replaced with; C 6-10 Ariel is 1-4R a It may also be substituted with; the heteroaryl group contains 5 to 10 cyclic atoms, and 1 to 4 of the cyclic atoms are each independently N, N(R) a ), selected from the group consisting of O and S, and the heteroaryl has 1 to 3 R a -N3;-CO2H;-OH;-SO 1-2 (R a );-NR a R a ;-SO 1-2 (NR a R a ); and may be substituted with thioalkoxy, R a Each is independently C 1-6 Alkyl, -OH, -halo, -NH2, -N(C) 1-4 Alkyl)2, C 1-4 Alkoxy, C 1-4 Haloalkoxy, -C(=O)(C 1-4 Alkyl), -C(=O)OH, -CON(C 1-4 Alkyl)2,-S(O) 1-2 (C 1-4 Selected from alkyl)2 and cyano.

[0130] Some of the compounds in this disclosure may exist in tautomerized forms, which are also intended to be included within the scope of this disclosure. A “tautomer” is a compound that has a significantly different atomic arrangement in its structure but exists in a readily and rapidly equilibrium state. It should be understood that the compounds in this disclosure may be described as different tautomers. When a compound has a tautomerized form, it should be understood that all tautomerized forms are intended to be within the scope of this disclosure, and that the naming of the compounds does not exclude any tautomerized form.

[0131] The compounds and pharmaceutically acceptable salts of this disclosure may exist in one or more tautomers, including ketone-enols, amide-nitriles, lactam-lactims, amide-imido acids (e.g., in the nucleic acid bases guanine, thymine, and cytosine), amine-enamines and enamine-enamines, as well as geometric isomers and mixtures thereof. Cyclic chain tautomerism is represented by glucose and other sugars, where the aldehyde group (-CHO) of the sugar chain molecule reacts with one of the hydroxyl groups (-OH) of the same molecule to form a cyclic (ring-shaped) form. All tautomers are included in the scope of this disclosure. Tautomers exist in solution as a mixture of sets of tautomers. In solid form, usually one tautomer is dominant. One tautomer can be described, but this disclosure includes all tautomers of the compounds disclosed herein. The concept of tautomers that can be interconverted by tautomerization is called tautomerism. In tautomerism, simultaneous shifts of electrons and hydrogen atoms occur.

[0132] Base-catalyzed tautomerism occurs through 1) deprotonation; 2) delocalized anion (e.g., enolate); and 3) protonation of the anion at different positions. Acid-catalyzed tautomerism occurs through 1) protonation; 2) delocalized cation; and 3) deprotonation at different positions adjacent to the cation.

[0133] As used herein, “protecting group” refers to an unstable chemical moiety known in the art that prevents a reactant group (e.g., hydroxyl, amino, carboxyl, and sulfhydryl groups) from undergoing undesirable reactions during the synthetic process. Protecting groups are typically used selectively and / or orthogonally to the protected site during reactions at other reaction sites, after which the protecting group may be removed, the deprotected group left as is, or it may be used for further reactions. In some embodiments, “substituted” groups or substituents include protecting groups.

[0134] The term "alkyl," as used herein, refers to a saturated aliphatic hydrocarbon group, either linear or branched, having 1 to 10 carbon atoms unless otherwise specified. For example, "C 1-6 The term "alkyl" includes alkyl groups having 1, 2, 3, 4, 5, or 6 carbon atoms in a linear or branched configuration. The term "aminoalkyl," as used herein, refers to the aforementioned alkyl groups, which are substituted at any position with one or more amino groups permitted by their normal valence. The amino groups may be unsubstituted, monosubstituted, or disubstituted.

[0135] The term "cyclic functional group" is intended to refer to a functional group that forms a cyclic structure. Examples of cyclic functional groups include, but are not limited to, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, and aryl groups.

[0136] The term "cycloalkyl," as used herein, means a saturated or unsaturated non-aromatic hydrocarbon cyclic group having 3 to 14 carbon atoms, unless otherwise specified. Examples of cycloalkyls include, but are not limited to, cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, or cyclohexyl. Cycloalkyls may contain multiple spiro or fused rings. Cycloalkyls may be mono-, di-, tri-, tetra-, or penta-substituted at positions permitted by their normal valence.

[0137] As used herein, the term “alkenyl” refers to a linear or branched non-aromatic hydrocarbon group having 2 to 10 carbon atoms, unless otherwise specified, and containing at least one carbon-carbon double bond. Up to 5 carbon-carbon double bonds may be present in this group. For example, a “C2-C6” alkenyl is defined as an alkenyl group having 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 portions of the alkenyl group may contain double bonds and may be mono-, di-, tri-, tetra-, or penta-substituted at positions permitted by normal valence. The term “cycloalkenyl” means a monocyclic hydrocarbon group having a specific number of carbon atoms and at least one carbon-carbon double bond.

[0138] The term "alkynyl," as used herein, refers, unless otherwise specified, to a linear or branched hydrocarbon group containing 2 to 10 carbon atoms and at least one carbon-carbon triple bond. Up to 5 carbon-carbon triple bonds may be present. Thus, "C2-C6 alkynyl" means an alkynyl group having 2 to 6 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 2-propynyl, and 2-butynyl. The linear or branched portion of the alkynyl group may contain triple bonds permissible by normal valence and may be mono-, di-, or tri-substituted at positions permissible by normal valence.

[0139] When used herein, "alkoxyl" or "alkoxy" refers to the aforementioned alkyl group having an indicated number of carbon atoms linked by oxygen bridges. For example, C 1-6 Alkoxy groups are intended to include C1, C2, C3, C4, C5, and C6 alkoxy groups. For example, C 1-8 Alkoxys include C1, C2, C3, C4, C5, C 6、It is intended to contain C7 and C8 alkoxy groups. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, t-butoxy, n-pentoxy, s-pentoxy, n-heptoxy, and n-octoxy.

[0140] As used herein, "keto" refers to an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, or aryl group linked by a carbonyl bridge, as defined herein. Examples of keto groups include, but are not limited to, alkanoyl (e.g., acetyl, propinoyl, butanoyl, pentanoyl, hexanoyl), alkenoyl (e.g., acryloyl), alkinoyl (e.g., ethinoyl, propinoyl, hexanoyl), aryloyl (e.g., benzoyl), and heteroaryloyl (e.g., pyrroloyl, imidazoloyl, quinolinoyl, pyridinoyl).

[0141] As used herein, "alkoxycarbonyl" refers to the aforementioned alkoxy group linked by a carbonyl bridge (i.e., -C(O)O-alkyl). Examples of alkoxycarbonyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-propoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, or n-pentoxycarbonyl.

[0142] When used herein, "aryloxycarbonyl" refers to the aryl group defined herein that is linked by an oxycarbonyl bridge (i.e., -C(O)O-aryl). Examples of aryloxycarbonyl groups include, but are not limited to, phenoxycarbonyl and naphthyloxycarbonyl.

[0143] When used herein, "heteroaryloxycarbonyl" refers to a heteroaryl group defined herein that is linked by an oxycarbonyl bridge (i.e., -C(O)O-heteroaryl). Examples of heteroaryloxycarbonyls include, but are not limited to, 2-pyridyloxycarbonyl, 2-oxazolyloxycarbonyl, 4-thiazolyloxycarbonyl, or pyrimidinyloxycarbonyl.

[0144] As used herein, "aryl" means a stable monocyclic or polycyclic carbocyclic ring in which at least one ring is aromatic and each ring contains up to seven atoms. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, tetrahydronaphthyl, indanyl, and biphenyl. When the aryl substituent is bicyclic and one ring is non-aromatic, the bond is understood to be via an aromatic ring. The aryl group may be mono-, di-, tri-, tetra-, or penta-substituted at positions permitted by normal valence.

[0145] The term "heteroaryl," as used herein, refers to a stable monocyclic or polycyclic ring system with up to seven atoms in each ring, where at least one ring is aromatic, and which contains one to four heteroatoms selected from groups chosen from the group consisting of O, N, and S. Examples of heteroaryl groups include, but are not limited to, acridinyl, carbazolyl, synnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, benzimidazolonyl, benzoxazolonyl, quinolinyl, isoquinolinyl, dihydroisoindonyl, imidazopyridinyl, isoindonyl, indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl, pyrazidyl, pyridadinyl, pyridinyl, pyrimidinyl, pyrrolyl, and tetrahydroquinoline. It is also understood that "heteroaryl" includes N-oxide derivatives of nitrogen-containing heteroaryls. If the heteroaryl substituent is bicyclic and one ring is non-aromatic or does not contain a heteroatom, the bond is understood to be via an aromatic ring or a heteroatom-containing ring. The heteroaryl group may be mono- or di-substituted at positions permitted by normal valence.

[0146] The terms “heterocyclic,” “heterocyclic formula,” and “heterocyclyl” as used herein mean a 3- to 14-membered aromatic or non-aromatic heterocyclic ring containing 1 to 4 heteroatoms selected from the group consisting of O, N, and S, and include polycyclic groups. The term “heterocyclic formula” as used herein is also considered synonymous with “heterocyclic” and “heterocyclyl,” and is understood to have the same definition as given herein. “Heterocyclyl” includes the aforementioned heteroaryls, as well as their dihydro and tetrahydro analogs.Examples of heterocyclils include azetidinil, benzimidazolyl, benzofuranil, benzoflazanil, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carborinil, cinnolinil, furanil, imidazolyl, indolinil, indolyl, indazinil, indazolyl, isobenzofuranil, isoindolyl, isoquinolyl, izothiazolyl, isoxazolyl, naphtopyridinil, oxadiazolyl, and oxoxazolidinyl. Oxazolyl, oxazoline, oxopiperazinil, oxopyrrolidinil, oxomorpholinil, isoxazoline, oxetanil, pyranil, pyrazinil, pyrazolyl, pyridadinil, pyridopyridinil, pyridadinil, pyridyl, pyridinonil, pyrimidyl, pyrimidinonil, pyrrolyl, quinazolinil, quinolyl, quinoxalinil, tetrahydropyranil, tetrahydrofuranil, tetrahydrothiopyranil, tetrahydroisoquinolinil, tetrazolyl, tetrazolopyridyl Thiazolyl, thiazolyl, thienyl, thiazolyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, pyridine-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl Examples include, but are not limited to, lyl, dihydrooxazolyl, dihydropyrazine, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl, dioxidethiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl, tetrahydrothienyl, and their N-oxides. The bonding of heterocyclyl substituents can be caused by carbon atoms or heteroatoms. The heterocyclyl group may be mono-, di-, tri-, tetra-, or penta-substituted at positions permitted by normal valence.

[0147] The phrase "consisting of" excludes elements, steps, or components not specified in the claims when used in the claims of this specification. The phrase "essentially consisting of" limits the scope of the claim to specific materials or steps and those that do not materially affect the basic novel features of the claimed invention.

[0148] Those skilled in the art will readily understand and interpret that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or unprotonated state, depending on the environment in which they are placed. Therefore, the structures disclosed herein may, when used herein, have certain functional groups, such as OH, SH, or NH, either protonated or unprotonated. This disclosure is intended to encompass the disclosed compounds and compositions regardless of their protonation state based on the pH of their environment, as will be readily understood by those skilled in the art.

[0149] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Methods and materials identical or equivalent to those described herein may be used in the execution or testing of the present invention, but suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated in their entirety by reference. In case of any inconsistency, the descriptions herein, including definitions, shall be adjusted. Furthermore, materials, methods, and examples are illustrative and not intended to be limiting.

[0150] Other features and advantages of the present invention will become apparent from the following detailed description and claims. [Brief explanation of the drawing]

[0151] [Figure 1]This graph shows the relative cF12 expression in cynomolgus monkeys after administration of an RNAi agent containing a sequence directed towards factor 12. Only AD04443 contained a 5-cyclopropylphosphonic acid modified nucleotide and was located at the 5' end of the antisense strand. [Figure 2] This graph shows the mean HBsAg levels normalized to pre-treatment and saline controls in pHBV model mice after administration of RNAi agents containing sequences targeting HBV. Only AD04580 contained a 5-cyclopropylphosphonic acid modified nucleotide and was located at the 5' end of the antisense strand. [Modes for carrying out the invention]

[0152] This document describes 5'-cyclophosphonate-modified nucleotides and RNAi agents (also called RNAi triggers) containing 5'-cyclophosphonate-modified nucleotides. In some embodiments, one or more 5'-cyclophosphonate-modified nucleotides bind to the end of the RNAi agent, forming a terminal nucleotide of the RNAi agent. In some embodiments, the 5'-cyclophosphonate-modified nucleotides bind to the 5' end or 5' terminus of the RNAi agent, forming a terminal nucleotide at the 5' end of the RNAi agent. In some embodiments, the 5'-cyclophosphonate-modified nucleotides bind to the 5' end of the antisense strand of a double-stranded RNAi agent, forming a terminal nucleotide at the 5' end of the antisense strand of the double-stranded RNAi agent.

[0153] The 5'-cyclophosphonic acid-modified nucleotides disclosed herein have a cyclic group or cyclic moiety located at the 5' carbon of the sugar of the nucleotide (or at an equivalent position to the sugar substitution portion).

