CN116829567A

CN116829567A - Oligonucleotide prodrugs based on cyclic disulfide-modified phosphates

Info

Publication number: CN116829567A
Application number: CN202180093361.3A
Authority: CN
Inventors: A·V·凯尔因; J·M·皮尔森; J·K·奈尔; M·A·迈尔; A·拜斯比; C·唐
Original assignee: Alnylam Pharmaceuticals Inc
Current assignee: Alnylam Pharmaceuticals Inc
Priority date: 2020-12-31
Filing date: 2021-12-30
Publication date: 2023-09-29

Abstract

The present application relates to compounds comprising the structure of formula (I): cyclic disulfide moiety-phosphorus coupling group (I). The cyclic disulfide moiety has the structure of (C-I), (C-II) or (C-III). The application also relates to oligonucleotides comprising one or more compounds comprising the structure of formula (I), wherein at least one phosphorus coupling group comprises a nucleoside or an oligonucleotide. The application also relates to pharmaceutical compositions comprising the oligonucleotides described herein and methods of reducing or inhibiting expression of a target gene by administering to a subject a therapeutically effective amount of the oligonucleotides described herein.

Description

Oligonucleotide prodrugs based on cyclic disulfide modified phosphates

Cross Reference to Related Applications

The present application claims U.S. provisional application No. 63/132,535 submitted on 31 th 12 th 2020; and U.S. provisional application No. 63/287,833 issued on month 9 of 2021, which is incorporated herein by reference in its entirety.

Technical Field

The present application relates generally to the field of modified phosphate-based oligonucleotide prodrugs.

Background

Phosphate is an important intermediate in the formation of nucleotides and their assembly into RNA and DNA. Within the cell, the phosphate group is typically used as a tunable leaving group. The phosphate is charged at physiological pH, which is used to bind the phosphate to the active site of the enzyme. However, in order for the phosphate to bind to the enzyme, it must first penetrate the membrane to access the enzyme, as charged molecules may have difficulty crossing the cell membrane, except by endocytosis. In the case of compounds having larger, more lipophilic substituents, this limitation can be ameliorated.

Alternatively, prodrug pathways have been investigated to temporarily mask any negative charge of phosphate on oligonucleotides at physiological pH. Prodrugs are agents that are administered in inactive or significantly less active forms and, under different stimuli, undergo chemical or enzymatic transformations in the body to produce the active parent drug. The prodrug approach of masking the negative charge of the phosphate group of an oligonucleotide with a cytocleavable protecting/masking group may provide a number of advantages over its unprotected counterpart, including, for example, enhancing cell penetration by cell isolation and avoiding or minimizing degradation in serum.

However, the prodrug approach remains a substantial challenge, in part because of the difficulty in selecting the optimal masking group. For example, cell lysis of the protecting group can often produce products that are considered disadvantageous or even toxic. Furthermore, the protecting group must reach a balance between allowing absorption in the intestine and allowing lysis in the blood or target cells.

Accordingly, there is a continuing need to develop new and improved modified phosphate prodrugs for masking internucleotide phosphate linkages of oligonucleotides to produce effective and efficient oligonucleotide-based drugs for effective in vivo delivery and improved in vivo efficacy of the oligonucleotides.

Disclosure of Invention

One aspect of the present invention relates to compounds comprising the structure of formula (I): this->The structure is as follows: In these formulae:

R ₁ is O or S, and is bonded toP atoms of (c);

R ₂ 、R ₄ 、R ₆ 、R ₇ 、R ₈ and R is ₉ Each independently is H, halogen, OR ¹³ OR alkylene-OR ¹³ N (R ') (R ') or alkylene-N (R ') (R "), alkyl, C (R) ¹⁴ )(R ¹⁵ )(R ¹⁶ ) Or alkylene-C (R) ¹⁴ )(R ¹⁵ )(R ¹⁶ ) Alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, each of which may optionally be substituted with one or more R ^sub Group substitution;

R ₃ and R is ₅ Each independently is H, halogen, OR ¹³ OR alkylene-OR ¹³ N (R ') (R ') or alkylene-N (R ') (R "), alkyl, C (R) ¹⁴ )(R ¹⁵ )(R ¹⁶ ) Or alkylene-C (R) ¹⁴ )(R ¹⁵ )(R ¹⁶ ) Alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, each of which may optionally be substituted with one or more R ^sub Group substitution; or R is ₃ And R is ₅ Together with the adjacent carbon atoms and the two sulfur atoms, form a second ring;

g is O, N (R'), S or C (R) ¹⁴ )(R ¹⁵ )；

n is an integer from 0 to 6;

R ¹³ independently at each occurrence is H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, ω -aminoalkyl, ω -hydroxyalkyl, ω -hydroxyalkenyl, alkylcarbonyl, or arylcarbonyl, each of which may optionally be substituted with one or more R ^sub Group substitution;

R ¹⁴ 、R ¹⁵ And R is ¹⁶ Each independently is H, halogen, haloalkyl, alkyl, alkylaryl, aryl, heteroaryl, aralkyl, hydroxy, alkoxy, aryloxy, N (R') (R ");

r 'and R' are each independently H, alkyl, alkenyl, alkynyl, aryl, hydroxy, alkoxy, omega-aminoalkyl, omega-hydroxyalkyl, omega-hydroxyalkenyl or omega-hydroxyalkynyl, each of which may optionally be substituted with one or more R ^sub Group substitution; and is also provided with

R ^sub Independently for each occurrence, halogen, haloalkyl, alkyl, alkylaryl, aryl, aralkyl, hydroxy, alkoxy, aryloxy, oxo, nitro, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, alkylamino, aminoalkyl, alkoxycarbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamido, aralkylsulfonamide, alkylcarbonyl, arylcarbonyl, acyloxy, cyano or ureido.

In some embodiments, inIn (a):

R ₁ is O;

g is CH ₂ ；

n is 0 or 1;

R ₂ 、R ₄ 、R ₆ 、R ₇ 、R ₈ and R is ₉ Each independently is H, halogen, OR ¹³ Or C ₁ -C ₆ alkylene-OR ¹³ N (R ') (R') or C ₁ -C ₆ alkylene-N (R') (R "), C ₁ -C ₆ Alkyl, aryl, heteroaryl, each of which may optionally be substituted with one or more R ^sub Group substitution;

R ₃ and R is ₅ Each independently is H, halogen, OR ¹³ Or C ₁ -C ₆ alkylene-OR ¹³ N (R ') (R') or C ₁ -C ₆ alkylene-N (R') (R "), C ₁ -C ₆ Alkyl, aryl, heteroaryl, each of which may optionally be substituted with one or more R ^sub Group substitution; or R is ₃ And R is ₅ A second ring of 6 to 8 atoms together with the adjacent carbon atoms and the two sulfur atoms;

R ¹³ at each occurrence independently H, C ₁ -C ₆ Alkyl, aryl, alkylcarbonyl or arylcarbonyl; and is also provided with

R 'and R' are each independently H or C ₁ -C ₆ An alkyl group.

In some embodiments of the present invention, in some embodiments,with->Is a structure of (a). R is R ₂ May be an optionally substituted aryl group, for example, an optionally substituted phenyl group. In some embodiments, R ₂ Is a mono-, di-or tri-substituted phenyl group. In one embodiment, R ₂ Is para-substituted phenyl. In some embodiments, R ₂ Is optionally substituted C _1-6 An alkyl group. In one embodiment, R ₂ Is halogenated C _1-6 An alkyl group. In one embodiment, R ₂ Is C _1-6 An alkyl group.

In some embodiments of the present invention, in some embodiments,having a structure selected from one of the following Ia), ib) and II) groups. Ia) the group contains the following structure:

ib) the group contains the following structure:

the group II) contains the following structure:

in some embodiments of the present invention, in some embodiments,the structure is as follows: In these formulae:

X ₁ and Z ₁ Each independently is H, OH, OM, OR ¹³ 、SH、SM、SR ¹³ C (O) H, S (O) H or alkyl, each of which may optionally be substituted with one or more R ^sub Group substitution, N (R'), B (R) ¹³ ) ₃ 、BH ₃ ^- Se; or D-Q, wherein D is independently at each occurrence absent, O, S, N (R'), alkylene, each of which may optionally be substituted with one or more R ^sub Group substitution, and Q is independently at each occurrence a nucleoside or oligonucleotide;

X ₂ and Z ₂ Each independently N (R') (R "), OR ¹⁸ Or D-Q, wherein D is independently at each occurrence absent, O, S, N, N (R'), alkylene, each of which may optionally be substituted with one or more R ^sub Group substitution, and Q is independently at each occurrence a nucleoside or oligonucleotide,

Y ₁ S, O or N (R');

m is an organic or inorganic cation; and is also provided with

R ¹⁸ Is H or alkyl, optionally substituted by one or more R ^sub And (3) group substitution.

In some embodiments of the present invention, in some embodiments,with->Is a structure of (a). In this formula: x is X ₁ And Z ₁ Each independently is OH, OM, SH, SM, C (O) H, S (O) H, C optionally substituted with one or more hydroxy or halogen groups ₁ -C ₆ Alkyl, or D-Q; d is independently at each occurrence C which is absent, O, S, NH, optionally substituted with one or more halogen groups ₁ -C ₆ An alkylene group; and Y is ₁ Is S or O. In one embodiment, X ₁ Is OH or SH; and Z is ₁ Is D-Q.

In some embodiments of the present invention, in some embodiments,with->Is a structure of (a). In this formula: x is X ₂ Is N (R') (R "); z is Z ₂ Is X ₂ 、OR ¹⁸ Or D-Q; r is R ¹⁸ Is H or C substituted by cyano ₁ -C ₆ An alkyl group; and R' are each independently C ₁ -C ₆ An alkyl group.

In one embodiment of the present invention, in one embodiment,has a structure selected from the group consisting of:the variables R', R "and Q are as defined above in formulas P-I and P-II.

In one embodiment, the compound has a structure selected from one of the following:

in one embodiment of the present invention, in one embodiment,has the structure of (P-I), and +.> -P(Y ₁ )(X ₁ ) -having a structure selected from:

x is O or S.

In some embodiments, the compounds contain one or more ligands, optionally linked to by one or more linkers R of (2) ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ 、R ₇ 、R ₈ And R is ₉ Any one of them.

In some embodiments, the ligand is selected from the group consisting of antibodies, ligand binding portions of receptors, ligands of receptors, aptamers, carbohydrate-based ligands, fatty acids, lipoproteins, folic acid, thyroid stimulating hormone, melanotropin, surface active protein a, mucins, glycosylated polyamino acids, transferrin, bisphosphonic acids, polyglutamic acid, polyaspartic acid, lipophilic portions (e.g., lipophilic portions that enhance plasma protein binding), cholesterol, steroids, bile acids, vitamin B12, biotin, fluorophores, and peptides.

In certain embodiments, at least one ligand is a carbohydrate-based ligand that targets liver tissue. In one embodiment, the carbohydrate-based ligand is selected from galactose, multivalent galactose, N-acetyl-galactosamine (GalNAc), multivalent GalNAc, mannose, multivalent mannose, lactose, multivalent lactose, N-acetyl-glucosamine (GlcNAc), multivalent GlcNAc, glucose, multivalent glucose, fucose, and multivalent fucose.

In certain embodiments, at least one ligand is a lipophilic moiety. In one embodiment, by logK _ow The lipophilicity of the lipophilic moiety measured is greater than 0, or the hydrophobicity of the compound measured by the unbound fraction of the compound in a plasma protein binding assay is greater than 0.2.

In one embodiment, the lipophilic moiety contains saturated or unsaturated C ₄ -C ₃₀ A hydrocarbon chain, and optionally a functional group selected from hydroxyl, amine, carboxylic acid, sulfonic acid, phosphoric acid, thiol, azide, and alkyne. For example, the lipophilic moiety contains saturated or unsaturated C ₆ -C ₁₈ A hydrocarbon chain.

In certain embodiments, at least one ligand targets a receptor that mediates delivery to CNS tissue. In one embodiment, the ligand is selected from the group consisting of Angiopep-2, a lipoprotein receptor-related protein (LRP) ligand, a bEnd.3 cell binding ligand, a transferrin receptor (TfR) ligand, a mannose receptor ligand, a glucose transporter, and an LDL receptor ligand.

In certain embodiments, at least one ligand targets a receptor that mediates delivery to ocular tissue. In one embodiment, the ligand is selected from the group consisting of trans retinol, RGD peptide, LDL receptor ligand and carbohydrate-based ligand.

Another aspect of the invention relates to oligonucleotides (e.g., single-stranded iRNA agents or double-stranded iRNA agents) comprising one or more structures of formula (I):

another aspect of the invention relates to oligonucleotides (e.g., single-stranded iRNA agents or double-stranded iRNA agents) comprising one or more structures of formula (II):

in the formulae (I) and (II), The structure is as follows: or a salt thereof.

In these formulae:

R ₁ is O or S, and is bonded to the formula (I)Or a P atom of formula (II);

g is O, N (R'), S or C (R) ¹⁴ )(R ¹⁵ )；

n is an integer from 0 to 6;

R ^sub Independently for each occurrence, halogen, haloalkyl, alkyl, alkylaryl, aryl, aralkyl, hydroxy, alkoxy, aryloxy, oxo, nitro, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, alkylamino, aminoalkyl, alkoxycarbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamide, arenesulfonamido, aralkylsulfonamide, alkylcarbonyl, arylcarbonyl, acyloxy, cyano or ureido;

wherein whenWith the structure of formula (C-III), at least one +.> Attached to the 5' end of the nucleoside or oligonucleotide.

In formula (I), at least one ofContaining nucleosides or oligonucleotides.

In formula (II), represents a bond to an oligonucleotide, Y is absent, =o or=s; x is OH, SH OR X ', wherein X' is OR ¹³ Or SR (S.J) ¹³ 。

All of the above-mentioned references to the first aspect of the invention in relation to compounds Formula (C-I) and formula (C-II), -, are described in the specification >All of the formulae, all of the variables defined in these formulae, all of the ligands and all of the subgenera and class structures associated with the compounds,/o> And->Is applicable to these aspects of the invention in relation to oligonucleotides.

ib) the group contains the following structure:

the group II) contains the following structure:

in some embodiments of the present invention, in some embodiments,a structure having one of the structures selected from the group III). III) the group contains the following structure:

In some embodiments, the oligonucleotide contains a structure selected from one of:

or a salt thereof, wherein X is O or S.

In some embodiments, the oligonucleotide comprises a structure having the formula: -P (O) (SH) -, or a salt thereof.

In some embodiments, the oligonucleotide comprises a structure having the formula: -P (O) (OH) -, or a salt thereof>

In some embodiments, the oligonucleotide comprises a structure having the formula: -P(O)(OR ¹³ ) A method for producing a composite material x-ray or (b) and salts thereof. Variable R ¹³ As defined above.

In some embodiments, the oligonucleotide comprises a structure having the formula: -P(S)(OR ¹³ ) A method for producing a composite material x-ray or (b) and salts thereof. Variable R ¹³ As defined above.

In some embodiments, the oligonucleotide comprises a structure having the formula: -P(OR ¹³ ) A method for producing a composite material x-ray or (b) and salts thereof. Variable R ¹³ As defined above.

In some embodiments of the present invention, in some embodiments,has a structure selected from the group consisting of:

wherein indicates a bond to the phosphorus atom of the-P (X) (Y) -, group.

in one embodiment, the oligonucleotide comprises a structure selected from the group consisting of: x is O or S.

In some embodiments, the oligonucleotide contains at least one at the 5' -end of the oligonucleotide

In some embodiments, the first nucleotide at the 5' -end of the oligonucleotide has the following structure:or a salt thereof. In these structures:

* Represents a bond to an internucleotide linkage which is optionally modified subsequently;

the base is an optionally modified nucleobase;

rs isAnd is also provided with

R is H, OH, O-methoxyalkyl, O-methyl, O-allyl, CH ₂ -allyl, fluoro, O-N-methylacetamido (O-NMA), O-dimethylaminoethoxyethyl (O-DMAEOE), O-aminopropyl (O-AP) or ara-F. Variables X, X' and Y are as defined in formula (II) above.

In some embodiments, the first nucleotide at the 5' -end of the oligonucleotide has the following structure:

or a salt thereof. Variable base, R ^S 、R ¹³ 、R ¹⁴ And Y is as defined above.

In some embodiments, the first nucleotide at the 5' -end of the oligonucleotide has the following structure: Or a salt thereof. Variable base, R ^S And R is as defined above.

In some embodiments, the base in these structures is uridine. In some embodiments, R in these structures is methoxy. In some embodiments, R in these structures is hydrogen.

In some embodiments, the oligonucleotide contains at least one at the 3' -end of the oligonucleotide

In some embodiments, the oligonucleotide contains at least one at the 5' -end of the oligonucleotideAnd at least one +.sup.f at the 3' -end of the oligonucleotide>

In some embodiments, the oligonucleotide contains at least one of the following positions within the oligonucleotide

In some embodiments, the oligonucleotide is a single stranded oligonucleotide.

In some embodiments, the oligonucleotide is a double-stranded oligonucleotide comprising a sense strand and an antisense strand.

In some embodiments, the sense strand and the antisense strand are each 15 to 30 nucleotides in length. In one embodiment, the sense strand and the antisense strand are each 19-25 nucleotides in length. In one embodiment, the sense strand and the antisense strand are each 21 to 23 nucleotides in length.

In some embodiments, the oligonucleotide comprises a single stranded overhang on at least one terminus, e.g., a 3 'and/or 5' overhang of 1-10 nucleotides in length, e.g., an overhang of 1, 2, 3, 4, 5, or 6 nucleotides in length. In some embodiments, both strands have at least one segment of 1-5 (e.g., 1, 2, 3, 4, or 5) single stranded nucleotides in the duplex region. In one embodiment, the single stranded overhang is 1, 2 or 3 nucleotides in length, optionally on at least one terminus.

In some embodiments, the oligonucleotide may also have a blunt end located at the 5 '-end of the antisense strand (or the 3' -end of the sense strand), or vice versa. In one embodiment, the oligonucleotide comprises a 3' overhang at the 3' -end of the antisense strand and optionally a blunt end at the 5' -end of the antisense strand. In one embodiment, the oligonucleotide has a 5' overhang at the 5' -end of the sense strand and optionally a blunt end at the 5' -end of the antisense strand. In one embodiment, the oligonucleotide has two blunt ends at both ends of the double-stranded iRNA duplex.

In one embodiment, the oligonucleotide has a sense strand length of 21 nucleotides and an antisense strand length of 23 nucleotides, wherein the strand forms a duplex region of 21 consecutive base pairs with a single stranded overhang of 2 nucleotides at the 3' -end.

In one embodiment, the sense strand contains at least one ofIn one embodiment, the antisense strand contains at least one +.>In one embodiment, the sense strand and the antisense strand each contain at least one ∈ ->

In one embodiment, the oligonucleotide contains at least one at the 5' -end of the antisense strand And at least one targeting ligand at the 3' -end of the sense strand.

In some embodiments, the sense strand further comprises at least one phosphorothioate linkage at the 3' -end. In some embodiments, the sense strand comprises at least two phosphorothioate linkages at the 3' -end.

In some embodiments, the sense strand further comprises at least one phosphorothioate linkage at the 5' -end. In some embodiments, the sense strand comprises at least two phosphorothioate linkages at the 5' -end.

In some embodiments, the antisense strand further comprises at least one phosphorothioate linkage at the 3' -end. In some embodiments, the antisense strand comprises at least two phosphorothioate linkages at the 3' -end.

In some embodiments, the oligonucleotide further comprises a phosphate or phosphate mimic at the 5' -end of the antisense strand. In one embodiment, the phosphate ester mimic is a 5' -Vinyl Phosphonate (VP).

In some embodiments, the 5 '-end of the antisense strand does not contain a 5' -Vinylphosphonate (VP).

In some embodiments, the oligonucleotide further comprises at least one terminal chiral phosphorus atom.

Site-specific, chiral modifications to internucleotide linkages can occur at the 5 'end, 3' end, or both the 5 'and 3' ends of the strand. This is referred to herein as "terminal" chiral modification. The terminal modification may occur at a 3 'or 5' -terminal position in the terminal region, e.g., at a position on the terminal nucleotide or within the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the strand. Chiral modifications may occur on the sense strand, the antisense strand, or both the sense and antisense strands. Each chiral pure phosphorus atom may be in the Rp configuration or the Sp configuration, and combinations thereof. Further details regarding chiral modifications and chirally modified dsRNA agents can be found in PCT/US18/67103 entitled "chirally modified double stranded RNA agent" submitted on date 12, 21, 2018, which is incorporated herein by reference in its entirety.

In some embodiments, the oligonucleotide further comprises a terminal chiral modification at a first internucleotide junction at the 3' end of the antisense strand, having a connecting phosphorus atom of Sp configuration; a terminal chiral modification at the first internucleotide junction at the 5' end of the antisense strand, having a linking phosphorus atom of Rp configuration; and a terminal chiral modification at the first internucleotide junction at the 5' -end of the sense strand, having a linking phosphorus atom in Rp configuration or Sp configuration.

In one embodiment, the oligonucleotide further comprises a terminal chiral modification at the junction between the first and second nucleotides that occurs at the 3' end of the antisense strand, having a linking phosphorus atom in the Sp configuration; a terminal chiral modification at the first internucleotide junction at the 5' end of the antisense strand, having a linking phosphorus atom of Rp configuration; and a terminal chiral modification at the first internucleotide junction at the 5' end of the sense strand, having a linking phosphorus atom of Rp or Sp configuration.

In one embodiment, the oligonucleotide further comprises a terminal chiral modification at the first, second and third internucleotide linkages of the 3' end of the antisense strand having a linked phosphorus atom of Sp configuration; a terminal chiral modification at the first internucleotide junction at the 5' end of the antisense strand, having a linking phosphorus atom of Rp configuration; and a terminal chiral modification at the first internucleotide junction at the 5' end of the sense strand, having a linking phosphorus atom of Rp or Sp configuration.