[0154] In some embodiments, the 5' terminal nucleotide of the RNAi agent includes or is a 5'-cyclophosphonic acid-modified nucleotide. In some embodiments, the 5' terminal nucleotide of the antisense strand of the double-stranded RNAi agent includes or is a 5'-cyclophosphonic acid-modified nucleotide. In some embodiments, the 5' terminal of an oligomeric compound such as a single-stranded RNAi agent, or the 5'-cyclophosphonic acid-modified nucleotide located on the antisense strand of a double-stranded RNAi agent, promotes the loading of the oligomeric compound onto RISC, thereby initiating the RNAi mechanism.

[0155] In some embodiments, one or more 5'-cyclophosphonic acid-modified nucleotides bind to the end of a single-stranded antisense oligonucleotide to form a terminal nucleotide of the single-stranded antisense oligonucleotide. In some embodiments, a 5'-cyclophosphonic acid-modified nucleotide binds to the 5' end of a single-stranded antisense oligonucleotide to form a terminal nucleotide at the 5' end of the single-stranded antisense oligonucleotide.

[0156] In some embodiments, the disclosed compounds have a structure represented by formula A, as described in the Summary of the Invention section.

[0157] In some embodiments, the compound of formula A disclosed has a 5'-cyclophosphonic acid structure represented by formula I,

[0158] [ka]

[0159] Cyclo is a 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, pyr) Razole, imidazole, thiophene, benzothiophene, thiazole, benzothiazole, furan, oxazole, isoxazole, benzofuran, indole, indazole, benzimidazole, oxadiazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, quinolinyl, isoquinolinyl, or quinoxalinyl), or heterocyclyl (e.g., tetrahydrofuran, tetrahydropyran, piperidine, pyrrolidine, dioxane, or dioxolane),

[0160] D is O, S, CH2-CH 2、 CH=CH, OCH 2、 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 ) and

[0161] R 1 These are 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.

[0162] R 2、 R3 and R 4 Each of these 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.

[0163] 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 When ), X is a heterocyclic base part, D is OC(H)(X 3 ) or OC(R 2 )(X 3 When ), 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 This is the heterocyclic base part,

[0164] 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-S CH3, -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 )], a substituted C1-C6 alkyl, a substituted C1-C6 alkoxy, a substituted C2-C6 alkenyl, or a substituted C2-C6 alkynyl, R 5 , R 6 , R 7 and R 8 Each of these is independently H or C1-C6 alkyl,

[0165] Y 1 , Y 2 , Y 3 , and Y 4 Each of these 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, or Y 4 is Y 1 Or Y 2 It is bonded to one of the following, and the bond is O, S, NR 9、 C(R 10 )(R 11 ), C(R 10 )=C(R 11 ), C[=C(R 10 )(R 11 The Y 1 , Y 2 and Y 3 The other two are 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 each R9 , R 10 and R 11 These are 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.

[0166] J is O, S, NR 12 , NN(R 13 )2, or N-OR 13 And,

[0167] R 12 H, OH, halogen, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, or the following formulas:

[0168] [ka]

[0169] R 14 R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, 15 H, C1-C 18 Selected from alkyl or aryl,

[0170] R 13 H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, or the following:

[0171] [ka]

[0172] R 14R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, 15 H, C1-C 18 Selected from alkyl or aryl,

[0173] K and L are OH and OR, respectively, independently. 16 , SR 16 , or NR 16 Selected from, R 16 H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, or selected from the following formulas:

[0174] [ka]

[0175] R 14 R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or -NH-C=(NH)NH2, 15 H, C1-C 18 Selected from alkyl or aryl,

[0176] Q is O, S, N(R 30 ), or C(R 31 )(R 32 The divalent part is selected from ) and R 30 R is H, an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 alkoxy, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl, 31 and R 32Each of these is independently H, a halogen, an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 alkoxy, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl.

[0177] A is (i) an internucleoside bond that connects the 5'-cyclophosphonic acid modified nucleotide of formula I to the remainder of the RNAi agent, or (ii) a phosphoramidite group.

[0178] In some embodiments, when A is a phosphoramidite group, A binds to Q of formula I by binding Q to a phosphoramidite-forming agent, thereby forming a phosphoramidite compound.

[0179] In some embodiments, Q in formula I is O.

[0180] In some embodiments, the disclosed compounds have a 5'-cyclophosphonic acid structure represented by formula II,

[0181] [ka]

[0182] Cyclo, D, X, Z, Y 1 , Y 2 , Y 3 , Y 4 J, K, L, Q, and A are defined in conjunction with the aforementioned formula I.

[0183] In some embodiments, the structure of Y of formulas I and II 1 , Y 2 , Y 3 and Y 4 These are H, respectively.

[0184] In some embodiments, the cyclo in the structures of formulas I and II is a cycloalkyl group containing 3, 4, 5, 6, or 7 carbon atoms.

[0185] In some embodiments, the cyclo in the structures of formulas I and II is a cycloalkenyl containing 4, 5, 6, or 7 carbon atoms.

[0186] In some embodiments, the cyclo in the structures of formulas I and II is a cycloalkynyl containing 5, 6, or 7 carbon atoms.

[0187] In some embodiments, the cyclo in the structures of formulas I and II is an aryl containing 3, 4, 5, 6, or 7 carbon atoms.

[0188] In some embodiments, the cyclo in the structures of formulas I and II is a heterocyclyl group comprising 2, 3, 4, 5, or 6 carbon atoms and one or more non-carbon atoms.

[0189] In some embodiments, the cyclo in the structures of formulas I and II is an aryl or heterocyclyl group comprising 2, 3, 4, 5, or 6 carbon atoms and one or more non-carbon atoms.

[0190] In some embodiments, the cyclo group in the structures of formulas I and II is a bicyclic group.

[0191] In some embodiments, the cyclo in the structures of formulas I and II is selected from the group consisting of the following:

[0192] [ka]

[0193] In some embodiments, the cyclo in the structures of formulas I and II is a cyclic functional group, and the cyclic functional group is attached to the phosphate moiety and sugar ring of formulas I and II at the following positions of the cyclic functional group, which are indicated using 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.

[0194] In some embodiments, the cyclo in the structures of formulas I and II is selected from the group consisting of the following:

[0195] [ka]

[0196] In some embodiments, the cyclo in the structures of formulas I and II is substituted. In some embodiments, the cyclo in the structures of formulas I and II is a substituted cyclic group attached to the phosphate and sugar rings of formulas I and II at the following positions of a substituted cyclic functional group, indicated using 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.

[0197] In some embodiments, the cyclo in the structures of formulas I and II is a cyclopropyl group having a specific stereochemistry, for example,

[0198] [ka]

[0199] In some embodiments, the cyclo group in the structures of formulas I and II is a substituted cyclopropyl functional group selected from the group consisting of the following:

[0200] [ka]

[0201] In some embodiments, the disclosed compounds have a structure represented by formula Ia or formula II-a,

[0202] [ka]

[0203] Cyclo, D, X, Z, Y 1 , Y 2 , Y 3 , Y 4 J, K, L, and A are defined in conjunction with the aforementioned formulas I and II, respectively.

[0204] As used herein, unless otherwise specified, references to Formula I include references to Formula Ia and Formula Ib, and references to Formula II include references to Formula II-a and Formula II-b, provided that such references are applicable to those skilled in the art in terms of the disclosures herein.

[0205] In some embodiments, the heterocyclic base moiety (e.g., X in structures of formulas I-VIII (including all subgroups or species of the formula (e.g., formulas Ib-5)) is a pyrimidine, a substituted pyrimidine, a purine, or a substituted purine. In some embodiments, the heterocyclic base moiety is a natural purine or a substituted purine. In some embodiments, the heterocyclic base moiety is a non-natural purine or a substituted purine. In some embodiments, the heterocyclic base moiety is a natural pyrimidine or a substituted pyrimidine. In some embodiments, the heterocyclic base moiety is a non-natural pyrimidine or a substituted pyrimidine.

[0206] In some embodiments, the heterocyclic base moiety (e.g., X in the structures of formulas I-VIII (including all subgroups or species of the formulas (e.g., formulas Ib-5)) is uracil, thymine, cytosine, 5-methylcytosine, adenine, guanine, or inosine.

[0207] In some embodiments, the heterocyclic base moiety (e.g., X in the structures of formulas I-VIII (including all subgroups or species of the formulas (e.g., formulas Ib-5)) is a substituted uracil, substituted thymine, substituted cytosine, substituted 5-methylcytosine, substituted adenine, substituted guanine, or substituted inosine. In some embodiments, the substituent is a protecting group.

[0208] In some embodiments, isotopes of hydrogen, such as deuterium or tritium, can be incorporated at one or more positions where hydrogen is present. In some embodiments, isotopes of other atoms are present (e.g., C, N, O, or F).

[0209] In some embodiments, the disclosed compounds have a 5'-cyclopropylphosphonic acid structure represented by the structure of formula Ib or formula II-b,

[0210] [ka]

[0211] D is O, S, CH2-CH 2、 CH=CH, OCH 2、 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 ) and

[0212] R 1 These are 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.

[0213] R 2 , R 3 and R 4 Each of these 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.

[0214] 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 When ), X is a heterocyclic base part, D is OC(H)(X 3 ) or OC(R 2 )(X 3 When ), 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 This is the heterocyclic base part,

[0215] 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-S CH3, -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(R5 )(R 6 )-O(CH2)2-N(R 7 )-C(=R 8 )[N(R 5 )(R 6 )], a substituted C1-C6 alkyl, a substituted C1-C6 alkoxy, a substituted C2-C6 alkenyl, or a substituted C2-C6 alkynyl, R 5 , R 6 , R 7 and R 8 Each of these is independently H or C1-C6 alkyl,

[0216] Y 1 , Y 2 , Y 3 , and Y 4 Each of these 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, or Y 4 is Y 1 Or Y 2 It is bonded to one of the following, and the bond is O, S, NR 9 , C(R 10 )(R 11 ), C(R 10 )=C(R 11 ), C[=C(R 10 )(R 11 The Y 1 , Y 2 and Y 3 The other two are 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 each R 9 , R 10 and R 11These are 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.

[0217] J is O, S, NR 12 , NN(R 13 )2, or N-OR 13 And,

[0218] R 12 H, OH, halogen, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, or the following:

[0219] [ka]

[0220] R 14 R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, 15 H, C1-C 18 Selected from alkyl or aryl,

[0221] R 13 H, C1-C 10 It is an alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or aryl, or has the following formula:

[0222] [ka]

[0223] R 14R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, 15 H, C1-C 18 Selected from alkyl or aryl,

[0224] K and L are OH and OR, respectively, independently. 16 , SR 16 , or NR 16 Selected from, R 16 H, C1-C 10 Selected from alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl, or having the following formulas:

[0225] [ka]

[0226] R 14 R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, 15 H, C1-C 18 Selected from alkyl or aryl,

[0227] Q is O, S, N(R 30 ), or C(R 31 )(R 32 The divalent part is selected from ) and R 30 R is H, an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 alkoxy, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl, 31 and R 32Each of these is independently H, a halogen, an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 alkoxy, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl.

[0228] G 1 , G 2 , G 3 and G 4 These are independently 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 Selected from the group consisting of ), R 33 is O(R 35 ), S(R 35 ), N(R 35 )(R 36 ) and R 34 , R 35 , and R 36 Each of these is independently selected from H, halogen, or C1-C6 alkyl.

[0229] A is (i) an internucleoside bond that connects a 5'-cyclophosphonic acid modified nucleotide of formula Ib or formula II-b to the remainder of the RNAi agent, or (ii) a phosphoramidite group.

[0230] When used herein, unless otherwise specified, references to formula Ib include references to formulas Ib-1, Ib-2, Ib-3, Ib-4, and Ib-5, and references to formula II-b include references to formulas II-b-1, II-b-2, II-b-3, II-b-4, and II-b-5, provided that such references are applicable to those skilled in the art in terms of the disclosures herein.

[0231] In some embodiments, the disclosed compounds have a 5'-cyclophosphonic acid structure represented by the structure of formula Ib-1 or formula II-b-1.

[0232] [ka]

[0233] D, X, Z, Y 1 , Y 2 , Y 3 , Y 4 J, K, L, A, and Q are defined by connecting them to the aforementioned formulas Ib and II-b, respectively.

[0234] In some embodiments, the disclosed compound has a structure represented by formula Ib-2 or formula II-b-2.

[0235] [ka]

[0236] D, X, Z, Y 1 , Y 2 , Y 3 , Y 4 J, K, L, A, G 1 , G 2 , G 3 , G 4 And A are defined in conjunction with the aforementioned formulas Ib and II-b, respectively.

[0237] In some embodiments, the disclosed compounds have a structure represented by formula Ib-3 or formula IIb-3.

[0238] [ka]

[0239] D, X, Z, Y 1 , Y 2 , Y 3 , Y 4 J, K, L, and A are defined in conjunction with the aforementioned formulas Ib and II-b, respectively.

[0240] In some embodiments, the disclosed compounds have a structure represented by formula Ib-4 or formula II-b-4.

[0241] [ka]

[0242] 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 These are defined by connecting them with the aforementioned formulas Ib and II-b, respectively.

[0243] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonic acid modified nucleotide represented by the following formula III or formula IV.

[0244] [ka]

[0245] X is the heterocyclic base part,

[0246] 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-S CH3, -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 )(R6 ), -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 )], a substituted C1-C6 alkyl, a substituted C1-C6 alkoxy, a substituted C2-C6 alkenyl, or a substituted C2-C6 alkynyl, R 5 , R 6 , R 7 and R 8 Each of these is independently H or C1-C6 alkyl,

[0247] J and J' are independently either O or S.

[0248] L, L', and K are OH and OR respectively, independently of each other. 16 , SR 16 , or NR 16 Selected from, R 16 H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, or selected from the following formulas:

[0249] [ka]

[0250] R 14 R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, 15 H, C1-C 18Selected from alkyl or aryl,

[0251] [ka]

[0252] The above formula includes the remaining RNAi agent.

[0253] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonic acid modified nucleotide represented by the following formula III-a or formula IV-a.

[0254] [ka]

[0255] X, Z, J, K, L, J', L', and the following equations are defined in conjunction with the aforementioned equations III and IV.

[0256] [ka]

[0257] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonic acid-modified nucleotide represented by the following formula III-b or formula IV-b:

[0258] [ka]

[0259] X, Z, J, J', L', and the following equations are defined in conjunction with the aforementioned equations III and IV.

[0260] [ka]

[0261] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonic acid modified nucleotide, the modified nucleotide comprising a thymine heterocyclic base moiety and a modification shown by the following structure i (2'-O-2-methoxyethyl or "2'-MOE").

[0262] [ka]

[0263] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonic acid modified nucleotide, the modified nucleotide comprising a thymine heterocyclic base moiety and a 2'-methoxyethyl (2'-O-2-methoxyethyl or "2'-MOE") modification (cPrp™) as shown in structure ii below.

[0264] [ka]

[0265] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonic acid modified nucleotide, the modified nucleotide comprising a thymine heterocyclic base moiety and a 2'-H modification (cPrpdT) as shown in structure iii below.

[0266] [ka]

[0267] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonic acid modified nucleotide, the 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.

[0268] [ka]

[0269] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonic acid modified nucleotide, the modified nucleotide comprising a uracil heterocyclic base moiety and a 2'-O-methyl modification (cPrpu) as shown in structure v below.

[0270] [ka]

[0271] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonic acid modified nucleotide, the modified nucleotide comprising a uracil heterocyclic base moiety and a 2'-O-methyl modification (cPrpus) as shown in structure vi below.

[0272] [ka]

[0273] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonic acid modified nucleotide, the modified nucleotide comprising a uracil heterocyclic base moiety and a 2'-deoxy modification (cPrpdU) as shown in structure vii below.

[0274] [ka]

[0275] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonic acid modified nucleotide, the modified nucleotide comprising a uracil heterocyclic base moiety and a 2'-O-methyl modification (cPrpdUs) as shown in structure viiii below.

[0276] [ka]

[0277] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonic acid modified nucleotide, the modified nucleotide comprising an adenine heterocyclic base moiety and a 2'-O-methyl modification (cPrpa) as shown in structure ix below.

[0278] [ka]

[0279] In some embodiments, the 5' end (or terminal nucleotide) of the antisense strand of the double-stranded RNAi agent is a 5'-cyclopropylphosphonic acid modified nucleotide, the modified nucleotide comprising an adenine heterocyclic base moiety and a 2'-O-methyl modification (cPrpas) as shown in structure x below.

[0280] [ka]

[0281] In some embodiments, structures i, ii, iii, iv, v, vi, vii, viiii, ix, or x may be located at the 5' end (terminal nucleotide) of the antisense strand of the double-stranded RNAi agent.

[0282] In some embodiments, structures i, ii, iii, iv, v, vi, vii, viiii, ix, or x may be located at the 5' end (terminal nucleotide) of a single-stranded antisense oligonucleotide.

[0283] Structure ix is ​​essentially merely illustrative. As discussed elsewhere in this specification, for example, the sugar substitution moiety may be used in conjunction with the aforementioned structure to change the five-membered furanose ring to a different structure that can substitute for the five-membered furanose ring, such as morpholino, cyclohexenyl, cyclohexitol, or an acyclic structure. Such changes are construed to be within the scope of the invention disclosed herein.

[0284] In some embodiments, the 5'-phosphate mimetic, for example, the 5'-C-malonyl group, is a cyclopropyl group, and the end (or terminal nucleotide) of the antisense strand of the RNAi agent is a 5'-cyclopropyl-C-malonyl modified nucleotide as shown in formulas V and VI below.

[0285] [ka]

[0286] X is the heterocyclic base part,

[0287] 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-S CH3, -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 )(R6 ), -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 )], a substituted C1-C6 alkyl, a substituted C1-C6 alkoxy, a substituted C2-C6 alkenyl, or a substituted C2-C6 alkynyl, R 5 , R 6 , R 7 and R 8 Each of these is independently H or C1-C6 alkyl,

[0288] J and J' are independently either O or S.

[0289] L' is OH, OR 16 , SR 16 , or NR 16 Selected from, R 16 H, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, or selected from the following formulas:

[0290] [ka]

[0291] R 14 R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, 15 H, C1-C 18 Selected from alkyl or aryl, The following formula includes the remaining RNAi agent.

[0292] [ka]

[0293] Those skilled in the art will readily understand that, under sufficiently basic conditions, the protic groups of formulas I, II, III, IV, V, VI, VII and / or VIII (subgroups or species of the formula (e.g., formula Ib-5)) and structures i, ii, iii, iv, v, vi, vii, viiii, ix, x, xi, xii and / or xiii) exist in a partially or completely deprotonated state. Similarly, under sufficiently acidic conditions, groups containing a base moiety or atom are protonated. All protonated and deprotonated versions of such groups disclosed herein are within the scope of the embodiments. For example, a carboxyl group is within the scope of the embodiments or claims, and the corresponding carboxylate salt is also within the scope of the embodiments or claims. For example, if an amino group is within the scope of the claims or embodiments, then the corresponding ammonium group is also within the scope of the embodiments or claims.

[0294] In some embodiments, the disclosed compounds are phosphoramidite compounds having a structure represented by formula Ib-5 or formula II-b-5.

[0295] [ka]

[0296] D is O, S, CH2-CH 2、 CH=CH, OCH 2、 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 ) and

[0297] R 1 These are 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.

[0298] R 2、 R 3 and R 4 Each of these 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.

[0299] D is O, S, CH2-CH 2、 CH=CH, OCH 2、 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 When ), X is a heterocyclic base part, D is OC(H)(X 3 ) or OC(R 2 )(X 3 When ), 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 This is the heterocyclic base part,

[0300] Z is H, -OH, F, OCH3,-O-(CH2)2-OCH3; halogen; -OCH2F,-OCHF 2、 -OCF 3、 -OCH2CH 3、 -O(CH2)2F, -OCH2CHF 2、 -OCH2CF 3、 -OCH2-CH=CH 2、 -O(CH2)2-OCH 3、 -O(CH2)2-SCH 3、 -O(CH2)2-OCF3, -O(CH2)2-O(CH2)2-N(CH3) 2、 -OCH2C(=O)-N(H)CH 3、 -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 )], a substituted C1-C6 alkyl, a substituted C1-C6 alkoxy, a substituted C2-C6 alkenyl, or a substituted C2-C6 alkynyl, R 5 , R 6 , R 7 and R 8 Each of these is independently H or C1-C6 alkyl,

[0301] Y 1 , Y 2 , Y 3 , and Y 4Each of these 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, or Y 4 is Y 1 Or Y 2 It is bonded to one of the following, and the bond is O, S, NR 9 , C(R 10 )(R 11 ), C(R 10 )=C(R 11 ), C[=C(R 10 )(R 11 The Y 1 , Y 2 and Y 3 The other two are 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 each R 9 , R 10 and R 11 These are 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.

[0302] J is O, S, NR 12 , NN(R 13 )2, or N-OR 13 And,

[0303] R 12 H, OH, halogen, C1-C 10 Alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, or the following formulas:

[0304] [ka]

[0305] R14 R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, 15 H, C1-C 18 Selected from alkyl or aryl,

[0306] R 13 H, C1-C 10 It is an alkyl, C2-C6 alkenyl, C2-C6 alkynyl, or aryl, or has the following formula:

[0307] [ka]

[0308] R 14 R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, 15 H, C1-C 18 Selected from alkyl or aryl,

[0309] K and L are OH and OR, respectively, independently. 16 , SR 16 , or NR 16 Selected from, R 16 H, C1-C 10 Selected from alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, or from the following formulas:

[0310] [ka]

[0311] R 14R is selected from C1-C4 alkyl groups which may be substituted with 1 to 3 substituents independently selected from H or SH, S-(C1-C4 alkyl), aryl which may be substituted with hydroxyl, heteroaryl which may be substituted with hydroxyl, amino, hydroxyl, oxo or NH-C=(NH)NH2, 15 H, C1-C 18 Selected from alkyl or aryl,

[0312] G 1 , G 2 , G 3 and G 4 These are independently 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 Selected from the group consisting of ), R 33 is O(R 35 ), S(R 35 ), N(R 35 )(R 36 ) and R 34 , R 35 , and R 36 Each of these is independently selected from H, halogen, or C1-C6 alkyl.

[0313] In some embodiments, X in formulas Ib-5 and IIb-5 comprises one or more protecting groups.

[0314] In some embodiments, the compounds disclosed by formula Ib-5 or formula II-b-5 have the following structure:

[0315] [ka] (2-Cyanoethyl((2R,3R,4R,5R)-2-(2-(diethoxyphosphoryl)cyclopropyl)-5-(2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl)(diisopropylphosphoramide acid);

[0316] [ka] (dimethyl[2-[(2R,3R,4R,5R)-3-([[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl]oxy)-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-l,2,3,4-tetrahydropyrimidine-1-yl)oxolan-2-yl]cyclopropyl]phosphonate); or

[0317] [ka] (2R,3R,4R,5R)-5-(6-benzamide-9H-purine-9-yl)-2-(2-(diethoxyphosphoryl)cyclopropyl)-4-methoxytetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphoramidite

[0318] In some embodiments, structures xi, xii, or xiii may be added to the 5' end (terminal nucleotide) of the antisense strand of the double-stranded RNAi agent.

[0319] In some embodiments, structures xi, xii, or xiii may be added to the 5' end (terminal nucleotide) of a single-stranded antisense oligonucleotide.

[0320] Structures xi, xii, and xiii are essentially illustrative. As discussed elsewhere in this specification, for example, sugar substitution moieties may be used in conjunction with the aforementioned structures, and the five-membered furanose ring may be changed to a different structure that can substitute for a five-membered furanose ring, such as morpholino, cyclohexenyl, cyclohexitol, or an acyclic structure. Furthermore, as discussed elsewhere in this specification, modifications at the 2' or 3' position of the modified nucleotide may be changed to a variety of modifications known in the art, and / or the heterocyclic base moiety may be modified from the particular structures described herein. Such changes are construed to be within the scope of the invention disclosed herein.

[0321] The 5'-cyclophosphonic acid-modified nucleotides in the form of phosphoramidite compounds disclosed herein may be useful for coupling 5'-cyclophosphonic acid-modified nucleotides using methods generally known in the art for the synthesis of phosphoramidites from oligonucleotides. The 5'-cyclophosphonic acid-modified nucleotides can be prepared as phosphoramidite compounds by coupling them with a phosphorus atom of a phosphoramidite-forming agent by a coupling reaction (e.g., phosphytilization), thereby forming the phosphoramidite compounds.

[0322] In some embodiments, a 5'-cyclophosphonic acid-modified nucleotide-phosphoramidite compound is used to attach the 5'-cyclophosphonic acid-modified nucleotide to the 5' end of the antisense strand of a double-stranded RNAi agent. In some embodiments, a 5'-cyclophosphonic acid-modified nucleotide-phosphoramidite compound is used to attach the 5'-cyclophosphonic acid-modified nucleotide to the 5' end of the antisense strand of a single-stranded RNAi agent.

[0323] When used herein, the RNAi agents and single-stranded antisense oligonucleotides containing 5'-cyclophosphonic acid-modified nucleotides described herein contain one or more asymmetric centers, thereby giving rise to enantiomers, diastereomers, and other stereoisomers. The binding structures described herein are selected individually for convenience and are not intended to be limited to any particular structure unless otherwise noted.

[0324] In some embodiments, the disclosed compounds have the structure of formula B represented by formula IX or formula X.

[0325] [ka]

[0326] Cyclo, D, X, Z, Y 1 , Y 2 , Y 3 , Y 4 J, K, L, Q, and A are defined in conjunction with the aforementioned formulas I and II, respectively.

[0327] RNAi agents, target ligands, and delivery polymers

[0328] Oligomer compounds having a sequence at least partially complementary to the target nucleic acid, such as RNAi agents, have been shown to alter the function and activity of the target nucleic acid both in vitro and in vivo. As discussed herein, RNAi agents may contain one or more 5'-cyclophosphonic acid modified nucleotides.