In one embodiment, the oligonucleotide further comprises a terminal chiral modification at the junction between the first and second nucleotides that occurs at the 3' end of the antisense strand, having a linking phosphorus atom in the Sp configuration; a terminal chiral modification at the third internucleotide linkage at the 3' end of the antisense strand, having a linking phosphorus atom of Rp configuration; a terminal chiral modification at the first internucleotide junction at the 5' end of the antisense strand, having a linking phosphorus atom of Rp configuration; and a terminal chiral modification at the first internucleotide junction at the 5' end of the sense strand, having a linking phosphorus atom of Rp or Sp configuration.

In one embodiment, the oligonucleotide further comprises a terminal chiral modification at the junction between the first and second nucleotides that occurs at the 3' end of the antisense strand, having a linking phosphorus atom in the Sp configuration; a terminal chiral modification at the junction between the first and second nucleotides that occurs at the 5' end of the antisense strand, having a linking phosphorus atom in the Rp configuration; and a terminal chiral modification at the first internucleotide junction at the 5' end of the sense strand, having a linking phosphorus atom of Rp or Sp configuration.

In some embodiments, the oligonucleotide has at least two phosphorothioate internucleotide linkages at the first five nucleotides (counting from the 5' end) on the antisense strand.

In some embodiments, the antisense strand comprises two blocks of one, two, or three phosphorothioate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages.

In some embodiments, the oligonucleotide contains one or more targeting ligands, optionally linked to the compound through one or more linkersR of (2) ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ 、R ₇ 、R ₈ And R is ₉ Any one of them.

In some embodiments, the targeting ligand is selected from the group consisting of antibodies, ligand binding portions of receptors, ligands of receptors, aptamers, carbohydrate-based ligands, fatty acids, lipoproteins, folic acid, thyroid stimulating hormone, melanotropin, surface active protein a, mucin, glycosylated polyamino acids, transferrin, bisphosphonic acids, polyglutamic acid, polyaspartic acid, lipophilic portions that enhance plasma protein binding, cholesterol, steroids, bile acids, vitamin B12, biotin, fluorophores, and peptides.

In some embodiments, at least one targeting ligand is a lipophilic moiety. In one embodiment, by logK _ow The lipophilicity of the lipophilic moiety measured is greater than 0, or the hydrophobicity of the compound measured by the unbound fraction of the compound in a plasma protein binding assay is greater than 0.2. In one embodiment, the lipophilic moiety contains saturated or unsaturated C ₄ -C ₃₀ A hydrocarbon chain and optionally a functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. For example, the lipophilic moiety contains saturated or unsaturated C ₆ -C ₁₈ A hydrocarbon chain.

In some embodiments, at least one targeting ligand targets a receptor that mediates delivery to a particular CNS tissue. In one embodiment, the targeting ligand is selected from the group consisting of Angiopep-2, lipoprotein receptor-related protein (LRP) ligand, bend.3 cell binding ligand, transferrin receptor (TfR) ligand, mannose receptor ligand, glucose transporter, and LDL receptor ligand.

In some embodiments, at least one wh targeting ligand targets a receptor that mediates delivery to ocular tissue. In one embodiment, the targeting ligand is selected from the group consisting of trans retinol, RGD peptide, LDL receptor ligand and carbohydrate based ligand. In one embodiment, the targeting ligand is an RGD peptide, such as H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH or cyclo (-Arg-Gly-Asp-D-Phe-Cys).

In some embodiments, at least one targeting ligand targets liver tissue. In some embodiments, the targeting ligand is a carbohydrate-based ligand. In one embodiment, the carbohydrate-based ligand is selected from galactose, multivalent galactose, N-acetyl-galactosamine (GalNAc), multivalent GalNAc, mannose, multivalent mannose, lactose, multivalent lactose, N-acetyl-glucosamine (GlcNAc), multivalent GlcNAc, glucose, multivalent glucose, fucose, and multivalent fucose. In one embodiment, the targeting ligand is a GalNAc conjugate. For example, galNAc conjugates are one or more GalNAc derivatives attached by a divalent or trivalent branched linker, such as:

in some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the antisense and sense strands of the oligonucleotide are modified. For example, when 50% of the oligonucleotides are modified, 50% of all nucleotides present in the oligonucleotides contain the modifications described herein.

In some embodiments, the antisense and sense strands of the oligonucleotide comprise at least 30%

35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or almost 100% of 2' o-methyl modified nucleotides.

In one embodiment, the oligonucleotide is a double-stranded dsRNA agent, and at least 50% of the nucleotides of the double-stranded dsRNA agent are independently modified with 2 '-O-methyl, 2' -O-allyl, 2 '-deoxy, or 2' -fluoro.

In one embodiment, the oligonucleotide is antisense and at least 50% of the nucleotides of the antisense oligonucleotide are independently modified with LNA, ceNA, 2 '-methoxyethyl or 2' -deoxy.

In some embodiments, the sense strand and the antisense strand comprise 12 or fewer, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 2 or fewer, or no 2' -F modified nucleotides. In some embodiments, the oligonucleotide has 12 or fewer, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 2 or fewer, or no 2' -F modifications on the sense strand. In some embodiments, the oligonucleotide has 12 or fewer, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 2 or fewer, or no 2' -F modifications on the antisense strand. In one embodiment, the sense strand and the antisense strand comprise no more than ten 2' -fluoro modified nucleotides.

In some embodiments, the oligonucleotide contains one or more 2'-O modifications selected from the group consisting of 2' -deoxy, 2 '-O-methoxyalkyl, 2' -O-methyl, 2 '-O-allyl, 2' -C-allyl, 2 '-fluoro, 2' -O-N-methylacetamido (2 '-O-NMA), 2' -O-dimethylaminoethoxyethyl (2 '-O-DMAEOE), 2' -O-aminopropyl (2 '-O-AP), and 2' -ara-F.

In some embodiments, the oligonucleotide contains one or more 2' -F modifications at any position of the sense strand or antisense strand.

In some embodiments, the oligonucleotide has less than 20%, less than 15%, less than 10%, less than 5% or is substantially free of non-natural nucleotides. Examples of unnatural nucleotides include acyclic nucleotides, LNA, HNA, ceNA, 2 '-O-methoxyalkyl (e.g., 2' -O-methoxymethyl, 2 '-O-methoxyethyl, or 2' -O-2-methoxypropyl), 2 '-O-allyl, 2' -C-allyl, 2 '-fluoro, 2' -O-N-methylacetamido (2 '-O-NMA), 2' -O-dimethylaminoethoxyethyl (2 '-O-DMAEOEE), 2' -O-aminopropyl (2 '-O-AP), 2' -ara-F, L-nucleoside modifications (e.g., 2 '-modified L-nucleosides, such as 2' -deoxy-L-nucleosides), BNA abasic sugars, abasic cyclic and open chain alkyl groups.

In some embodiments, the oligonucleotide has greater than 80%, greater than 85%, greater than 90%, greater than 95%, or nearly 100% of the natural nucleotides. For the purposes of these embodiments, natural nucleotides may include those having 2' -OH, 2' -deoxy, and 2' -OMe.

In some embodiments, the antisense strand contains at least one Unlocked Nucleic Acid (UNA) or Glycerol Nucleic Acid (GNA) modification, e.g., in the seed region of the antisense strand. In one embodiment, the seed region is located at position 2-8 (or position 5-7) of the 5' -end of the antisense strand.

In one embodiment, the oligonucleotide comprises a sense strand and an antisense strand each having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplex region is between 19 and 25 base pairs (preferably 19, 20, 21 or 22); wherein the oligonucleotide has less than 20%, less than 15%, less than 10%, less than 5% or is substantially free of non-natural nucleotides.

In one embodiment, the oligonucleotide comprises a sense strand and an antisense strand each 15-30 nucleotides in length; at least two phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplex region is between 19 and 25 base pairs (preferably 19, 20, 21 or 22); wherein the oligonucleotide has greater than 80%, greater than 85%, greater than 95% or nearly 100% of natural nucleotides, such as those having 2' -OH, 2' -deoxy, or 2' -OMe.

One aspect of the invention provides an oligonucleotide comprising a sense strand and an antisense strand, each strand independently having a length of 15 to 35 nucleotides; at least two phosphorothioate internucleotide linkages between the first five nucleotides counted from the 5' end of the antisense strand; at least three, four, five or six 2' -deoxy modifications on the sense strand and/or the antisense strand; wherein the oligonucleotide has a double-stranded (duplex) region of 19 to 25 base pairs; wherein the oligonucleotide comprises a ligand.

In one embodiment, the sense strand does not comprise a Glycol Nucleic Acid (GNA).

It will be appreciated that the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference. In other words, the oligonucleotide is capable of inhibiting the expression of the target gene.

In one embodiment, the oligonucleotide comprises at least three 2' -deoxy modifications. Counting from the 5' -end of the antisense strand, the 2' -deoxy modifications are located at positions 2 and 14 of the antisense strand, and counting from the 5' -end of the antisense strand, at position 11 of the sense strand.

In one embodiment, the oligonucleotide comprises at least five 2' -deoxy modifications. Counting from the 5' -end of the antisense strand, the 2' -deoxy modifications are located at positions 2, 12 and 14 of the antisense strand, and counting from the 5' -end of the antisense strand, at positions 9 and 11 of the sense strand.

In one embodiment, the oligonucleotide comprises at least seven 2' -deoxy modifications. Counting from the 5' -end of the antisense strand, the 2' -deoxy modifications are located at positions 2, 5, 7, 12 and 14 of the antisense strand, and counting from the 5' -end of the antisense strand, at positions 9 and 11 of the sense strand.

In one embodiment, the antisense strand comprises at least five 2 '-deoxy modifications, counting from the 5' -end of the antisense strand, at positions 2, 5, 7, 12 and 14. The antisense strand is 18-25 nucleotides or 18-23 nucleotides in length.

In one embodiment, the oligonucleotide comprises less than 20%, e.g., less than 15%, less than 10%, or less than 5% of or no non-natural nucleotides.

In one embodiment, the sense strand does not comprise a Glycol Nucleic Acid (GNA); and wherein the oligonucleotide comprises less than 20%, e.g., less than 15%, less than 10% or less than 5% of non-natural nucleotides or all comprise natural nucleotides.

In one embodiment, at least one of the sense strand and the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, or at least seven or more 2' -deoxy modifications in the central region of the sense strand or the antisense strand.

In one embodiment, the sense strand and/or antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, or at least seven or more 2' -deoxy modifications in the central region of the sense strand and/or antisense strand.

In some embodiments, the sense strand has a length of 18 to 30 nucleotides and comprises at least two 2' -deoxy modifications in the central region of the sense strand. For example, the sense strand has a length of 18 to 30 nucleotides and comprises at least two 2 '-deoxy modifications within positions 7, 8, 9, 10, 11, 12 and 13, counted from the 5' end of the sense strand.

In one embodiment, the antisense strand has a length of 18 to 30 nucleotides and comprises at least two 2' -deoxy modifications in the central region of the antisense strand. For example, the antisense strand has a length of 18 to 30 nucleotides and comprises at least two 2 '-deoxy modifications within positions 10, 11, 12, 13, 14, 15 and 16, counted from the 5' end of the antisense strand.

In one embodiment, the oligonucleotide comprises a sense strand and an antisense strand; wherein the sense strand has a length of 17-30 nucleotides and comprises at least one 2' -deoxy modification in the central region of the sense strand; and wherein the antisense strand independently has a length of 17-30 nucleotides and comprises at least two 2' -deoxy modifications in a central region of the antisense strand.