[0329] In some embodiments, the RNAi agents containing 5'-cyclophosphonic acid-modified nucleotides disclosed herein are double-stranded. For double-stranded RNAi agents, the lengths of the RNAi agent and antisense strand described herein are independently 16 to 30 nucleotides long. In some embodiments, the double-stranded RNAi agent comprises a sense strand and an antisense strand that are at least partially complementary (at least 70% complementary) to each other. The antisense strand contains a region having a sequence that is fully complementary (100% complementary) or at least substantially complementary (at least 85% complementary) to the sequence of the target mRNA. The lengths of the sense strand and antisense strand of the double-stranded RNAi agent may each be 16 to 30 nucleotides long. The sense strand and antisense strand may be the same length or of different lengths. In some embodiments, the sense strand and antisense strand are independently 17 to 26 nucleotides long. In some embodiments, the sense strand and antisense strand are independently 17 to 21 nucleotides long. In some embodiments, both the sense strand and antisense strand are 21 to 26 nucleotides long. In some embodiments, the sense strand is approximately 19 nucleotides long and the antisense strand is approximately 21 nucleotides long. In some embodiments, the sense strand is approximately 21 nucleotides long and the antisense strand is approximately 23 nucleotides long. In some embodiments, both the sense strand and the antisense strand are 26 nucleotides long. In some embodiments, the RNAi agent sense strand and antisense strand are independently 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides long. In some embodiments, the double-stranded RNAi agent has a double length of approximately 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides. The region of complete or substantially complementary sense and antisense strands is typically 15–25 nucleotides long (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) and appears at or near the 5' end of the antisense strand (e.g., this region is separated from the 5' end of the antisense strand by 1, 2, 3, or 4 nucleotides that are not completely or substantially complementary).In some embodiments, the 5'-cyclophosphonic acid-modified nucleotide is the 5' terminal nucleotide of the antisense strand of a double-stranded RNAi agent.

[0330] In some embodiments, the RNAi agent containing a 5'-cyclophosphonic acid-modified nucleotide is a single-stranded antisense oligonucleotide. In some embodiments, the length of each single-stranded antisense oligonucleotide is independently about 8 to about 40 nucleotides.

[0331] In some embodiments, the RNAi agent containing a 5'-cyclophosphonic acid-modified nucleotide is a double-stranded molecule having a sense strand and an antisense strand. In some embodiments, the RNAi agent containing a 5'-cyclophosphonic acid-modified nucleotide is a single-stranded antisense oligonucleotide.

[0332] In some embodiments, 5'-cyclophosphonic acid-modified nucleotides bind to the terminus of the RNAi agent, enhancing its nuclease stability. In some embodiments, RNAi agents disclosed herein, comprising 5'-cyclophosphonic acid-modified nucleotides at their terminus, can, upon delivery to cells, repress or knock down the expression of target genes in vitro or in vivo through the biological process of RNA interference (RNAi).

[0333] For double-stranded RNAi agents, the sense strand and / or antisense strand may optionally and independently contain an additional 1, 2, 3, 4, 5, or 6 nucleotides (extended) at the 3' end, 5' end, or both the 3' and 5' ends of the core sequence. If present, the additional sense strand nucleotides may be identical or different from the corresponding sequence of the target mRNA. If present, the additional sense strand nucleotides may be complementary or non-complementary to the corresponding additional nucleotides of the sense strand. For single-stranded RNAi agents, if additional nucleotides are present, they may be complementary or non-complementary to the corresponding sequence of the target mRNA.

[0334] In some embodiments, the sense and antisense strands of a double-stranded RNAi agent containing the terminal 5'-cyclophosphonic acid modified nucleotides described herein contain the same number of nucleotides. In some embodiments, the sense and antisense strands of the RNAi agent described herein contain different numbers of nucleotides. In some embodiments, the 5' end of the sense strand and the 3' end of the antisense strand of the RNAi agent form a blunt 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 blunt end. In some embodiments, both ends of the RNAi agent form a blunt end. In some embodiments, neither end of the RNAi agent is blunt-terminated. The term “blunt end” as used herein refers to the end of a double-stranded RNAi agent where the terminal nucleotides of the two annealed strands are complementary (form complementary base pairs).

[0335] In some embodiments, the 5' end of the sense strand and the 3' end of the antisense strand of the RNAi agent form frayed ends. In some embodiments, the 3' end of the sense strand and the 5' end of the antisense strand of the RNAi agent form frayed ends. In some embodiments, both ends of the RNAi agent form frayed ends. In some embodiments, neither end of the RNAi agent is a frayed end. As used herein, a frayed end refers to the end of a double-stranded trigger molecule where the terminal nucleotides of the two annealed strands form a pair (i.e., no overhang) but are not complementary (i.e., form a non-complementary pair). As used herein, an overhang is an extension of one or more unpaired nucleotides on one strand of a double-stranded RNAi agent. The unpaired nucleotides may be on the sense strand or the antisense strand and form either a 3' or 5' overhang. In some embodiments, the RNAi agent includes a blunt end and a frayed end, a blunt end and a 5' overhanging end, a blunt end and a 3' overhanging end, a frayed end and a 5' overhanging end, a frayed end and a 3' overhanging end, two 5' overhanging ends, two 3' overhanging ends, a 3' overhanging end and a 5' overhanging end, two frayed ends, or two blunt ends.

[0336] In some embodiments, the RNAi agent comprises at least one nucleotide (also referred to herein as an internucleoside bond) having a modified backbone. In some embodiments, the modified backbone or internucleoside bond is one or more phosphorothioate bonds.

[0337] In some embodiments, the sense strand of the RNAi agent contains 1 to 4 phosphorothioate bonds. In other embodiments, the antisense strand of the RNAi agent contains 1 to 4 phosphorothioate bonds. In some embodiments, both the sense strand and the antisense strand contain 1 to 4 phosphorothioate bonds.

[0338] In some embodiments where the RNAi agent is single-stranded, the RNAi agent includes phosphorothioate bonds for all or substantially all of the modified nucleotides of the nucleotide or molecule.

[0339] In some embodiments, RNAi agents comprising 5'-cyclophosphonic acid-modified nucleotides have the structures of formula I, II, III, IV, V, VI, VII, and / or formula VIII (including all subgroups or species of the formula (e.g., formula Ib-5)) and / or structures i, ii, iii, iv, v, vi, vii, viiii, viiii, ix, x, xi, xii, and / or structure xiii, and are double-stranded. Double-stranded RNAi agents can be formed by annealing an antisense strand with a sense strand. In some embodiments, RNAi agents comprising 5'-cyclophosphonic acid-modified nucleotides are single-stranded oligonucleotides having the structures of formula I, II, III, IV, V, VI, VII and / or VIII (including all subgroups or species of the formula (e.g., formulas Ib-5)) and / or structures i, ii, iii, iv, v, vi, vii, viiii, viiii, ix, x, xi, xii and / or xiii. The RNAi agents described herein are synthesized using methods commonly used in the art.

[0340] In some embodiments, the RNAi agent contains one or more modified nucleotides. “Modified nucleotide” as used herein refers to a nucleotide other than a ribonucleotide (2'-hydroxylnucleotide). Deoxyribonucleotides are considered a type of modified nucleotide as used herein. In some embodiments, at least 50% (e.g., 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 in the RNAi agent are modified. Modified nucleotides as used herein include deoxyribonucleotides, nucleotide mimes, debasalized nucleotides (represented herein as X, Ab), 2'-modified nucleotides, 3'-3' linked (inverted) nucleotides (represented herein as invdN, invN, invn, invX, invAb), non-natural base-containing nucleotides, cross-linked nucleotides, peptide nucleic acids (PNA), and 2',3'-seconucleotide mimes (unlocked nucleic acid base analogs, represented herein as N UNA (or represented as NUNA), loctonucleotide (N in this specification) LNA (represented as NLNA), 3'-O-methoxy(2'-nucleoside-linked) nucleotide (represented as 3'-OMen), 2'-F-arabinonucleotide (represented herein as NfANA or Nf ANAThis includes, but is not limited to, 5'-Me, 2'-fluoronucleotides (represented herein as 5Me-Nf), morpholinonucleotides, vinyl phosphonate deoxyribonucleotides (represented herein as vpdN), vinyl phosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleotides (cPrpN). Examples of 2'-modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2' position of a 5-membered sugar ring) include, but are not limited to, 2'-O-methylnucleotides (represented herein as the lowercase letter "n" in the nucleotide sequence), 2'-deoxy-2'-fluoronucleotides (represented herein as Nf, or also as 2'-fluoronucleotide), 2'-deoxynucleotides (represented herein as dN), 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides (represented herein as NM or 2'-MOE), 2'-aminonucleotides, and 2'-alkylnucleotides. Not all positions of a given compound are uniformly modified. Conversely, two or more modifications may be incorporated into a single RNAi agent or one of its nucleotides. The RNAi agent may be synthesized and / or modified by methods known in the art. Modifications in one nucleotide are independent of modifications in another nucleotide.

[0341] Modified nucleotides include synthetic and natural nucleic acid bases, 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, adenine and guanine 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl or 2-n-butyl) and other alkyl groups of adenine and guanine. Examples include ulo derivatives, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azocymine, 5-uracil (pseudracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoroguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

[0342] In some embodiments, all or substantially all of the nucleotides of the RNAi agent are modified nucleotides.

[0343] In some embodiments, one or more nucleotides in the RNAi agent are ribonucleotides. Ribonucleotides are referred to herein as "N" (uppercase letter without further symbols).

[0344] The nucleotides of the RNAi agents described herein may be linked by phosphate-containing or non-phosphate-containing covalent nucleoside bonds. Examples of modified nucleoside bonds or skeletons include, but are not limited to, 5'-phosphorothioate groups (lowercase "s" before nucleotides, e.g., sN, sn, sNf, or sdN), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkylphosphonates (e.g., methylphosphonate or 3'-alkylenephosphonate), chiral phosphonates, phosphinates, phosphoramidic acids (e.g., 3'-aminophosphoramic acid, aminoalkylphosphoramic acid, or thiophosphoramic acid), thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino bonds, boranophosphates having a normal 3'-5' bond, a 2'-5' bond analogue of boranophosphate, or boranophosphates with inverted polarity where adjacent pairs of nucleic acid units are bonded together in a 3'-5' to 5'-3' or 2'-5' to 5'-2' configuration. In some embodiments, the modified nucleoside bond or skeleton lacks a phosphate atom. Examples of modified nucleoside bonds lacking a phosphate atom include, but are not limited to, short-chain alkyl or cycloalkyl sugar bonds, mixed heteroatoms and alkyl or cycloalkyl sugar bonds, or one or more short-chain heteroatoms or heterocyclic sugar bonds. Examples of modified nucleoside skeletons in some embodiments include, but are not limited to, siloxane skeletons, sulfide skeletons, sulfoxide skeletons, sulfone skeletons, formacetyl and thioformacetyl skeletons, methyleneformacetyl and thioformacetyl skeletons, alkene-containing skeletons, sulfamic acid skeletons, methyleneimino and methylenehydrazino skeletons, sulfonic acid and sulfonamide skeletons, amide skeletons, and other skeletons having mixed N, O, S and CH2 components.

[0345] In some embodiments, RNAi agents containing 5'-cyclophosphonic acid-modified nucleotides suppress the expression of target mRNA in cells, cell populations, tissues, or of interest. In some embodiments, a therapeutically effective dose of the RNAi agent containing 5'-cyclophosphonic acid-modified nucleotides is administered to the target, thereby suppressing the expression of target mRNA.

[0346] In some embodiments, the RNAi agents described are used to treat, prevent, or manage clinical symptoms associated with the expression of a target mRNA. In some embodiments, one or more RNAi agents are administered in a therapeutic or prophylactic dose to a subject in need of treatment, prevention, or management.

[0347] The RNAi agents and methods comprising the 5'-cyclophosphonic acid-modified nucleotides described can be used to treat or prevent at least one symptom of a disease or disorder in a subject that benefits from reducing or suppressing the expression of a target mRNA. In some embodiments, the subject is administered a therapeutically effective dose of one or more RNAi agents to treat at least one symptom. In other embodiments, the subject is administered a prophylactically effective dose of one or more RNAi agents to treat at least one symptom.

[0348] In some embodiments, the gene expression level and / or target mRNA level of subjects administered with the target ligand conjugated to the repressive oligomer compound is reduced 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% compared to subjects before administration or subjects not administered with the target ligand complex. The gene expression level and / or mRNA level of the subjects may be reduced in the target cells, cell populations, and / or tissues. In some embodiments, the protein levels of subjects administered with the described target ligand conjugated to the repressive oligomer compound are reduced 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%, compared to subjects before administration of the target ligand complex or subjects not administered the target ligand complex. The protein levels of the subjects may be reduced in the target cells, cell populations, and / or tissues. The reduction in gene expression, mRNA, or protein levels can be assessed by methods known in the art. The reduction or decrease in mRNA levels and / or protein levels is expressed herein collectively as repression, reduction, or decrease in the expression of the target gene.

[0349] RNAi agents disclosed herein, comprising 5'-cyclophosphonic acid-modified nucleotides, and compositions comprising such RNAi agents can be delivered to cells, cell populations, tumors, tissues, or subjects using oligonucleotide delivery techniques known in the art. Generally, suitable methods recognized in the art for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with RNAi agents comprising one or more 5'-cyclophosphonic acid-modified nucleotides as described herein. For example, they can be administered topically (e.g., by direct injection, transplantation, or local administration), systemically, or by subcutaneous, intravenous, oral, intraperitoneal, parenteral routes, intracranial (e.g., intraventricular, intraparenchymal, and subarachnoid), intramuscular, percutaneous, airway (aerosol), nasal, rectal, or topically (buccal and sublingual). In certain embodiments, the compositions are administered by subcutaneous or intravenous infusion or injection.

[0350] In some embodiments where the RNAi agent is double-stranded, the RNAi agent may contain a non-nucleotide group that binds to either the 3' or 5' end of the sense strand or the antisense strand. In some embodiments, the target ligand or target group, binding group, or delivery vehicle is covalently bound to the sense strand. In some embodiments, the target ligand, binding group, and / or delivery vehicle binds to the 3' and / or 5' ends of the sense strand. In some embodiments, the target ligand, binding group, and / or delivery vehicle binds to the 5' end of the sense strand. In some embodiments, the target ligand, binding group, and / or delivery vehicle binds directly or indirectly to the 3' and / or 5' ends of the sense strand by a linker. In some embodiments, the target ligand binds to the RNAi agent by an unstable, cleavable, or reversible bond or a linker / spacer.

[0351] In some embodiments where the RNAi agent is single-stranded, the RNAi agent may include a target ligand or target group, binding group, or delivery vehicle that binds to the end where the terminal 5'-cyclophosphonic acid modified nucleotide is absent. In some embodiments where the RNAi agent is single-stranded, the 5'-cyclophosphonic acid modified nucleotide binds to the 5' end of the RNAi agent, and the target ligand, binding group, or delivery vehicle binds to the 3' end of the RNAi agent.

[0352] In some embodiments, a delivery vehicle can be used to deliver RNAi agents to cells or tissues. The delivery vehicle is a compound that improves the delivery of RNAi agents to cells or tissues. Examples of delivery vehicles include, but are not limited to, polymers, such as amphiphilic polymers, membrane-active polymers, peptides, melittin peptides, melittin-like peptides, lipids, reversibly modified polymers or peptides, or reversibly modified membrane-active polyamines.

[0353] RNAi agents containing 5'-cyclophosphonic acid-modified nucleotides can be combined with lipids, nanoparticles, polymers, liposomes, micelles, dynamic polycomplexes (DPCs), or other delivery systems available in the art. RNAi agents can also be chemically coupled with target groups or target moieties, lipids (including, but not limited to, cholesterol or cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, for example, International Publication No. 2000 / 053722, International Publication No. 2008 / 0022309, International Publication No. 2011 / 104169, International Publication No. 2012 / 083185, International Publication No. 2013 / 032829, International Publication No. 2013 / 158141, respectively, as incorporated herein by reference), or other delivery systems available in the art.

[0354] In some embodiments, one or more 5'-cyclophosphonic acid-modified nucleotides bound to the RNAi agent are included in a pharmaceutical composition for in vivo delivery to cells. Such pharmaceutical compositions include, but are not limited to, RNAi agents comprising one or more 5'-cyclophosphonic acid-modified nucleotides bound to a delivery polymer to form an RNAi trigger delivery polymer complex. In some embodiments, the delivery polymer is a membrane-active polyamine. In some embodiments, the delivery polymer is a reversibly modified membrane-active polyamine.

[0355] In some embodiments, the target ligand or target group is a galactose cluster that binds to an RNAi agent comprising one or more 5'-cyclophosphonic acid-modified nucleotides. In some embodiments, the RNAi agent described herein binds to a galactose cluster. When used herein, a galactose cluster comprises a molecule having 2 to 4 terminal galactose derivatives. When used herein, a terminal galactose derivative comprises both galactose and a derivative of galactose having an affinity equal to or greater than that of galactose for asialoclycoprotein receptors. Terminal galactose derivatives are typically bound to the molecule by a C-1 carbon. In some embodiments, a galactose cluster has three terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine) each having an affinity for asialoclycoprotein receptors. In some embodiments, a galactose cluster has three terminal N-acetyl-galactosamines. Other terms common in the art include triantennae galactose, trivalent galactose, and galactose trimer. It is known that triantennae galactose derivative clusters bind to ASGPr, which has a higher affinity than biantennae or monoantennae galactose derivative structures (Baenziger and Fiete, Cell, 1980, 22, 611-620; Connolly et al., J. Biol. Chem. 1982, 257, 939-945).

[0356] In some embodiments, the galactose cluster comprises three galactose derivatives, each bound to a central branch point. In some embodiments, the galactose cluster comprises four galactose derivatives, each bound to a central branch point. The galactose derivatives are bound to the central branch point by the C-1 carbon of the sugar. In some embodiments, the galactose derivatives are bound to the branch point by a linker or spacer.

[0357] In some embodiments, the galactose derivative includes N-acetyl-galactosamine (GalNAc or NAG). Other polysaccharides having affinity for asialocrypoprotein receptors are selected from a list including galactose, galactosamine, N-formyl-galactosamine, N-propionyl-galactosamine, Nn-butanoylgalactosamine, and N-iso-butanoylgalactosamine. The affinity of many galactose derivatives for asialocrypoprotein receptors has been studied (see, e.g., Iobst, ST and Drickamer, KJBC 1996, 271, 6686) or is well known in the art and can be readily measured using commonly used methods.

[0358] Known target ligands suitable for targeted RNAi agents containing 5'-cyclophosphonic acid-modified nucleotides are well known, for example, in U.S. Patent Nos. 14 / 452,626, 15 / 452,324, 15 / 452,423, and 62 / 415,752, the contents of which are incorporated herein by reference.

[0359] Pharmaceutical compositions and preparations The RNAi agents and other oligomeric compounds, comprising 5'-cyclophosphonic acid-modified nucleotides disclosed herein, can be used to treat subjects (e.g., humans or animals, e.g., apes, monkeys, pigs, sheep, goats, cattle, horses, dogs, cats, rabbits, rats, or mice) with diseases or disorders that are expected to benefit from administration of the compound. In some embodiments, at least one of the RNAi agents described herein, comprising 5'-cyclophosphonic acid-modified nucleotides, is used when preparing a pharmaceutical composition (i.e., a drug) to treat a subject that is expected to benefit from reducing or suppressing gene expression. These pharmaceutical compositions are useful for suppressing gene expression in cells, tissues, or organisms. In some embodiments, the pharmaceutical compositions described herein are used to treat a subject with diseases or disorders that are expected to benefit from reducing or suppressing gene expression.

[0360] In some embodiments, RNAi agents comprising 5'-cyclophosphonic acid-modified nucleotides(s) can be used to treat subjects (e.g., humans) with a disease or disorder in which it is expected that reducing or suppressing the expression of a target mRNA would be beneficial. The subject is administered a therapeutically effective dose of one or more RNAi agents. The subject may be a human, a patient, or a human patient. The subject may be an adult, adolescent, child, or infant. The pharmaceutical compositions described herein, comprising a target ligand bound to an expression-repressing oligomer compound, can be used to provide a method for treating a disease. Such a method may involve administering the pharmaceutical compositions described herein to a human or animal.

[0361] Accordingly, in some embodiments, the pharmaceutical compositions described herein may contain one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions described herein can be formulated for administration to a subject.

[0362] A pharmaceutical composition or drug, when used herein, contains a pharmacologically effective amount of the RNAi agent and / or RNAi agent complex described herein, and one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance other than the active pharmaceutical ingredient (API, therapeutic product, e.g., RNAi agent or RNAi agent trigger) that has been appropriately evaluated for safety and is intentionally included in the drug delivery system. Excipients are not intended to exert, or produce, a therapeutic effect at the intended dose. Excipients may act to a) assist in the processing of the drug delivery system during manufacturing, b) protect, support, or enhance the stability, bioavailability, or patient tolerance of the API, c) assist in product identification, and / or d) improve one or more of the overall safety, efficacy, or delivery of the API during storage or use. A pharmaceutically acceptable excipient may be an inactive substance or not.

[0363] Excipients include, but are not limited to, absorption enhancers, anti-tacks, defoamers, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, dextran, glucose, diluents, disintegrants, emulsifiers, bulking agents, fillers, fragrances, lubricants, water-retaining agents, oils, polymers, preservatives, physiological saline, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, viscosity enhancers, isotonic agents, vehicles, water-repellent agents, and wetting agents.

[0364] A pharmaceutical composition may include other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to, antipruritic agents, astringents, topical anesthetics, anti-inflammatory agents, and antihistamines (e.g., diphenhydramine, deoxylamine, acribastine, or cetirizine). Cells, tissues, or isolated organs expressing or containing RNAi triggers comprising 5'-cyclophosphonic acid-modified nucleotides as defined herein may also be used as a "pharmaceutical composition." "Pharmacologically effective amount," "therapeutic effective amount," or simply "effective amount" refers to the amount of RNAi agent that, when used herein, produces the intended pharmacological, therapeutic, or prophylactic effect.

[0365] In some embodiments, the RNAi trigger described herein is used in combination with one or more additional therapeutic agents, or a second RNAi trigger or other therapeutic agents, including but not limited to small molecule drugs, antibodies, antibody fragments, and / or vaccines.

[0366] RNAi triggers containing 5'-cyclophosphonic acid-modified nucleotides and pharmaceutical compositions containing RNAi triggers disclosed herein may be packaged in or contained in kits, containers, packs, or dispensers. RNAi triggers and pharmaceutical compositions containing RNAi triggers may be packaged in pre-filled syringes or vials.

[0367] Cells, tissues, and non-human organisms comprising at least one RNAi trigger containing a 5'-cyclophosphonic acid-modified nucleotide as described herein are considered. Cells, tissues, and non-human organisms are prepared by delivering the RNAi trigger to the cells, tissues, or non-human organisms by methods available in the art. In some embodiments, the cells are mammalian cells, but not limited to human cells. The cells or non-human organisms are useful for investigations or as investigation tools (e.g., drug testing or diagnostics).

[0368] In some embodiments, RNAi agents comprising an RNAi trigger containing a 5'-cyclophosphonic acid-modified nucleotide are used to treat subjects with a disease or disorder in which it is expected that they would benefit from reducing or suppressing the expression of a target gene. In some embodiments, the RNAi agents described herein are used to treat or prevent a subject with a disease or disorder in which it is expected that they would benefit from reducing or suppressing the expression of a target gene. The subject is administered a therapeutically effective dose of one or more RNAi agents described herein, thereby treating the symptoms.

[0369] In some embodiments, an RNAi agent comprising a 5'-cyclophosphonic acid-modified nucleotide is used to treat or manage a clinical condition, and to administer one or more of the RNAi agents described herein in therapeutic or prophylactic doses to subjects requiring treatment, prevention, or management. In some embodiments, the method includes administering the RNAi trigger molecules described herein to a mammal, such as a human, for treatment.

[0370] In some embodiments, RNAi triggers containing 5'-cyclophosphonic acid-modified nucleotides can be used to suppress the expression of target genes in target cells, cell populations, or tissues. In some embodiments, RNAi triggers can be used to formulate compositions for, for example, suppressing the expression of target genes in target cells, cell populations, or tissues. In some embodiments, a therapeutically effective dose of one (or more) RNAi agents described herein is administered to a target to suppress the expression of the target gene (e.g., an effective dose to suppress the expression of the target gene).

[0371] In some embodiments, the gene expression level and / or target mRNA level of subjects administered with the RNAi trigger comprising a 5'-cyclophosphonic acid-modified nucleotide is reduced 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% compared to subjects before administration or subjects not administered the RNAi trigger. The gene expression level and / or mRNA level of subjects may be reduced in the cells, cell populations, and / or tissues of the subjects. In some embodiments, the protein levels of subjects administered with the RNAi trigger comprising a 5'-cyclophosphonic acid-modified nucleotide are reduced 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% compared to subjects before administration or subjects not administered the RNAi trigger. The protein levels of the subjects may be reduced in the cells, cell populations, and / or tissues of the subjects. The reduction in gene expression, mRNA, or protein levels can be assessed by methods known in the art. The reduction or decrease in mRNA levels and / or protein levels is expressed herein collectively as suppression, reduction, or decrease in the expression of the target gene or target mRNA.

[0372] The route of administration is a route through which the RNAi trigger containing the 5'-cyclophosphonic acid-modified nucleotide described herein is brought into contact with the body. In general, methods for administering drugs and nucleic acids for the treatment of a target are known in the art and can be applied to the administration of the compositions described herein. The compounds described herein can be administered by appropriate routes of preparations appropriately tailored to a specific route. Thus, the compounds described herein can be administered, for example, by intravenous, intramuscular, intradermal, percutaneous, or intraperitoneal injection.

[0373] In some embodiments, the RNAi trigger molecules or compositions described herein can be delivered to cells, cell populations, tissues, or subjects using oligonucleotide delivery techniques known in the art. Generally, suitable methods recognized in the art for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with RNAi triggers comprising 5'-cyclophosphonic acid-modified nucleotides described herein. For example, they can be delivered by topical administration (e.g., direct injection, transplantation, or local administration), systemic administration, or by subcutaneous, intravenous, oral, intraperitoneal, parenteral routes, intracranial (e.g., intraventricular, intraparenchymal, and subarachnoid), intramuscular, percutaneous, airway (i.e., aerosol), nasal, rectal, or topical (buccal and sublingual) administration. In certain embodiments, the compositions are administered by subcutaneous or intravenous infusion or injection.