In one embodiment, the oligonucleotide comprises a sense strand and an antisense strand; wherein the sense strand has a length of 17-30 nucleotides and comprises at least two 2' -deoxy modifications in a central region of the sense strand; and wherein the antisense strand independently has a length of 17-30 nucleotides and comprises at least one 2' -deoxy modification in the central region of the antisense strand.

In one embodiment, the sense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more 2' -deoxy modifications in the central region of the sense strand.

In one embodiment, the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more 2' -deoxy modifications in the central region of the antisense strand.

In one embodiment, the oligonucleotide comprises less than 20%, e.g., less than 15%, less than 10% or less than 5% of the non-natural nucleotides or the oligonucleotides all comprise natural nucleotides; and wherein the sense strand and/or the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more 2' -deoxy modifications in the central region of the sense strand and/or the antisense strand.

In one embodiment, the oligonucleotide comprises less than 20%, e.g., less than 15%, less than 10% or less than 5% of the non-natural nucleotides or the oligonucleotides all comprise natural nucleotides; and wherein the sense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more 2' -deoxy modifications in the central region of the sense strand.

In one embodiment, the oligonucleotide comprises less than 20%, e.g., less than 15%, less than 10% or less than 5% of the non-natural nucleotides or the oligonucleotides all comprise natural nucleotides; and wherein the antisense strand comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more 2' -deoxy modifications in the central region of the antisense strand.

In one embodiment, when the oligonucleotide comprises less than 8 non-2' ome nucleotides, the antisense strand comprises at least one DNA. For example, in any embodiment of the invention, when the oligonucleotide comprises less than 8 non-2' ome nucleotides, the antisense strand comprises at least one DNA.

In one embodiment, when the antisense strand comprises two deoxynucleotides and the nucleotides are located at positions 2 and 14 counted from the 5 '-end of the antisense strand, the oligonucleotide comprises 8 or fewer (e.g., 8, 7, 6, 5, 4, 3, 2, 1, or 0) non-2' ome nucleotides. For example, in any of the embodiments of the invention, when the antisense comprises two deoxynucleotides and the nucleotides are at positions 2 and 14 counted from the 5 '-end of the antisense strand, the oligonucleotide comprises 0, 1, 2, 3, 4, 5, 6, 7 or 8 non-2' -OMe nucleotides.

Another aspect of the invention relates to a pharmaceutical composition comprising an oligonucleotide as described herein and a pharmaceutically acceptable excipient.

All of the above embodiments relating to oligonucleotides in the above aspects of the invention relating to oligonucleotides are suitable in this aspect of the invention relating to pharmaceutical compositions.

In another aspect, the invention further provides a method of delivering an oligonucleotide of the invention to a specific target in a subject by subcutaneous or intravenous administration. The invention further provides an oligonucleotide of the invention for use in a method of delivering the agent to a specific target in a subject by subcutaneous or intravenous administration.

Another aspect of the invention relates to a method of reducing or inhibiting expression of a target gene in a subject comprising administering an oligonucleotide described herein to the subject in an amount sufficient to inhibit expression of the target gene.

All of the above embodiments of the invention relating to oligonucleotides in the above aspects of the invention relating to oligonucleotides are suitable in this aspect of the invention relating to methods of reducing expression of a target gene in a subject.

Another aspect of the invention relates to a method for modifying an oligonucleotide comprising contacting the oligonucleotide with a compound described herein under conditions suitable for reacting the compound with the oligonucleotide, wherein the oligonucleotide comprises a free hydroxyl group.

In some embodiments, the free hydroxyl is part of the 5' -terminal nucleotide. In some embodiments, the free hydroxyl group is part of a 3' -terminal nucleotide.

In some embodiments, the oligonucleotide comprises a 5' -OH group. In some embodiments, the oligonucleotide comprises a 3' -OH group.

In some embodiments, suitable conditions for reacting a compound with an oligonucleotide include an acidic catalyst. For example, the acid catalyst may be a substituted tetrazole. Suitable acidic catalysts include, but are not limited to, 1H-tetrazole, 5-ethylthio-1H-tetrazole, 2-benzylthiotetrazole, 4, 5-dicyanoimidazole, 5-nitrophenyl-1H-tetrazole, 5- (bis-3, 5-trifluoromethylphenyl) -1H-tetrazole, 5-benzylthio-1H-tetrazole, 5-methylthio-1H-tetrazole, 1-hydroxybenzotriazole, 1-hydroxy-6-trifluoromethylbenzotriazole, 4-nitro-1-hydroxy-6-trifluoromethylbenzotriazole, pyridinium chloride, pyridinium bromide, pyridinium 4-methylbenzenesulfonate, 2, 6-di (tert-butyl) pyridinium chloride, pyridinium trifluoroacetate, N- (phenyl) imidazolium triflate (N-PhIMT), N- (phenyl) -imidazolium perchlorate (N-Ph), N- (methyl) benzimidazolium triflate (NMeBIT), N- (p-acetylphenyl) imidazolium triflate (N- (p-acetyl) triflate (NMeBIT), N- (p-acetyl) imidazolium triflate (IMT), IMTK (IMTK) and (IMTK) TK Benzimidazolium triflate (BIT), 2- (phenyl) imidazolium triflate (2-PhIMT), N- (methyl) imidazolium triflate (N-MeIMT), 4- (methyl) imidazolium triflate (4-MeIMT), saccharin-1-methylimidazole, N- (cyanomethyl) pyrrolidine triflate, trichloroacetic acid (TCA), trifluoroacetic acid (TFA), dichloroacetic acid (DCA) and 2, 4-dinitrobenzoic acid (2, 4-DNBA), ferric chloride (FeCl 3), aluminum chloride (AlCl 3), trifluoroboron etherate (BF 3-OEt 2), zirconium (IV) chloride (ZrCl 4) and bismuth (III) chloride (BiCl 3), trimethylchlorosilane, 2, 4-dinitrophenol, 1-methyl-5-mercaptotetrazole and 1-phenyl-5-mercaptotetrazole.

All the above embodiments relating to compounds and oligonucleotides of the above aspects of the invention are applicable to this aspect of the invention relating to methods of modifying oligonucleotides.

Another aspect of the invention relates to a method for preparing a modified oligonucleotide comprising: under conditions suitable for forming a modified oligonucleotide comprising a group of formula (B):

wherein Y is O or S; and X is-OH, -SH or X',

oxidizing a first oligonucleotide comprising a group of formula (a):

or a salt thereof, wherein:

RS is

X' is-OR ¹³ or-SR ¹³ Wherein R is ¹³ Is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, ω -aminoalkyl, ω -hydroxyalkyl, ω -hydroxyalkenyl, alkylcarbonyl or arylcarbonyl, each of which may optionally be substituted with one or more R ^sub And (3) group substitution.

Variable base, R ^S X, X' and Y are as defined above.

In some embodiments, the first nucleotide at the 5 '-end of the first oligonucleotide comprises a group of formula (a), and the first nucleotide at the 5' -end of the modified oligonucleotide comprises a group of formula (B). In some embodiments, the last nucleotide at the 3 '-end of the first oligonucleotide comprises a group of formula (a), and the last nucleotide at the 3' -end of the modified oligonucleotide comprises a group of formula (B).

In some embodiments, the first nucleotide at the 5' -end of the first oligonucleotide is according to formula (C):

or a salt thereof, wherein:

the base is an optionally modified nucleobase; and is also provided with

R is H, OH, O-methoxyalkyl, O-methyl, O-allyl, CH ₂ -allyl, fluoro, O-N-methylacetamido (O-NMA), O-dimethylaminoethoxyethyl (O-DMAEOE), O-aminopropyl (O-AP) or ara-F. Variable R ^S And X' is as defined above.

In some embodiments, the first nucleotide at the 5' -end of the modified oligonucleotide has the structure of formula (D):

variable base, R, R ^S X and Y are as defined above.

In some embodiments, the first nucleotide at the 5' -end of the modified oligonucleotide has the structure of formula (E) or (F):

or a salt thereof, wherein x represents a bond to a subsequent nucleotide. Variable base, R, R ^S X and Y are as defined above.

In some embodiments, the conditions suitable for forming the modified oligonucleotide comprise the use of an oxidizing agent selected from the group consisting of: iodine; sulfur; a peroxide; peracid; phenylacetyl disulfide; 3H-1, 2-benzodithiol-3-one 1, 1-dioxide; dixanthogen (dixanthogen); 5-ethoxy-3H-1, 2, 4-dithiazol-3-one; 3- [ (dimethylaminomethylene) amino ] -3H-1,2, 4-dithiazole-5-thione (DDTT); dimethyl sulfoxide; and N-bromosuccinimide. For example, the oxidizing agent may be a peracid (e.g., m-chloroperbenzoic acid) or a peroxide (e.g., t-butyl hydroperoxide or trimethylsilyl peroxide).

All the above embodiments relating to compounds and oligonucleotides of the above aspects of the invention are applicable to this aspect of the invention relating to a method of preparing a modified oligonucleotide.

Drawings

FIG. 1 is a graph depicting the in vitro activity of F12siRNA containing a modified phosphate prodrug at the 5' end in primary mouse hepatocytes after transfection with RNAiMAX at concentrations of 0.1, 1, 10 and 100nm and analyzed 24 hours after transfection. The percentage of residual F12 messenger was determined by qPCR and plotted against the control.

FIG. 2 is a graph depicting the in vitro activity of F12siRNA duplex containing modified phosphate prodrug at the 5' end in primary mouse hepatocytes incubated at concentrations of 0.1, 1, 10 and 100nm and analyzed 48 hours after incubation. The percentage of residual F12 messenger was determined by qPCR and plotted against the control.

FIG. 3 is a graph depicting the in vitro activity of F12siRNA duplex containing modified phosphate prodrug at the 5' end in primary mouse hepatocytes after transfection with RNAiMAX at concentrations of 0.1, 1 and 10nm and analyzed 24 hours after transfection. The percentage of residual F12 messenger was determined by qPCR and plotted against the control.

FIGS. 4A-J show representative LCMS spectra of oligonucleotides tested in a DTT reduction assay.

FIG. 5 is a graph depicting relative mF12 protein in circulation in mice as determined by ELISA after subcutaneous administration of F12 siRNA duplex containing modified phosphate prodrug at the 5' end at a single dose of 0.3mg/kg as compared to PBS control.

FIG. 6 is a graph depicting relative mF12 protein in circulation in mice as determined by ELISA after subcutaneous administration of F12 siRNA duplex containing modified phosphate prodrug at the 5' end at a single dose of 0.1mg/kg or 0.3mg/kg as compared to PBS control.

Figure 7 shows a possible in vivo cytoplasmic unmasking mechanism for 5 '-phosphate exhibited by 5' cyclic disulfide modified phosphate prodrugs.

FIG. 8 is a graph depicting relative SOD1 mRNA remaining in the thoracic spinal cord, hippocampus, frontal cortex, striatum and heart of rats as determined by qPCR after 14 days of Intrathecal (IT) administration of SOD1 siRNA duplex containing modified phosphate prodrug at the 5' end at a single dose of 0.1 mg.

FIG. 9 is a graph depicting relative SOD1 mRNA remaining in the thoracic spinal cord, cerebellum, frontal cortex, striatum and heart of rats as determined by qPCR after 84 days of Intrathecal (IT) administration of SOD1 siRNA duplex containing modified phosphate prodrug at the 5' end at a single dose of 0.3 mg.

FIG. 10 is a graph depicting relative SOD1mRNA remaining in the thoracic spinal cord, hippocampus, frontal cortex, striatum and heart of rats as determined by qPCR after 14 days of Intrathecal (IT) administration of SOD1 siRNA duplex containing modified phosphate prodrug at the 5' end at a single dose of 0.9 mg.

FIG. 11 is a graph depicting relative SOD1mRNA remaining in the thoracic spinal cord, cerebellum, frontal cortex, striatum and heart of rats as determined by qPCR after 84 days of Intrathecal (IT) administration of SOD1 siRNA duplex containing modified phosphate prodrug at the 5' end at a single dose of 0.9 mg.

FIG. 12 is a graph depicting the relative SOD1mRNA remaining in the thoracic spinal cord, hippocampus, frontal cortex, striatum and heart of rats as determined by qPCR 14 days after intrathecal administration of SOD1 siRNA duplex containing modified phosphate prodrug at the 5' end at a single dose of 0.9 mg.

FIG. 13 is a graph depicting relative SOD1mRNA remaining in the thoracic spinal cord, hippocampus, frontal cortex, striatum and heart of rats as determined by qPCR after intrathecal administration of SOD1 siRNA duplex containing modified phosphate prodrug at 5' end at a single dose of 0.3mg or 0.9mg for 14 days.

FIG. 14 is a graph depicting the relative SOD1mRNA remaining in the right hemispheres of mice as determined by qPCR 7 days after administering SOD1 siRNA duplex containing modified phosphate prodrug at the 5' end at a single dose of 100 μg to the intracranial ventricle.

Detailed Description

The inventors have discovered a new class of cyclic disulfide moieties that can be introduced to the phosphate groups of oligonucleotides (e.g., single stranded iRNA agents, double stranded iRNA agents) to temporarily mask the phosphate groups, and which can be cleaved in vivo by cell activation. Cell activation is by glutathione or dithiothreitol mediated reduction/bioconversion mechanisms to release the active anionic form of the phosphate group from the masking group. The inventors have found that cyclic disulfide moieties may be introduced at the 5 'end, 3' end and/or internal positions of the strand, either at the sense strand or at the antisense strand or both. The incorporation of a cyclic disulfide moiety modified phosphate prodrug at the 5' end of the antisense strand provides particularly good results.

Modified phosphate prodrug compounds

One aspect of the invention relates to modified phosphate ester prodrug compounds. The compounds comprise the structure of formula (I):

the structure is as follows:

In the formulae (C-I), (C-II) and (C-III):

R ₁ is O or S, and is bonded toP atoms of (c);

R ₃ and R is ₅ Each independently is H, halogenElement OR ¹³ OR alkylene-OR ¹³ N (R ') (R ') or alkylene-N (R ') (R "), alkyl, C (R) ¹⁴ )(R ¹⁵ )(R ¹⁶ ) Or alkylene-C (R) ¹⁴ )(R ¹⁵ )(R ¹⁶ ) Alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, each of which may optionally be substituted with one or more R ^sub Group substitution; or R is ₃ And R is ₅ Together with the adjacent carbon atoms and the two sulfur atoms, form a second ring;

g is O, N (R'), S or C (R) ¹⁴ )(R ¹⁵ )；

n is an integer from 0 to 6;

In some embodiments, inIn (a):

R ₁ is O;

g is CH ₂ ；

n is 0 or 1;

R 'and R' are each independently H or C ₁ -C ₆ An alkyl group.

May have the following structure:

in the formulae (P-I) and (P-II):

X ₁ and Z ₁ Each independently is H, OH, OM, OR ¹³ 、SH、SM、SR ¹³ C (O) H, S (O) H or alkyl, each of which may optionally be substituted with one or moreR is a number of ^sub Group substitution, N (R') (R), B (R) ¹³ ) ₃ 、BH ₃ ^- Se; or D-Q, wherein D is independently at each occurrence absent, O, S, N (R'), alkylene, each of which may optionally be substituted with one or more R ^sub Group substitution, and Q is independently at each occurrence a nucleoside or oligonucleotide;

Y ₁ s, O or N (R');

m is an organic or inorganic cation; and is also provided with

In some embodiments of the present invention, in some embodiments,with->Structure is as follows. In this formula: x is X ₁ And Z ₁ Each independently is OH, OM, SH, SM, C (O) H, S (O) H, C optionally substituted with one or more hydroxy or halogen groups ₁ -C ₆ Alkyl, or D-Q; d is independently at each occurrence C which is absent, O, S, NH, optionally substituted with one or more halogen groups ₁ -C ₆ An alkylene group; and Y is ₁ Is S or O. In one embodiment, X ₁ Is OH or SH; and Z is ₁ Is D-Q.

In one embodiment of the present invention, in one embodiment,with->Wherein X is ₁ Is OH or SH.

In some embodiments of the present invention, in some embodiments,with->Structure is as follows. In this formula: x is X ₂ Is N (R') (R "); z is Z ₂ Is X ₂ 、OR ¹⁸ Or D-Q; r is R ¹⁸ Is H or C substituted by cyano ₁ -C ₆ An alkyl group; and R' are each independently C ₁ -C ₆ Alkyl (e.g., isopropyl).

In one embodiment of the present invention, in one embodiment,has a structure selected from the group consisting of:The variables R', R "and Q are as defined above for the formulae P-I and P-II. In one embodiment, each of R' and R "is isopropyl.

In some embodiments of the present invention, in some embodiments,having the structure-P (Z) (X), wherein:

x is selected from-OCH ₃ 、-OCH ₂ CH ₃ 、-OCH ₂ CH ₂ CH ₃ 、-OCH ₂ CH(CH ₃ ) ₂ 、-OCH ₂ CH ₂ CN、-OCH ₂ CH ₂ Si(CH ₃ ) ₃ 、-OCH ₂ CH ₂ Si(CH ₂ CH ₃ ) ₃ 、-OC(H)＝CH ₂ 、-OCH ₂ C(H)＝CH ₂ 、

And is also provided with

Z is selected from: or (b)

X and Z together with the phosphorus atom to which they are attached form a cyclic structure selected from:

in one embodiment of the present invention, in one embodiment,with->Is a structure of (a). />

Can have a structure->Exemplary 5-membered Cyclic Compounds of formula (C-I)>Comprising the following steps:

In some embodiments, the compound has the formula (la) Wherein: r is R ₂ 、R ₃ 、R ₄ And R is ₅ Each independently is H, alkyl (e.g., CH ₃ ) Heterocycle, CH ₂ R ¹⁵ Aryl (e.g., phenyl), heteroaryl, CHFR ¹⁵ 、CF ₂ R ¹⁵ 、CF ₃ The method comprises the steps of carrying out a first treatment on the surface of the And may be of any stereoisomeric configuration; and R is ¹⁵ Is alkyl, heterocyclyl, aryl, OH, O-alkyl, NH ₂ NH (alkyl), N (alkyl) ₂ 、CF ₂ R ¹⁵ Or CF (CF) ₃ The method comprises the steps of carrying out a first treatment on the surface of the And may be of any stereoisomeric configuration.

Exemplary compounds of formula (I) having a 5-membered cyclic disulfide moiety are shown in table 1.

TABLE 1 Compounds having a 5-membered cyclic disulfide moiety

In some embodiments of the present invention, in some embodiments,having the structure-> Wherein R is ₃ And R is ₅ Together with the adjacent carbon atoms and the two sulfur atoms, form a second ring. In one embodiment, the second ring has 6 to 8 atoms.

Exemplary bicyclic compounds of formula (C-I)Comprising the following steps:

In some embodiments, the compound has the formula (la) Wherein R is ₃ And R is ₅ Together with the adjacent carbon atoms and the two sulfur atoms, form a second ring (e.g., having 6-8 atoms).

Exemplary compounds of formula (I) having a bicyclic disulfide moiety are shown in table 2.

TABLE 2 Compounds with bicyclic disulfide moieties

May also have a structure->

Exemplary Cyclic Compounds of formula (C-II)Comprising the following steps:

In some embodiments, the compound has the formula:

in these formulae, n is 1, 2, 3, 4, 5 or 6; g is O, NR ¹⁵ S or any other heteroatom; r is R ₂ 、R ₃ 、R ₄ 、R ₅ And R is ₆ Each independently is H, alkyl (e.g., CH ₃ ) Heterocycle, CH ₂ R ¹⁵ Aryl (e.g., phenyl), heteroaryl, CHFR ¹⁵ 、CF ₂ R ¹⁵ 、CF ₃ The method comprises the steps of carrying out a first treatment on the surface of the And may be of any stereoisomeric configuration; and R is ¹⁵ Is alkyl, heterocyclyl, aryl, OH, O-alkyl, NH ₂ NH (alkyl), N (alkyl) ₂ 、CF ₂ R ¹⁵ Or CF (CF) ₃ The method comprises the steps of carrying out a first treatment on the surface of the And may be of any stereoisomeric configuration.

Exemplary compounds of formula (I) having a larger (7 membered or greater) cyclic disulfide moiety are shown in table 3.

TABLE 3 Compounds having 7-or 8-membered cyclic disulfide moieties

It may also have the structure:Exemplary 6-membered Cyclic Compounds of formula (C-III)>Comprising the following steps:

In some embodiments, the compound has the formula (la) In the formula, R ₂ 、R ₃ 、R ₄ 、R ₅ And R is ₆ Each independently is H, alkyl (e.g., CH ₃ ) Heterocycle, CH ₂ R ¹⁵ Aryl (e.g., phenyl), heteroaryl, CHFR ¹⁵ 、CF ₂ R ¹⁵ 、CF ₃ The method comprises the steps of carrying out a first treatment on the surface of the And may be of any stereoisomeric configuration; and R is ¹⁵ Is alkyl, heterocyclyl, aryl, OH, O-alkyl, NH ₂ NH (alkyl), N (alkyl) ₂ 、CF ₂ R ¹⁵ Or CF (CF) ₃ The method comprises the steps of carrying out a first treatment on the surface of the And may be of any stereoisomeric configuration.

Exemplary compounds of formula (I) having a 6 membered cyclic disulfide moiety are shown in table 4.

TABLE 4 Compounds with 7-or 8-membered cyclic disulfide moieties

As is familiar to those skilled in the art, throughout this application, certain terms within chemical structures are abbreviated, including, for example, methyl (Me), benzoyl (Bz), phenyl (Ph), and Piv.

The term "halo" or "halogen" refers to any group of fluorine, chlorine, bromine or iodine.