[0374] Generally, the effective dose of the active compound is in the range of approximately 0.1 to 100 mg / kg of body weight per day, for example, approximately 1.0 to 50 mg / kg of body weight per day. In some embodiments, the effective dose of the active compound is in the range of approximately 0.25 to 5 mg / kg of body weight per dose. In some embodiments, the effective dose of the active ingredient is in the range of approximately 0.5 to 3 mg / kg of body weight per dose. The dosage may also depend on variables such as the patient's health condition, the relative biological efficacy of the compound being delivered, the formulation of the drug, the presence and type of excipients in the formulation, and the route of administration. Furthermore, the initial dose may be increased beyond the upper limit to rapidly achieve the desired blood or tissue level, or the initial dose may be smaller than the optimal dose.

[0375] The pharmaceutical compositions described herein, which include an expression-inhibiting oligomer compound such as an RNAi agent containing one or more 5'-cyclophosphonic acid-modified nucleotides for the treatment of a disease or for forming a pharmaceutical or composition for the treatment of a disease, can be combined with a second or other expression-inhibiting oligomer compound, small molecule drug, antibody, antibody fragment and / or vaccine, etc., but are not limited to these.

[0376] The RNAi agent described herein, comprising one or more 5'-cyclophosphonic acid-modified nucleotides, can be packaged in kits, containers, packs, or dispensers when added to pharmacologically acceptable excipients or adjuvants. The pharmaceutical compositions described herein may be packaged in pre-filled syringes or vials.

[0377] The embodiments and items provided above will be explained in the following non-limiting examples. [Examples]

[0378] The following examples are intended to illustrate specific embodiments disclosed herein, and are not limiting.

[0379] Some of the abbreviations used in the details of the following experiments for the synthesis of the examples are defined below: h or hr = hours; min = minutes; mol = moles; mmol = millimoles; M = molar concentration; μM = micromoles; g = grams; μg = micrograms; rt or RT = room temperature; L = liters; mL = milliliters; wt = weight; Et2O = diethyl ether; THF = tetrahydrofuran; DMSO = dimethyl sulfoxide; Â = ethyl acetate; Et3N or TEA = triethylamine; i-Pr2NEt or DIPEA or DIEA = diisopropylethylamine; CH2CI2 or DCM = methylene chloride; CHCl3 = chloroform; CDCl3 = deuterated chloroform; CCl4 = carbon tetrachloride; MeOH = methanol; EtOH = ethanol; DMF = dimethylformamide; BOC = t-butoxycarbonyl; CBZ = benzyloxycarbonyl; TB S = t-butyldimethylsilyl; TBSCl or TBDMSCl = t-butyldimethylsilyl chloride; 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 carbon; HCl = hydrogen chloride or hydrochloric acid; NMM = N-methylmorpholine; H2 = hydrogen gas; KF = potassium fluoride; EDC-HCl = N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; MTBE = methyl-tert-butyl ether; MeOH = methanol; Ar = argon; N2 = nitrogen; SiO2 = silica; RT = retention time.

[0380] Furthermore, examples of RNAi agents suitable for use with the 5'-cyclophosphonic acid-modified nucleotides disclosed herein are described in various tables of the following examples.

[0381] The following symbols are used to indicate modified nucleotides for the sequences described in the table disclosed herein. Those skilled in the art will readily understand that, when present in oligonucleotides, unless otherwise specified, monomers are linked to each other by 5'-3'-phosphodiester bonds. N=2'-OH (unmodified) ribonucleotide (uppercase letter without f or d) n=2'-OMe modified nucleotide Nf=2'-Fluoro-modified nucleotide dN=2'-deoxynucleotide N UNA =2',3'-seconucleotide mimetics (unlocked nucleic acid base analogs) N LNA =locked nucleotide Nf ANA =2'-F-arabinonucleotide NM = 2'-methoxyethylnucleotide X or Ab = non-basic ribose R = Ribitol (invdN) = inverted deoxyribonucleotide (3'-3' linked nucleotide) (invAb) = inverted non-base nucleotide (invX) = inverted non-base nucleotide (invn) = inverted 2'-OMe nucleotide s = phosphorothioate-linked nucleotide vpdN = vinyl deoxyribonucleotide phosphonate (3'OMen)=3'-OMenucleotide (5Me-Nf) = 5'-Me,2'-fluoronucleotide cPrp = Cyclopropyl phosphonate

[0382] The compounds of this disclosure can be prepared using synthetic chemical techniques known in the art and described herein.

[0383] Example 1: Synthesis of Compound 4 (dimethyl[2-[(2R,3R,4R,5R)-3-([[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl]oxy)-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-1-yl)oxolan-2-yl]cyclopropyl]phosphonate)

[0384] A. Synthesis of Compound 2 (dimethyl[2-[(2R,3R,4R,5R)-3-[(tert-butyldiphenylsilyl)oxy]-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-l,2,3,4-tetrahydropyrimidine-l-yl)oxolan-2-yl]cyclopropyl]phosphonate)

[0385] [ka]

[0386] 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-tetrahydropyrimidine-1-yl)oxolan-2-yl]ethenyl]phosphonate) was synthesized following the same procedure as Whittaker, B. et al., Tetrahedron Lett. 49, 6984-6987 (2008) and Abbas, S. et al., Org. Lett. 3(21), 3365-3367 (2001).

[0387] A 1000 mL three-necked round-bottom flask, purged and held under a nitrogen inactivating atmosphere, was filled with a solution of sodium hydride in dimethyl sulfoxide (500 mL) (8.2 g, 341.67 mmol, 3.00 equivalent). Next, a solution of trimethylsulfoxonium iodide (45 g, 204.48 mmol, 3.00 equivalent) was added. The resulting solution was stirred at 25°C for 0.5 hours. Then, a solution of compound 1 in dimethyl sulfoxide (50 mL) (45 g, 68.31 mmol, 1.00 equivalent) was added dropwise while stirring at 25°C. The resulting solution was stirred at 25°C for 20 hours. The reaction mixture was quenched by adding 50 mL of saturated ammonium chloride aqueous solution.

[0388] The obtained solution was extracted with 3 × 1000 mL of dichloromethane, and the organic layer was combined with it. The combined organic material was washed with 1 × 2000 mL of saturated sodium chloride aqueous solution. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was flash-chromatographed on silica gel and eluted with dichloromethane / methanol (100:1-10:1). 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-tetrahydropyrimidine-1-yl)oxolan-2-yl]cyclopropyl]phosphonate) was obtained as a white solid. (HNMR:(CDCb,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),3.70 -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))

[0389] 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-tetrahydropyrimidine-1-yl)oxolan-2-yl]cyclopropyl]phosphonate)

[0390] [ka]

[0391] A 100 mL three-necked round-bottom flask, purged and held under a nitrogen inactivating atmosphere, was filled with tetrahydrofuran (60 mL) and 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-tetrahydropyrimidine-1-yl)oxolan-2-yl]cyclopropyl]phosphonate) (6.0 g, 8.92 mmol, 1.00 equivalent) from triethylamine (4.18 g, 41.31 mmol, 3.00 equivalent). Triethylamine trihydrofluoride (13.34 g, 82.86 mmol, 6.00 equivalent) was then added dropwise while stirring at 0°C. The resulting solution was stirred at 25°C for 18 hours. The resulting mixture was concentrated under vacuum and diluted with 60 mL of dichloromethane.

[0392] The obtained solution was washed with 100 mL of saturated sodium bicarbonate aqueous solution and 100 mL of saturated sodium chloride aqueous solution. The organic solution was dried over sodium sulfate, filtered, and concentrated under vacuum. The residue was flash-chromatographed on silica gel and eluted with dichloromethane / methanol (100:1-10:1). 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-tetrahydropyrimidine-1-yl)oxolan-2-yl]cyclopropyl]phosphonate) was obtained as a white solid. (LC-MS:(ES,m / z):[M+H] +=435.(H-NMR:(CDCb,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)).

[0393] C. Synthesis of compound 4 (dimethyl[2-[(2R,3R,4R,5R)-3-([[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl]oxy)-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-1-yl)oxolan-2-yl]cyclopropyl]phosphonate)

[0394] [ka]

[0395] A 50 mL three-necked round-bottom flask, purged and held under a nitrogen inactivating atmosphere, was filled with dichloromethane (50 mL) and compound 3 (dimethyl[2-[(2R,3R,4R,5R)-3-hydroxy-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-1-yl)oxolan-2-yl]cyclopropyl]phosphonate) (2.5 g, 5.76 mmol, 1.00 equivalent) from 4,5-dicyanoimidazole (810 mg, 6.86 mmol, 1.20 equivalent). Next, 3-(bis(diisopropylamino)phosphinoxy)propanenitrile (2.25 g, 7.46 mmol, 1.30 equivalent) was added dropwise while stirring at 0°C. The resulting solution was stirred at 25°C for 3 hours. The resulting solution was diluted with 25 mL of dichloromethane.

[0396] The obtained solution was washed with 2 × 50 mL saturated sodium bicarbonate aqueous solution and 50 mL saturated sodium chloride aqueous solution. The organic solution was dried over sodium sulfate, filtered, and concentrated under vacuum. The residue was flash-chromatographed on silica gel and eluted with dichloromethane / ethyl acetate (5:1-1:5) (containing 0.5% triethylamine). 2.1 g (57%) of compound 4 (dimethyl[2-[(2R,3R,4R,5R)-3-([[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl]oxy)-4-(2-methoxyethoxy)-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-1-yl)oxolan-2-yl]cyclopropyl]phosphonate) was obtained 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=2.8H z,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, 3H).P-NMR:(CD3COCD3,161MHz,ppm):δ149.67,149.51,149.21,31.59,31.55,31.41,31.29.)

[0397] Compound 4 described above is a phosphoramidite compound that can be used to form the termini of double-stranded RNAi agents and / or single-stranded antisense oligonucleotides by adding a 5'-cyclopropylphosphonic acid-2'-MOE modified nucleotide. As a general subject, phosphoramidites that can be used to form the termini of the disclosed double-stranded RNAi agents and / or single-stranded antisense oligonucleotides can be produced by adding a 5'-cyclophosphonic acid modified nucleotide using a similar synthetic process. For example, those skilled in the art will recognize and understand that Compound 1 of Example 1 can be synthesized with different groups at the 2' position, such as a 2'-F, 2'-H, or 2'-O-methyl group. Similarly, as a non-limiting example, those skilled in the art will recognize that different heterocyclic base moieties (e.g., uracil, cytosine, guanine, 5-methylcytosine, etc.) can be used instead of thymine shown in Example 1.

[0398] Example 2: Synthesis of Compound 15 (2-cyanoethyl((2R,3R,4R,5R)-2-(2-(diethoxyphosphoryl)cyclopropyl)-5-(2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl)diisopropylphosphoramidite)

[0399] 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)

[0400] [ka]

[0401] Commercially available compound 5 was purchased. DMT-Cl (276 g, 813 mol) was added to a solution of compound 5 (200 g, 775 mmol) in pyridine (1.5 mL). The reaction mixture was stirred at 25°C for 8 hours under an N2 atmosphere. TLC (petroleum ether / ethyl acetate = 1 / 1, Rf = 0.2) indicated that the reaction was complete. The solvent was removed under reduced pressure. The residue was diluted with HCl (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 relative to ethyl acetate) to obtain compound 6 (448 g, containing HCl) as a pale yellow rubber. 1 H NMR:400MHz CDCl39.01(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))

[0402] 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)

[0403] [ka]

[0404] To a solution of compound 6 (448 g, 799 mmol) in DCM (2.8 L), imidazole (163 g, 2.4 mol) and TBSCl (241 g, 1.6 mol) were added. The reaction mixture was stirred at 25°C for 8 hours. 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 obtain compound 7 (620 g, crude) as a colorless rubber. The crude product was used in the next step without further purification.

[0405] 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)

[0406] [ka]

[0407] A mixture of compound 7 (620 g, 919 mmol) and AcOH (4 L) in H2O (1 L) was degassed, purged three times with N2, and then stirred at 25°C for 16 hours under an N2 atmosphere. TLC (petroleum ether / ethyl acetate = 1 / 1, Rf = 0.2) indicated 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 the pH was 7-8, and then diluted with ₹ (5 L × 3). The combined organic layer was 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 = 5:1-1:1) to obtain compound 8 (296 g, yield 87%) as a white solid. 1H NMR:400MHz CDCl3.δ9.27(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. 09(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)).

[0408] D. Synthesis of compound 9((2S,3S,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-methoxytetrahydrofuran-2-carbaldehyde)

[0409] [ka]

[0410] A solution of compound 8 (60 g, 161 mmol) in DCM (3 L) was mixed with 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 hour, and then at 25°C for 3 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, Rf = 0.16) indicated that the reaction was complete. The resulting mixture was quenched with a 4 L solution of saturated Na2S2O3 and saturated NaHCO3 (1:1), and then extracted with SiO3 (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 obtain compound 9 (48 g, crude) as an orange oil. The crude product was used in the next step without further purification.