The term "aliphatic" or "aliphatic group" as used herein means a straight or branched, substituted or unsubstituted hydrocarbon chain that is saturated or contains one or more units of unsaturation, or a mono-or bi-or multi-cyclic hydrocarbon that is saturated or contains one or more units of unsaturation but is not aromatic, having a single point of attachment to the remainder of the molecule. In some embodiments, the aliphatic group contains 1 to 50 aliphatic carbon atoms, for example, 1 to 10 aliphatic carbon atoms, 1 to 6 aliphatic carbon atoms, 1 to 5 aliphatic carbon atoms, 1 to 4 aliphatic carbon atoms, 1 to 3 aliphatic carbon atoms, or 1 to 2 aliphatic carbon atoms. In some embodiments, "cycloaliphatic" refers to a monocyclic or bicyclic C that is saturated or contains one or more units of unsaturation, but which is not aromatic ₃ -C ₁₀ Hydrocarbons (e.g. monocyclic C ₃ -C ₆ Hydrocarbons) that have a single point of attachment to the remainder of the molecule. Suitable aliphatic groups include, but are not limited to, straight or branched chain, substituted or unsubstituted alkyl, alkenyl, alkynyl, and hybrids thereof, such as (cycloalkyl) alkyl, (cycloalkenyl) alkyl, or (cycloalkyl) alkenyl.

The term "alkyl" refers to a hydrocarbon chain, which may be straight or branched, containing the indicated number of carbon atoms. For example, C ₁ -C ₁₂ Alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms. Unless otherwise indicated, "alkyl" generally refers to C ₁ -C ₂₄ Alkyl (e.g., C ₁ -C ₁₂ Alkyl, C ₁ -C ₈ Alkyl or C ₁ -C ₄ Alkyl). The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced with a halogen, and includes alkyl moieties in which all hydrogens are replaced with a halogen (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may optionally be inserted into O, N or S. The term "aralkyl" refers to an alkyl moiety in which an alkyl hydrogen atom is replaced with an aryl group. Aralkyl groups include groups in which more than one hydrogen atom is replaced with an aryl group. Examples of "aralkyl" include benzyl, 9-fluorenyl, benzhydryl, and trityl.

The term "alkenyl" refers to a straight or branched hydrocarbon chain containing 2 to 8 carbon atoms and characterized by having one or more double bonds. Unless otherwise indicated, "alkenyl" generally refers to C ₂ -C ₈ Alkenyl (e.g., C ₂ -C ₆ Alkenyl, C ₂ -C ₄ Alkenyl or C ₂ -C ₃ Alkenyl). Examples of typical alkenyl groups include, but are not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl, and 3-octenyl. The term "alkynyl" refers to a straight or branched hydrocarbon chain containing from 2 to 8 carbon atoms and characterized by one or more triple bonds. Unless otherwise indicated, "alkynyl" generally refers to C ₂ -C ₈ Alkynyl groups (e.g.,

C ₂ -C ₆ alkynyl, C ₂ -C ₄ Alkynyl or C ₂ -C ₃ Alkynyl). Some examples of typical alkynyl groups are ethynyl, 2-propynyl and 3-methylbutynyl and propargyl. sp (sp) ² And sp (sp) ³ Carbon may optionally be used as the point of attachment for alkenyl and alkynyl groups, respectively.

The term "alkoxy" refers to an-O-alkyl group. The term "alkylene" refers to a divalent alkyl group (i.e., -R-). The term "aminoalkyl" refers to an alkyl group substituted with an amino group. The term "mercapto" refers to a-SH group. The term "thioalkoxy" refers to an-S-alkyl group.

The term "alkylene" refers to a divalent alkyl group. "alkylene chain" is polymethylene, i.e., - (CH) ₂ ) _n -wherein n is a positive integer, preferably 1 to 6, 1 to 4, 1 to 3, 1 to 2 or 2 to 3. The substituted alkylene chain being in which one or more methylene hydrogen atoms are substitutedSubstituted polymethylenes. Suitable substituents include those described below.

The term "alkenylene" refers to a divalent alkenyl group. Substituted alkenylene chains are polymethylene groups containing at least one double bond in which one or more hydrogen atoms are replaced by substituents. Suitable substituents include those described below.

The term "aryl" refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system in which 0, 1, 2, 3, or 4 atoms of each ring may be substituted with substituents. The term "aryl" may be used interchangeably with the term "aryl ring". Examples of aryl groups include phenyl, biphenyl, naphthyl, anthracenyl, and the like, which may bear one or more substituents. Also included within the scope of the term "aryl" as used herein are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthalimidyl, phenanthridinyl, tetrahydronaphthyl, and the like. The term "arylalkyl" or the term "aralkyl" refers to an alkyl group substituted with an aryl group. The term "arylalkoxy" refers to an alkoxy group substituted with an aryl group.

The term "cycloalkyl" or "cyclic group" as used herein includes saturated and partially unsaturated, but not aromatic, cyclic hydrocarbon groups having 3 to 12 carbons, e.g., 3 to 8 carbons and e.g., 3 to 6 carbons, wherein cycloalkyl groups may additionally be optionally substituted. Cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

The term "heteroaryl" or "heteroaryl-" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic or 11-14 membered tricyclic ring system having 1-3 heteroatoms (if monocyclic), 1-6 heteroatoms (if bicyclic) or 1-9 heteroatoms (if tricyclic) selected from O, N or S (e.g., carbon atoms and 1-3, 1-6 or 1-9 heteroatoms N, O or S (if monocyclic, bicyclic or tricyclic, respectively), wherein 0, 1, 2,3 or 4 atoms of each ring may be substituted with substituents, the term also includes groups in which the heteroaromatic ring is fused to one or more aryl, cycloalkyl or heterocyclyl rings, examples of heteroaryl groups include pyrrolyl, pyridyl, pyridazinyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, pyrazinyl, indolizinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, isothiazolyl, thiadiazolyl, purinyl, naphthyridinyl, pteridinyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzothiazolyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl and pyrido [2,3-b ] -1, 4-oxazin-3 (4H) -one, and the like. The term "heteroarylalkyl" or the term "heteroarylalkyl" refers to an alkyl group substituted with a heteroaryl group. The term "heteroarylalkoxy" refers to an alkoxy group substituted with a heteroaryl group.

The term "heterocyclyl", "heterocycle", "heterocyclic" or "heterocyclic ring" refers to a non-aromatic 5-8 membered monocyclic, 8-12 membered bicyclic or 11-14 membered tricyclic ring system having 1-3 heteroatoms (if monocyclic), 1-6 heteroatoms (if bicyclic) or 1-9 heteroatoms (if tricyclic) selected from O, N or S (e.g., carbon atoms and 1-3, 1-6 or 1-9 heteroatoms N, O or S (if monocyclic, bicyclic or tricyclic, respectively), examples of heterocyclic groups include triazolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, dioxolanyl, diaza, and the like, in saturated or partially unsaturated rings having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, nitrogen may be N (as in 3, 4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl)Radical, oxazal->Radical, thiazal->A group, morpholinyl, tetrahydrofuranyl, tetrahydrothiophenylpyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, quinuclidinyl and the like. The term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl group, wherein the alkyl and heterocyclyl moieties are independently optionally substituted.

The term "oxo" refers to an oxygen atom that forms a carbonyl group when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl or heteroarylcarbonyl substituent, any of which may be further substituted with a substituent.

The term "substituted" means that one or more hydrogen groups in a given structure are replaced with groups of specified substituents, including but not limited to: halogen, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthio alkyl, arylthio alkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclyl and aliphatic. It is understood that the substituents may be further substituted.

Suitable divalent substituents on the saturated carbon atoms of an "optionally substituted" group include the following: ═ O, ═ S, ═ NNR ₂ 、═NNHC(O)R*、═NNHC(O)OR*、═NNHS(O) ₂ R*、═NR*、═NOR*、-O(C(R* ₂ )) _2-3 O-or-S (C (R) ₂ )) _2-3 S-, wherein each occurrence of R is independently selected from hydrogen, which may be as followsC of defined substitution _1-6 Aliphatic or unsubstituted 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. Suitable divalent substituents bonded to the ortho-substitutable carbon of an "optionally substituted" group include: -O (CR) ₂ ) _2-3 O-, wherein each occurrence of R is independently selected from hydrogen, C which may be substituted as defined below _1-6 Aliphatic or unsubstituted 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

Oligonucleotide prodrugs

Another aspect of the invention relates to oligonucleotides (e.g., single-stranded iRNA agents or double-stranded iRNA agents) comprising one or more compounds comprising the structure of formula (I): in formula (I), at least one +.>Containing nucleosides or oligonucleotides.

Relates toAll, & gt>All of the above-described embodiments of all of the formulae, all of the variables defined in these formulae and all subgenera and species structures of the compounds, cyclic disulfide moieties and phosphorus coupling groups of the first aspect of the invention in relation to the compounds (or modified phosphate prodrug compounds) are suitable in this aspect of the invention which relates to oligonucleotides.

In some embodiments, the oligonucleotide contains at least one at an internal position of the oligonucleotide

In some embodiments, whenHaving the structure of formula (C-III), at least oneAttached to the 5' end of the nucleoside or oligonucleotide.

Additional structures of modified phosphate prodrug compounds include those disclosed in WO 2014/088920 published at 2014, month 06, 12, the contents of which are incorporated herein by reference in their entirety. In particular, these modified phosphate prodrug compounds are introduced at the 5' end into oligonucleotides.

In some embodiments, the oligonucleotide is a single stranded oligonucleotide, e.g., a single stranded iRNA agent (e.g., a single stranded siRNA).

In some embodiments, the oligonucleotide is a double-stranded oligonucleotide, e.g., a double-stranded iRNA agent (e.g., a double-stranded siRNA), comprising a sense strand and an antisense strand.

In one embodiment, the sense strand contains at least one ofIn one embodiment, the antisense strand contains at least one +.>In one embodiment, both the sense strand and the antisense strand contain at least one ∈ - >

On the sense strand or the antisense strand or both the sense and antisense strands, will The introduction of the phosphate group as a temporary protecting group is illustrated in schemes 10-15 of example 9 below.

Oligonucleotide definition and design

Unless specifically defined otherwise, nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, and the procedures and techniques thereof, are those well known and commonly employed in the art. Standard techniques can be used for chemical synthesis and chemical analysis. Some of these techniques and procedures can be found, for example, in "Carbohydrate Modifications in Antisense Research", american Chemical Society, washington d.c.,1994, edited by Sangvi and Cook; "Remington's Pharmaceutical Sciences," Mack Publishing Co., easton, pa., 18 th edition, 1990; and "Antisense Drug Technology, principles, structures, and Applications," CRC Press, boca Raton, fla, edited by Stanley t.rooke; and Sambrook et al, "Molecular Cloning, A laboratory Manual," 2 nd edition, cold Spring Harbor Laboratory press, 1989, which are incorporated herein by reference for any purpose. All patents, applications, published applications and other publications mentioned throughout the disclosure herein, and other data, are incorporated herein by reference in their entirety, where permitted.

As used herein, the term "target nucleic acid" refers to any nucleic acid molecule whose expression or activity can be modulated by an siRNA compound. Target nucleic acids include, but are not limited to, RNAs transcribed from DNA encoding the target protein (including, but not limited to, pre-mrnas and mrnas or portions thereof), as well as cdnas derived from such RNAs, and mirnas. For example, a target nucleic acid may be a cellular gene whose expression is associated with a particular disorder or disease state (or mRNA transcribed from the gene). In some embodiments, the target nucleic acid may be a nucleic acid molecule from an infectious agent.

As used herein, the term "iRNA" refers to an agent that mediates targeted cleavage of an RNA transcript. These agents are associated with a cytoplasmic polyprotein complex called RNAi-induced silencing complex (RISC). Agents effective to induce RNA interference are also referred to herein as siRNA, RNAi agents, or iRNA agents. Accordingly, these terms may be used interchangeably herein. As used herein, the term iRNA includes micrornas and pre-micrornas. Furthermore, as used herein, the "compound" or "compounds" of the invention also refer to and are used interchangeably with iRNA agents.

The iRNA agent should include a region of sufficient homology to the target gene and be of sufficient length in terms of nucleotides such that the iRNA agent or fragment thereof can mediate down-regulation of the target gene. (for ease of illustration, the term nucleotide or ribonucleotide is sometimes used herein to refer to one or more monomeric subunits of an iRNA agent, it is to be understood herein that in the case of modified RNAs or nucleotide proxies, the use of the term "ribonucleotide" or "nucleotide" herein may also refer to modified nucleotides or proxy substitution portions at one or more positions.) thus, an iRNA agent is or includes a region that is at least partially, and in some embodiments, fully, complementary to a target RNA. There need not be complete complementarity between the iRNA agent and the target, but the correspondence must be sufficient for the iRNA agent or cleavage product thereof to be capable of directing sequence-specific silencing, e.g., by RNAi cleavage of the target RNA (e.g., mRNA). The degree of complementarity or homology to the target strand is most critical in the antisense strand. Although perfect complementarity is often required, particularly in the antisense strand, some embodiments may include, particularly in the antisense strand, one or more, or e.g., 6, 5, 4, 3, 2, or fewer mismatches (relative to the target RNA). The sense strand need only be sufficiently complementary to the antisense strand to maintain the overall double stranded character of the molecule.

iRNA agents include: molecules long enough to elicit an interferon response (which can be cleaved by Dicer (Bernstein et al 2001.Nature,409: 363-366) and enter RISC (RNAi-induced silencing complex)); and molecules short enough not to trigger an interferon reaction (which may also be cleaved by Dicer and/or enter RISC), e.g., molecules having a size that allows entry into RISC, e.g., molecules resembling Dicer-cleavage products. Molecules that are short enough not to elicit an interferon response are referred to herein as siRNA agents or short iRNA agents. As used herein, "siRNA agent or short iRNA agent" refers to an iRNA agent, e.g., a double-stranded RNA agent or a single-stranded agent, that is sufficiently short that it does not induce a detrimental interferon response in human cells, e.g., that has a duplex region of less than 60, 50, 40, or 30 nucleotide pairs. The siRNA agent or cleavage product thereof may down-regulate the target gene, for example, by inducing RNAi against the target RNA, where the target may comprise endogenous or pathogen target RNA.

As used herein, a "single-stranded iRNA agent" is an iRNA agent that consists of a single molecule. It may comprise duplex regions formed by intra-strand pairing, for example it may be or comprise a hairpin or disc handle structure. The single stranded iRNA agent may be antisense to the target molecule. The single stranded iRNA agent may be long enough that it can enter RISC and participate in RISC-mediated cleavage of target mRNA. The single stranded iRNA agent is at least 14 nucleotides in length, and in other embodiments is at least 15, 20, 25, 29, 35, 40, or 50 nucleotides in length. In certain embodiments, it is less than 200, 100, or 60 nucleotides in length.

A loop refers to a region of an iRNA strand that does not pair with an opposing nucleotide in a duplex when one segment of the iRNA strand forms a base pair with the other strand or with another segment of the same strand.

Hairpin iRNA agents will have a duplex region equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region may have a length equal to or less than 200, 100, or 50. In certain embodiments, the duplex region ranges in length from 15-30, 17-23, 19-23, and 19-21 nucleotide pairs. The hairpin may have a single stranded overhang or a terminal unpaired region, in some embodiments on the 3' side, and in certain embodiments on the antisense side of the hairpin. In some embodiments, the length of the overhang is 2-3 nucleotides.

As used herein, a "double-stranded (ds) iRNA agent" is an iRNA agent that includes more than one strand, and in some cases two strands, where strand hybridization can form a region of duplex structure.

As used herein, the terms "siRNA activity" and "RNAi activity" refer to gene silencing by siRNA.

As used herein, "gene silencing" by an RNA interference molecule refers to a decrease in mRNA level of a target gene in a cell of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% up to and including 100%, and any integer therebetween, of the mRNA level found in the cell in the absence of the miRNA or RNA interference molecule. In a preferred embodiment, the mRNA level is reduced by at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, up to and including 100%, and any integer between 5% and 100%.

As used herein, the term "modulating gene expression" means that the expression of a gene or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits is up-regulated or down-regulated such that the expression, level or activity is greater or less than that observed in the absence of a modulator. For example, the term "modulate" may mean "inhibit," but the use of the word "modulate" is not limited to this definition.

As used herein, modulation of gene expression occurs when expression of a gene or an RNA molecule encoding one or more proteins or protein subunits or equivalent RNA molecule differs by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold or more from the level observed in the absence of an siRNA, e.g., RNAi agent. The% and/or fold difference may be calculated relative to a control or non-control, e.g.,

or (b)

As used herein, the terms "inhibit," "down-regulate," or "reduce" in relation to gene expression means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is reduced below that observed in the absence of a modulator. Gene expression is down-regulated when the expression of the gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is reduced by at least 10%, and preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or most preferably 100% (i.e., no gene expression) relative to a corresponding unregulated control.

As used herein, the term "increase" or "up-regulation" in connection with gene expression means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is increased above that observed in the absence of a modulator. Gene expression is up-regulated when the expression of the gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, or the activity of one or more proteins or protein subunits, is increased by at least 10%, preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 100%, 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more relative to a corresponding unregulated control.

As used herein, the term "increased" or "increase" generally means an increase in a static significant amount; for the avoidance of any doubt, "increased" means increased by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including 100% increase or any increase between 10-100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold or at least about 10-fold increase, or any increase between 2-fold and 10-fold or more, as compared to a reference level, as compared to the reference level.

As used herein, the term "reduced" or "reduction" generally means a statistically significant amount of reduction. However, for the avoidance of doubt, "reduced" means reduced by at least 10% from the reference level, for example by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including 100% reduction (i.e. no level present as compared to the reference sample), or any reduction between 10-100%.

The double-stranded iRNA comprises two oligonucleotide strands that are sufficiently complementary to hybridize to form a duplex structure. Typically, the duplex structure is between 15 and 30 base pairs in length, more typically between 18 and 25 base pairs, still more typically between 19 and 24 base pairs, and most typically between 19 and 21 base pairs. In some embodiments, longer double stranded iRNA between 25 and 30 base pairs in length is preferred. In some embodiments, shorter double stranded iRNA between 10 and 15 base pairs in length is preferred. In another embodiment, the double stranded iRNA is at least 21 nucleotides in length.

In some embodiments, the double-stranded iRNA comprises a sense strand and an antisense strand, wherein the antisense RNA strand has a region of complementarity to at least a portion of the target sequence, and the duplex region is 14-30 nucleotides in length. Similarly, the length of the region complementary to the target sequence is between 14 and 30, more typically between 18 and 25, still more typically between 19 and 24, and most typically between 19 and 21 nucleotides.

As used herein, the phrase "antisense strand" refers to a strand of an oligonucleotide that is substantially or 100% complementary to a target sequence of interest. The phrase "antisense strand" includes the antisense region of two oligonucleotide strands formed from two separate strands, as well as single molecule oligonucleotide strands capable of forming a hairpin or dumbbell structure. The terms "antisense strand" and "guide strand" are used interchangeably herein.

The phrase "sense strand" refers to an oligonucleotide strand having a nucleotide sequence that is wholly or partially identical to a target sequence, such as a messenger RNA or DNA sequence. The terms "sense strand" and "passenger strand" are used interchangeably herein.

"specifically hybridizable" and "complementary" means that a nucleic acid can form hydrogen bonds with another nucleic acid sequence by conventional Watson-Crick or other non-conventional types. With respect to the nucleic acid molecules of the invention, the free energy of binding of the nucleic acid molecule to its complementary sequence is sufficient to allow performance of the relevant function of the nucleic acid, such as RNAi activity. Determination of the binding free energy of nucleic acid molecules is well known in the art (see, e.g., turner et al, 1987,CSH Symp.Quant.Biol.LII pp.123-133; frier et al, 1986,Proc.Nat.Acad.Sci.USA 83:9373-9377; turner et al, 1987,/. Am. Chem. Soc. 109:3783-3785). Percent complementarity indicates the percentage of consecutive residues in a nucleic acid molecule that can form hydrogen bonds (e.g., watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90% and 100% complementary). "complete complementarity" or 100% complementarity means that all consecutive residues of a nucleic acid sequence will form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. Incomplete complementarity refers to the situation in which some, but not all, of the nucleoside units of both strands can form hydrogen bonds with each other. "substantially complementary" refers to polynucleotide strands exhibiting 90% or greater complementarity, excluding regions, e.g., overhangs, of polynucleotide strands selected as non-complementary. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to the non-target sequence under conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, or in the case of in vitro assays under conditions in which the assay is performed. Non-target sequences typically differ by at least 5 nucleotides.

In some embodiments, the double stranded region is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.

In some embodiments, the antisense strand is equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In some embodiments, the sense strand has a length equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

27. 28, 29 or 30 nucleotides.

In one embodiment, the sense strand and the antisense strand are each 15 to 30 nucleotides in length. In one embodiment, the sense strand and the antisense strand are each 19-25 nucleotides in length. In one embodiment, the sense strand and the antisense strand are each 21 to 23 nucleotides in length.

In some embodiments, one strand has at least one segment of 1-5 single stranded nucleotides in the double stranded region. By "single stranded nucleotide segment in a double stranded region" is meant that there is at least one nucleotide base pair at both ends of the single stranded segment. In some embodiments, both strands have at least one segment of 1-5 (e.g., 1, 2, 3, 4, or 5) single stranded nucleotides in the double stranded region. When two strands have a segment of 1-5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double-stranded region, such single-stranded nucleotides can be opposite each other (e.g., mismatched segments), or they can be positioned such that the second strand does not have a single-stranded nucleotide opposite the single-stranded iRNA of the first strand, and vice versa (e.g., single-stranded loops). In some embodiments, single stranded nucleotides are present within 8 nucleotides from either end, e.g., 8, 7, 6, 5, 4, 3, or 2 nucleotides from the 5 'or 3' end of the region of complementarity between the two strands.

In one embodiment, the oligonucleotide comprises a single stranded overhang at least one terminus. In one embodiment, the single stranded overhang is 1, 2 or 3 nucleotides in length.

In one embodiment, the sense strand of the iRNA agent is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, wherein the strand forms a double-stranded region of 21 consecutive base pairs with a single-stranded overhang of 2 nucleotides in length at the 3' -end.

In some embodiments, each strand of the double-stranded iRNA has a ZXY structure, e.g., as described in PCT publication No. 2004080406, which is incorporated herein by reference in its entirety.