[0411] E. Synthesis of Compound 10

[0412] [ka]

[0413] 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 minutes, then cooled to 5°C. The solution was treated with compound 10-2 (110 g, 583 mmol) and heated while maintaining 25°C. The solution was cooled to 5°C, treated with additional LiHMDS (1M in THF, 723 mL), and heated to 2°C. The solution was quenched at 5°C by adding H2O (50 mL) and diluted with ₹ (1.5 L). The organic layer was separated, dried over Na2SO4, and concentrated under reduced pressure to obtain compound 10-3 as an oil. The crude oil was dissolved in THF (2.4 L) and cooled to 5°C. The solution was treated with NaH (57 g, 1.43 mol, 60% dispersed in mineral oil) and heated to 25°C. The slurry was stirred at 25°C for 18 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, Rf = 0.3) indicated that the reaction was complete. NaH was removed using a Celite pad, and the cake was washed with THF (600 mL x 2). The filtrate was concentrated to obtain compound 10 (250 g, crude) as a yellow oil.

[0414] F. Synthesis of compound 11 (O,O-diethyl((E)-2-((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl)vinyl)phosphonothioate)

[0415] [ka]

[0416] A solution of compound 9 in THF (1.6 L) (165 g, 445 mmol) was added dropwise to a solution of compound 10 in THF (0.8 L) (250 g, 583 mmol) at 5°C. The reaction mixture was then warmed to 25°C and stirred for 1 hour. TLC (petroleum ether / ethyl acetate = 1 / 1, Rf = 0.6) indicated completion of the reaction. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10:1~1:1) to obtain compound 11 as a clear oil (89 g, purity 85%; 72 g, purity 65%; 59 g, purity 48%, yield 65%). (Impurities were identified as triphenylphosphine oxide (Ph3P = 0)). 1 H NMR:400MHz CDCl3δ8.46(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))

[0417] G. Synthesis of compound 12(O,O-diethyl(2-((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl)cyclopropyl)phosphonothioate)

[0418] [ka]

[0419] To a solution of Me3SOI (95.9 g, 436 mmol) in DMSO (800 mL), sodium hydroxide (17.4 g, 436 mmol, 60% dispersed in mineral oil) was added as a THF slurry. The resulting white, foamy slurry was stirred at 25°C for 15 minutes, and the resulting white solution was packed into a flask containing compound 11 (89 g, 85% purity, 145 mmol). The temperature was raised to 22-26°C, the yellow solution was stirred for 2 hours, and then heated to 50°C for 2 hours. LC-MS indicated that the reaction was complete. The reaction mixture was cooled to 5°C, quenched with ice, and the internal temperature was maintained below 30°C. The product was extracted with ethyl acetate (3 L), and the organic layer was washed with water (5 × 1 L). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 12 (206 g, crude) as a white solid. (The impurity was Ph3P=O, and the crude product was used in the next step without further purification.)

[0420] Synthesis of compound 13 (O,O-diethyl(2-((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)cyclopropyl)phosphonothioate)

[0421] [ka]

[0422] To a stirred solution of compound 12 (206 g, 385 mmol) in THF (1.3 L), 12 M HCl (340 mL, 4.1 mol) was added dropwise. The resulting mixture was stirred at 25°C for 1.5 hours. TLC (DCM / ethyl acetate = 1 / 1, Rf = 0.2) indicated that the reaction was complete. The reaction mixture was cooled in an ice bath and quenched with a saturated sodium bicarbonate solution until the pH became 8. The product was extracted with RINKAN (3.9 L × 2), and the combined organic layers were washed with brine (1.3 L). The organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was subjected to column chromatography (S₂). iThe compound was purified using O2, DCM / ethyl acetate (1:1 ratio) to obtain compound 13 (76 g, 47% yield) as a white solid. 1 H NMR:400MHz CDCl3δ8.37(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))

[0423] I. Synthesis of Compound 14 (Diethyl(2-((2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-Dihydropyrimidine-1(2H)-yl)-3-Hydroxy-4-Methoxytetrahydrofuran-2-yl)Cyclopropyl)Phosonate)

[0424] [ka]

[0425] A solution of compound 13 (76 g, 181 mmol) in 1.4 L of THF:H2O (1:1) was cooled to 5°C, and Oxone® (194 g, 316 mmol) was added to the solution. The reaction mixture was stirred at 5°C for 2 hours. LC-MS indicated that the reaction was complete. The reaction mixture was diluted with 200 mL of H2O and extracted with 2.5 L of DCM. The organic layer was collected, and the aqueous layer was further extracted with DCM (2.5 L x 4). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, DCM / MeOH=DCM, 1:1) to obtain compound 14 (Cy3P-U-OMe-3-OH) (40 g, crude) as a white solid. Finally, the crude product was separated by preparative HPLC (Gemini150). * 4.6mm (Luna200) * 25mm (C18, 10um, 100Å) + Geminil50 *The compound was purified using 30°C (18°C, 5 μm, 110 Å), 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 CDCl3δ9.54(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,3 H),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))

[0426] J. Synthesis of Compound 15 (2-cyanoethyl((2R,3R,4R,5R)-2-(2-(diethoxyphosphoryl)cyclopropyl)-5-(2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl)diisopropylphosphoramidite)

[0427] [ka]

[0428] To a solution of compound 14 (463 mg, 1.1 mmol) in DCM (6 mL), 4,5-dicyanoimidazole (54 mg, 0.46 mmol) was added, followed by 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (518 mg, 1.7 mmol, 1.5 equivalents) in dichloromethane (3 mL). The reaction mixture was stirred overnight. After confirming that all starting materials had been digested by HPLC, the reaction mixture was concentrated under reduced pressure to obtain a volume of approximately 2 mL. The crude solution was packed into a silica column and purified using a uniform concentration gradient (DCM: Â: triethylamine: methanol (6:4:0.1:0.05), Rf=0.25). Yield: 421 mg (61%) as a mixture of cyclopropyl and phosphoramidite diastereomers (total 4). 1H NMR:400MHz DMSO-d6. δ11.41(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))

[0429] Compound 15 described above is a phosphoramidite that can be used to form the ends of double-stranded RNAi agents and / or single-stranded antisense oligonucleotides by adding a 5'-cyclopropylphosphonic acid-2'-O-Me modified nucleotide. As a general subject, phosphoramidites that can be used to form the ends of the disclosed double-stranded RNAi agents and / or single-stranded antisense oligonucleotides can be produced by adding a 5'-cyclophosphonic acid modified nucleotide disclosed herein using a similar synthetic process. For example, those skilled in the art will recognize and understand that compound 5 of Example 2 can be synthesized with different groups at the 2' position, such as a 2'-F, 2'-deoxy group. Similarly, as a non-limiting example, those skilled in the art will recognize that different heterocyclic base moieties (e.g., thymine, cytosine, guanine, 5-methylcytosine, etc.) can be used instead of uracil as shown in Example 2.

[0430] Example 3: Synthesis of RNAi agents

[0431] The synthesis of the RNAi agent began with the separate synthesis of the sense and antisense strands. The two complementary strands were first synthesized in a solid support resin, subjected to cleavage and deprotection, then purified by either reverse-phase or ion-exchange chromatography, and finally annealed to form the RNAi agent.

[0432] A) Solid-state synthesis. RNAi agents were synthesized on the solid-state used for oligonucleotide synthesis according to phosphoramidite techniques. Depending on the size, commercially available automated oligosynthesizers, e.g., MerMade96E® (Bioautomation), MerMadel2® (Bioautomation), or AKTA Oligopilot (GE), were used. Solid-state synthesis began from the 3' end of the sequence, and phosphoramidite constituent blocks were added sequentially in each synthesis cycle to increase the oligomer in the required order. Each synthesis cycle included four chemical steps: 1) deprotection or detritylation; 2) coupling; 3) oxidation; and 4) capping. Phosphoramidites were derived from nucleotides of either natural, chemically modified, or small molecules such as N-acetylgalactosamine target ligands. Synthesis was performed on solid supports made from controlled porosity glass (CPG, 500 Å or 600 Å, available from Prime Synthesis, Aston, PA, USA). All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). In particular, the following 2'-O-methylphosphoramidite was used: (5'-O-dimethoxytrityl-N 6 -(benzoyl)-2'-O-methyladenosine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino)phosphoramidite, 5'-O-dimethoxy-trityl-N 4 -(acetyl)-2'-O-methylcytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino)phosphoramidite, (5'-O-dimethoxytrityl-N 2 -(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyano-ethyl-N,N-diisopropylamino)phosphoramidite, and 5'-O-dimethoxy-trityl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite. 2'-deoxy-2'-fluoro-phosphoramidite possessed the same protecting group as 2'-O-methylRNAamidite.

[0433] Target ligands containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), and all other amidites were dissolved in anhydrous acetonitrile (50 mM), to which molecular sieves (3 Å) were added. 5-benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) were used as the active solution. Coupling times were 10 minutes (RNA), 15 minutes (target ligand), 90 seconds (2'OMe), and 60 seconds (2'F). To introduce phosphorothioate binding, a 100 mM solution of 3-phenyl 1,2,4-dithiazolin-5-one (POS, Poly Org, Inc., Leominster, MA, USA) in anhydrous acetonitrile was used.

[0434] B) Hydrolysis of 5'-cyclopropylphosphonic acid-modified nucleotides. Hydrolysis of 5'-cyclopropylphosphonic acid-supported oligonucleotides was performed on an AKTA Oligopilot (GE) synthesizer. A mixture of trimethylsilaneiodide / pyridine / acetonitrile (1:13:40 v / v) was passed through the reactor column at a flow rate of 2 mL / min for 30 to 90 minutes, depending on the reaction scale. The column was washed with acetonitrile and removed from the synthesizer. The supported oligonucleotides were transferred to a funnel and washed with acetonitrile / water (1:1 v / v).

[0435] C) Cleavage and deprotection of the supporting oligonucleotide. After the final treatment of solid phase synthesis and deprotection of the 5'-cyclopropylphosphonic acid supporting oligonucleotide, the dried solid support was treated with a 1:1 solution of aqueous methylamine in 40% by weight water and 28% by weight aqueous ammonium hydroxide (Aldrich) at 30°C for 2 hours. The solution was evaporated, and the solid residue was reconstituted in water (see below).

[0436] D) Purification. Crude oligonucleotides were purified by reverse-phase HPLC or ion-exchange chromatography. The fractions containing the product were stored, and the purity and identification of each single-strand was confirmed by LC-MS. The concentration and yield of the oligonucleotides were evaluated using the theoretical extinction coefficient at UV (260 nm).

[0437] E) Annealing. Complementary strands were mixed by combining equimolar solutions of the sense and antisense strands. These solutions were left at ambient temperature or placed in a thermomixer at 70°C, heated to 95°C, held at 95°C for 5 minutes, and slowly cooled to room temperature. Several RNAi agents were lyophilized and stored at -15 to -25°C. Dual concentrations were measured by measuring the solution absorbance at 260 nm using a UV-Vis spectrometer. Unless otherwise noted, all conversion factors were 0.037 mg / (mL·cm). For some experiments, the conversion factors were calculated from experimentally measured extinction coefficients.

[0438] Example 4: Exemplary RNAi agent sequence containing a 5'-cyclophosphonic acid modified nucleotide target LP(a) (human Apo(a) gene)

[0439] The sequences listed in Table 1 below are for illustrative purposes only. The 5'-cyclophosphonic acid-modified nucleotides disclosed herein can be incorporated into RNAi agents or single-stranded antisense oligonucleotide target genes.

[0440] [Table 1]

[0441] The RNAi agents listed in Table 1 above were prepared according to the synthesis described in Example 3. As used in the tables herein, 2'-O-methyl nucleotides are represented by the lowercase letter "n" of the nucleotide sequence, 2'-deoxy-2'-fluoronucleotides by Nf, 2'-deoxy nucleotides by dN, 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides by NM herein, 3'-3' linked (inverted) nucleotides by invdN, invN, invn, invX, or invAb herein, 5'-cyclopropylphosphonates by cPrpN, cPrpn, cPrpNf, cPrpdN, or cPrpNM, with cPrp preceding the nucleotide, non-basic nucleotides (represented herein as Ab), n-acetyl-galactosamine cluster target ligands by NAG, and S'-phosphorothioate groups by sN, sn, sNf, or sdN, with a lowercase letter "s" preceding the nucleotide.

[0442] Example 5: In vivo analysis of RNAi agent sequence target LP(a) (human Apo(a) gene) in transgenic mice

[0443] A) To evaluate the in vivo efficacy of LP(a) RNAi agents, 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 a complete LPA gene with both additional 5' and 3' sequences (encoding the apo(a) protein). On day 1, mice were administered an RNAi agent conjugated to an N-acetyl-galactosamine target ligand attached to the 5' end of the sense strand. Each mouse received either a single subcutaneous (SC) injection of physiological saline (n=4) or individual treatment groups (n=3 for all treatment groups). Control serum (pre-treatment) samples were collected from mice before injection on day 1. Post-injection serum samples were collected from mice on days 8, 15, 22, 29, and 36.

[0444] B) Apo(a) protein levels. Human apo(a) protein levels in serum were monitored by analyzing mouse serum using ELISA for apo(a)(Abeam). For normalization, the apo(a) levels of each mouse at each time point were separated by the pre-treatment level of expression in the mouse (in this case, day -1) to determine the "day -1 normalized" expression ratio. Then, the expression at each time point was normalized to the saline group by dividing the "day -1 normalized" ratio for each individual mouse by the "day -1 normalized" ratio for all mice in the saline control group. This resulted in the expression for each time point normalized to the expression of the control group, as shown in Table 2.

[0445] [Table 2]

[0446] As shown in Table 2 above, the nadir for AD03541 reached day 8, while the nadir for AD03158 did not reach day 22. Furthermore, AD03541 achieved greater knockdown at all time points.

[0447] Example 6: Factor 12 knockdown (KD) in cynomolgus monkeys

[0448] We synthesized an RNAi agent having a sequence directed towards factor 12 (F12) and binding to an N-acetyl-galactosamine target ligand at the 5' end of the sense strand, and combined it in a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection.

[0449] On day 1, 3 mg / kg of either the F12 RNAi agent AD04254 or AD04443 was subcutaneously injected into cynomolgus macaques (Macaca fascicularis) primates. Two monkeys received the treatment group AD04443, and three monkeys received the treatment group AD04254.

[0450] [Table 3]

[0451] As shown in Table 3 above, AD04254 and AD04443 consisted of the same sense strand, and the only difference between the RNAi agents was whether the 5' end of the antisense strand contained a 2'-O-methylu modified nucleotide (AD04254) or a 5'-cyclopropyl acid modified nucleotide that also contained a 2'-O-methylu modification (AD04443).

[0452] Serum samples from treated cynomolgus monkeys were collected on days -29, -7, and 1 (pre-administration), as well as on days 8, 15, 22, and 29, and knockdown was monitored. Knockdown was measured by quantifying circulating cynoF12 protein (cF12) levels in the serum using a human F12 ELISA kit (Molecular Innovations). The cF12 level of each monkey at each time point was divided by the pre-treatment expression level of the monkey (mean of day -29, day -7, and day 1) to determine the "pre-administration normalized" expression ratio.

[0453] Figure 1 shows the normalized relative expression of the mean of cF12. As shown in Figure 1, based on a single dose, the F12 RNAi agent containing the 5'-cyclopropyl acid-modified nucleotide of the present invention at the 5' end of the antisense strand of this sequence shows numerically increased ability and knockdown slightly faster than the same F12 RNAi agent without the 5'-cyclopropyl acid-modified nucleotide.

[0454] Example 7: Factor 12 knockdown (KD) in wild-type mice

[0455] F12 double-stranded RNAi agents were prepared with an N-acetyl-galactosamine target ligand bound to the 5' end of the sense strand. Each double-stranded RNAi agent was directed to F12 and combined in a pharmaceutically acceptable buffer known in the art for subcutaneous (SC) injection.

[0456] F12 RNAi agents (AD04162 and AD04649) were delivered to wild-type mice by subcutaneous injection. On day 1, 200 μl of a solution containing either one RNAi agent at a dose of 0.5 mg / kg (mpk) in physiological saline, one RNAi agent at a dose of 0.5 mg / kg (mpk) in buffered saline, or one RNAi agent at a dose of 1.0 mg / kg (mpk) in buffered saline was subcutaneously injected into the loose skin on the back between the shoulders of 20 g mice. Four wild-type mice were present in each of the five treatment groups.

[0457] [Table 4]

[0458] As shown in Table 4 above, AD04162 and AD04649 consisted of the same sense strand, and the only difference between the RNAi agents was whether the 5' end of the antisense strand contained a 2'-O-methyluracil-modified nucleotide (AD04162) or a 5'-cyclopropylate uracil-modified nucleotide with 2'-deoxy modification (AD04649).

[0459] Serum samples from treated mice were collected on days -1 (pre-administration), 8, 15, 22, and 29 to monitor knockdown. Knockdown was measured by quantifying the circulating mouse F12 protein (mF12) levels in the serum using the internally developed mF12 alphaLISA® (Perkin Elmer). The mF12 level of each mouse at each time point was divided by the pre-treatment expression level to determine the "pre-administration normalized" expression ratio. Then, the expression at a specific time point was normalized to the saline group by dividing the "pre-administration normalized" ratio for each individual mouse by the "pre-administration normalized" ratio of all mice in the saline control group. This obtained the expression for each time point normalized to the control group expression. Experimental errors are shown as standard deviations in Table 5.

[0460] [Table 5]

[0461] As shown in Table 5, these experimental data support the idea that adding the 5'-cyclophosphonic acid-modified nucleotide of the present invention can increase the efficacy of the F12 RNAi agent, particularly at a dose of 1.0 mg / kg, in wild-type mice.

[0462] Example 8: Reduction of HBsAg in pHBV model mice

[0463] The reduction of HBV surface antigen (HBsAg) was evaluated using a pHBV model mouse. Female NOD.CB17-Prkdscid / NcrCrl (NOD-SCID) mice aged 6-8 weeks were transiently transfected in vivo with MC-HBV1.3 by hydrodynamic tail vein injection (Yang PL et al., "Hydrodynamic injection of viral DNA: a mouse model of acute hepatitis B virus infection," PNAS USA 2002 Vol. 99: p.13825-13830) and administered for 30-45 days before administration of an HBV RNAi agent or control. MC-HBV1.3 is a plasma-derived sphere containing redundant human hepatitis B virus sequence HBV 1.3 for a certain period, similar to HBV1.3.32 transgenic mice (GenBank accession #V01460) (Guidotti LG et al., "High-level hepatitis B virus replication in transgenic mice," J Virol 1995 Vol. 69, p6158-6169). A pHBV model of chronic HBV infection was created by injecting 5 μg of MC-HBV1.3 in Ringer solution in a total volume equivalent to 10% of the mouse's body weight into the mouse's tail vein. As previously described (Zhang G et al., "High levels of foreign gene expression in hepatocytes after tail vein injection of naked plasmid DNA," Human Gene Therapy 1999 Vol.10, pl735-1737), the solution was injected using a 27-gauge needle over 5-7 seconds. -On day 1, the HBsAg expression level of hepatitis B surface antigen (HBsAg) in the serum was measured by ELISA, and the mice were grouped according to their mean HBsAg expression level.

[0464] i) Serum collection: Mice were anesthetized with 2-3% isofran, and blood samples were collected from the submandibular region into serum separation tubes (Sarstedt AG & Co., Numbrecht, Germany). The blood was allowed to coagulate at ambient 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.

[0465] ii) Serum hepatitis B surface antigen (HBsAg) levels: Serum was collected and diluted 10 to 2000 times in skim milk powder containing 5% PBS. Secondary HBsAg standards diluted in skim milk powder solution were prepared from serum of ICR mice (Harlan Sprague Dawley) transfected with 10 μg of HBsAg-expressing plasma pRc / CMV-HBs (Aldevron, Fargo, ND). HBsAg levels were measured using the GS HBsAg EIA 3.0 kit (Bio-Rad Laboratories, Inc., Redmond, WA) as described by the manufacturer. The ayw subtype of recombinant HBsAg protein was also diluted in skim milk powder in PBS and used as a primary standard (Aldevron).

[0466] HBsAg expression in each mouse was normalized to a control group of mice injected with normal saline to account for the decrease in MC-HBV1.3 expression associated with non-treatment. First, to determine the "pre-treatment normalized" expression ratio, the HBsAg level of each mouse at a given time point was divided by the mouse's pre-treatment expression level (-1 day). Then, the expression at a specific time point was normalized to the control group by dividing the "pre-treatment normalized" ratio for each individual mouse by the "pre-treatment normalized" ratio for all mice in the normal saline control group.

[0467] On day 1, each mouse received a single subcutaneous dose of either 200 μl containing 2 mg / kg (mpk) of an HBV double-stranded RNAi agent bound to an N-acetyl-galactosamine target ligand at the 5' end of the sense strand, or 200 μl of phosphate-buffered saline without the HBV RNAi agent as a control. The administered HBV RNAi agents included AD04580 (containing a 5'-cyclopropylphosphonic acid uracil-modified nucleotide with a 2'-O-methyl-modified nucleotide located at the 5' end of the antisense strand) and AD04178 (containing a 2'-O-methyl-modified nucleotide located at the 5' end of the antisense strand) in phosphate-buffered saline. The RNAi agents AD04580 and AD04178 are otherwise identical, the only difference being 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, which is common to standard nucleotides.

[0468] The injection was administered subcutaneously (i.e., between the skin and muscle) into the loose skin of the neck and shoulder region. Three mice were tested in each group (n=3). Serum was collected on days 8, 15, 22, and 29, and serum hepatitis B surface antigen (HBsAg) levels were measured.

[0469] The experimental data is shown in Figure 2, where the mean HBsAg reflects the normalized mean value of HBsAg. As shown in Figure 2, in pHBV model mice, the RNAi agent containing 5'-cyclopropylphosphonate-modified nucleotides (AD04580) was significantly superior to the unmodified RNAi agent (AD04178).

[0470] Other embodiments The present invention has been described in conjunction with its detailed description, but the foregoing description is intended to be illustrative and does not limit the scope of the invention as defined by the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

1. below 【Chemistry 1】 [In the formula, TBDPS means tert-butyl-diphenylsilyl] A compound selected from the group consisting of the following.

2. below 【Chemistry 2】 [In the formula, TBDMS means tert-butyl-dimethylsilyl] A compound selected from the group consisting of the following.

3. The following three compounds 【Transformation 3】 A method for manufacturing, comprising the following steps: (a) Compound 1 【Chemistry 4】 [In the formula, TBDPS means tert-butyl-diphenylsilyl] This was reacted with trimethylsulfonium iodide in the presence of sodium hydride to form compound 2. 【Transformation 5】 The process of manufacturing; and (b) A step of reacting compound 2 with triethylamine trifluoride to produce compound 3; Methods that include...

4. Compound 14 【Transformation 6】 A method for manufacturing, comprising the following steps: Compound 13 【Transformation 7】 A step of reacting with oxone to produce compound 14; Methods that include...

5. The method according to claim 4, further comprising the following steps: Compound 12 【Transformation 8】 [In the formula, TBDMS means tert-butyl-dimethylsilyl] A step of reacting with hydrochloric acid to produce compound 13; Methods that include...

6. The method according to claim 5, further comprising the following steps: Compound 11 【Chemistry 9】 [In the formula, TBDMS means tert-butyl-dimethylsilyl] A step to produce compound 12 by reacting with trimethylsulfonium iodide in the presence of sodium hydride; Methods that include...

7. The method according to claim 6, further comprising the following steps: Compound 9 【Chemistry 10】 [In the formula, TBDMS means tert-butyl-dimethylsilyl] compound 10 【Chemistry 11】 A step of reacting with to produce compound 11; Methods that include...

8. The method according to claim 7, further comprising the following steps: Compound 8 【Chemistry 12】 [In the formula, TBDMS means tert-butyl-dimethylsilyl] A step to produce compound 9 by reacting with des-martinperiodinane; Methods that include...

9. The method according to claim 8, further comprising the following steps: Compound 7 【Chemistry 13】 [In the formula, TBDMS means tert-butyl-dimethylsilyl] A step of reacting with acetic acid in water to produce compound 8; Methods that include...

10. The method according to claim 9, further comprising the following steps: Compound 6 【Chemistry 14】 A step to produce compound 7 by reacting with tert-butyl-dimethylsilyl chloride (TBDMSCl) in the presence of imidazole; Methods that include...

11. The method according to claim 10, further comprising the following steps: Compound 5 【Chemistry 15】 A step to produce compound 6 by reacting with 4,4-dimethoxytrityl chloride (DMT-Cl) in the presence of pyridine; Methods that include...

12. Compound 14 【Chemistry 16】 A method for manufacturing, comprising the following steps: (a) Compound 5 【Chemistry 17】 Compound 6 was formed by reacting it with 4,4-dimethoxytrityl chloride (DMT-Cl) in the presence of pyridine. [Chemistry 18] The process of manufacturing; (b) Compound 6 is reacted with tert-butyl-dimethylsilyl chloride (TBDMSCl) in the presence of imidazole to obtain compound 7 【Chemistry 19】 [In the formula, TBDMS means tert-butyl-dimethylsilyl] The process of manufacturing; (c) Compound 7 is reacted with acetic acid in water to form compound 8 【Chemistry 20】 [In the formula, TBDMS means tert-butyl-dimethylsilyl] The process of manufacturing; (d) Compound 8 is reacted with des-martinperiodinane to form compound 9 【Chemistry 21】 [In the formula, TBDMS means tert-butyl-dimethylsilyl] The process of manufacturing; (e) Compound 9 to Compound 10 【Chemistry 22】 Reacting with compound 11 【Chemistry 23】 [In the formula, TBDMS means tert-butyl-dimethylsilyl] The process of manufacturing; (f) Compound 11 is reacted with trimethylsulfonium iodide in the presence of sodium hydride to obtain compound 12 【Chemistry 24】 [In the formula, TBDMS means tert-butyl-dimethylsilyl] The process of manufacturing; (g) Compound 12 is reacted with hydrochloric acid to form compound 13 【Chemistry 25】 The process of manufacturing; and (h) A step of reacting compound 13 with oxone to produce compound 14; Methods that include...

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