In certain embodiments, the two strands of a double-stranded oligonucleotide may be joined together. The two strands may be linked to each other at both ends or at only one end. By linked at one end is meant that the 5 '-end of the first strand is linked to the 3' -end of the second strand or that the 3 '-end of the first strand is linked to the 5' -end of the second strand. When the two strands are linked to each other at both ends, the 5 '-end of the first strand is linked to the 3' -end of the second strand, and the 3 '-end of the first strand is linked to the 5' -end of the second strand. The two strands may be joined together by an oligonucleotide linker, including but not limited to (N) _n The method comprises the steps of carrying out a first treatment on the surface of the Wherein N is independently a modified or unmodified nucleotide and N is 3-23. In some embodiments, n is 3-10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from GNRA, (G) ₄ 、(U) ₄ And (dT) ₄ Wherein N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide. Some nucleotides in the linker may be involved in base pair interactions with other nucleotides in the linker.The two strands may also be joined together by a non-nucleoside linker, such as the linkers described herein. Those of skill in the art will appreciate that any oligonucleotide chemical modification or variation described herein may be used for the oligonucleotide adaptors.

Hairpin and dumbbell oligonucleotides will have duplex regions equal to or at least 14, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24 or 25 nucleotide pairs. The duplex region may have a length equal to or less than 200, 100, or 50. In some embodiments, the duplex region ranges in length from 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs.

Hairpin oligonucleotides may have a single stranded overhang or terminal unpaired region, in some embodiments on the 3' side, and in some embodiments on the antisense side of the hairpin. In some embodiments, the length of the overhang is 1-4, more typically 2-3 nucleotides. Hairpin oligonucleotides that induce RNA interference are also referred to herein as "shRNA".

In certain embodiments, when a sufficient degree of complementarity is present, the two oligonucleotide strands specifically hybridize to avoid non-specific binding of the antisense strand to the non-target nucleic acid sequence under conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, and under conditions where the assay is performed in vitro.

As used herein, "stringent hybridization conditions" or "stringent conditions" refer to conditions under which the antisense strand hybridizes to its target sequence, but hybridizes to a minimum number of other sequences. Stringent conditions are sequence dependent and in different cases, and the "stringent conditions" for hybridization of the antisense strand to the target sequence are determined by the nature and composition of the antisense strand and the assays in which it is studied.

It is understood in the art that the introduction of nucleotide affinity modifications may allow for a greater number of mismatches than unmodified oligonucleotides. Similarly, certain oligonucleotide sequences may be more tolerant of mismatches than others. One of ordinary skill in the art can determine the appropriate number of mismatches between oligonucleotides or between an oligonucleotide and a target nucleic acid, for example, by determining the melting temperature (Tm). Tm or Δtm may be calculated by techniques familiar to those of ordinary skill in the art. For example, freier et al (Nucleic Acids Research,1997,25, 22:4429-4443) describe techniques that allow one of ordinary skill in the art to evaluate the ability of nucleotide modifications to increase the melting temperature of RNA: DNA duplex.

Additional dsRNA design

In one embodiment, the iRNA agent is a blunt end at 19nt in length, wherein the sense strand contains at least one three 2'-F modified motifs on three consecutive nucleotides at positions 7, 8, 9 of the 5' -end. The antisense strand contains at least one three 2 '-O-methyl modified motifs on three consecutive nucleotides at positions 11, 12, 13 of the 5' -end.

In one embodiment, the iRNA agent is a 20nt long, double-ended blunt end in which the sense strand contains at least one three 2'-F modified motifs on three consecutive nucleotides at positions 8, 9, 10 of the 5' -end. The antisense strand contains at least one three 2 '-O-methyl modified motifs on three consecutive nucleotides at positions 11, 12, 13 of the 5' -end.

In one embodiment, the iRNA agent is a double-ended blunt end of length 21nt, wherein the sense strand contains at least one three 2'-F modified motifs on three consecutive nucleotides at positions 9, 10, 11 of the 5' end. The antisense strand contains at least one three 2 '-O-methyl modified motifs on three consecutive nucleotides at positions 11, 12, 13 of the 5' -end.

In one embodiment, the iRNA agent comprises a sense strand of 21 nucleotides (nt) and an antisense strand of 23 nucleotides (nt), wherein the sense strand contains at least one three 2'-F modified motifs on three consecutive nucleotides at positions 9, 10, 11 of the 5' end; the antisense strand contains at least one three 2 '-O-methyl modified motifs on three consecutive nucleotides at positions 11, 12, 13 of the 5' end, wherein one end of the iRNA is blunt and the other end comprises a 2nt overhang. Preferably, the 2nt overhang is at the antisense 3' -end. Optionally, the iRNA agent further comprises a ligand (e.g., galNAc ₃ )。

In one embodiment, the iRNA agent comprises sense and antisense strands, wherein: the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5' -terminal nucleotide (position 1), positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3' -terminal nucleotide, comprises at least 8 ribonucleotides in the position that pairs with positions 1-23 of the sense strand to form a duplex; wherein at least the 3' -terminal nucleotide of the antisense strand is unpaired with the sense strand and at most 6 consecutive 3' -terminal nucleotides are unpaired with the sense strand, thereby forming a 1-6 nucleotide 3' single stranded overhang; wherein the 5 '-end of the antisense strand comprises 10-30 consecutive nucleotides that are unpaired with the sense strand, thereby forming a single-stranded 5' -overhang of 10-30 nucleotides; wherein when the sense strand and the antisense strand are aligned for maximum complementarity, at least the 5 '-end and 3' -end nucleotides of the sense strand pair with the nucleotide bases of the antisense strand, thereby forming a region of substantial duplex between the sense strand and the antisense strand; and when the double stranded nucleic acid is introduced into a mammalian cell, at least 19 ribonucleotides of the antisense strand along the length of the antisense strand are sufficiently complementary to the target RNA to reduce target gene expression; and wherein the sense strand contains at least one three 2' -F modified motifs on three consecutive nucleotides, wherein at least one motif occurs at or near the cleavage site. The antisense strand contains at least one three 2' -O-methyl modified motifs on three consecutive nucleotides at or near the cleavage site.

In one embodiment, an iRNA agent comprises a sense strand and an antisense strand, wherein the iRNA agent comprises a first strand of at least 25 and at most 29 nucleotides in length and a second strand of at most 30 nucleotides in length having at least one three 2 '-O-methyl modified motifs on three consecutive nucleotides from positions 11, 12, 13 of the 5' end; wherein said 3' end of said first strand and said 5' end of said second strand form a blunt end and said second strand is 1-4 nucleotides longer at its 3' end than the first strand length, wherein the duplex region is at least 25 nucleotides in length and said second strand is sufficiently complementary to the target mRNA along at least 19nt of said second strand length to introduce mammalian fines at said iRNA agentReducing target gene expression while in the cell, and wherein Dicer cleavage of the iRNA preferentially generates siRNA comprising the 3' end of the second strand, thereby reducing target gene expression in the mammal. Optionally, the iRNA agent further comprises a ligand (e.g., galNAc ₃ )。

In one embodiment, the sense strand contains at least one three identical modified motifs on three consecutive nucleotides, wherein one motif occurs at the cleavage site of the sense strand. For example, the sense strand may contain at least one three 2'-F modified motifs on three consecutive nucleotides within positions 7-15 of the 5' -end.

In one embodiment, the antisense strand may also contain at least one three identical modified motifs on three consecutive nucleotides, wherein one motif occurs at or near the cleavage site in the antisense strand. For example, the antisense strand may contain at least one three 2 '-O-methyl modified motifs on three consecutive nucleotides within positions 9-15 from the 5' -end.

For iRNA agents having duplex regions of 17-23nt in length, the cleavage sites of the antisense strand are typically around positions 10, 11 and 12 of the 5' -end. Thus, three identical modified motifs may occur at positions 9, 10, 11; 10. 11 and 12 positions; 11. 12, 13 positions; 12. 13 and 14 positions; or positions 13, 14, 15 of the antisense strand, counting from nucleotide 1 at the 5 '-end of the antisense strand, or counting from nucleotide 1 paired in the duplex region at the 5' -end of the antisense strand. The cleavage site in the antisense strand can also vary depending on the length of the duplex region of the iRNA starting from the 5' -end.

In some embodiments, the iRNA agent comprises a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand contains at least two motifs that are identically modified on three consecutive nucleotides, wherein at least one motif occurs at or near a cleavage site within the strand, and at least one motif occurs at another portion of the strand that is separated from the motif at the cleavage site by at least one nucleotide. In one embodiment, the antisense strand further comprises at least one motif with three identical modifications over three consecutive nucleotides, wherein at least one motif occurs at or near the cleavage site within the strand. The modification in the motif present at or near the cleavage site in the sense strand differs from the modification in the motif present at or near the cleavage site in the antisense strand.

In some embodiments, the iRNA agent comprises a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand contains at least one three 2' -F modified motifs on three consecutive nucleotides, wherein at least one motif occurs at or near a cleavage site in the strand. In one embodiment, the antisense strand further comprises at least one three 2' -O-methyl modified motifs on three consecutive nucleotides at or near the cleavage site.

In some embodiments, the iRNA agent comprises a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand comprises at least one three 2'-F modified motifs on three consecutive nucleotides from positions 9, 10, 11 of the 5' end, and wherein the antisense strand comprises at least one three 2 '-O-methyl modified motifs on three consecutive nucleotides from positions 11, 12, 13 of the 5' end.

In one embodiment, the iRNA agent comprises a mismatch to the target or a combination thereof within the duplex. Mismatches may occur in the overhang region or duplex region. Base pairs may be ordered based on their propensity to promote dissociation or melting (e.g., based on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairing on a single pairing basis, although secondary neighbors or similar assays may also be used). In promoting dissociation: a is better than G and C; g is better than G and C; and C is superior to G: C (i=inosine). Mismatches, e.g., non-canonical pairs or pairs outside of the canonical (as described elsewhere herein) are better than canonical (A: T, A: U, G: C) pairs; and pairing including universal bases is preferred over canonical pairing.

In one embodiment, the iRNA agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs from the 5' -end of the antisense strand within the duplex region, which may be independently selected from: a U, G: U, I:C and mismatch pairs, e.g., non-canonical or non-canonical pairs or pairs that include universal bases, to facilitate dissociation of the antisense strand at the 5' -end of the duplex.

In one embodiment, the nucleotide at position 1 from the 5' -end of the antisense strand within the duplex region is selected from A, dA, dU, U and dT. Alternatively, at least one of the first 1, 2 or 3 base pairs from the 5' -end of the antisense strand within the duplex region is an AU base pair. For example, the first base pair from the 5' -end of the antisense strand within the duplex region is an AU base pair.

In one embodiment, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the dsRNA agent is modified. For example, when 50% of the dsRNA agent is modified, 50% of all nucleotides present in the dsRNA agent contain modifications as described herein.

In some embodiments, the oligonucleotide contains one or more 2'-O modifications selected from the group consisting of 2' -deoxy, 2 'O-methoxyalkyl, 2' -O-methyl, 2 '-O-allyl, 2' -C-allyl, 2 '-fluoro, 2' -O-N-methylacetamido (2 '-O-NMA), 2' -O-dimethylaminoethoxyethyl (2 '-O-DMAEOE), 2' -O-aminopropyl (2 '-O-AP), and 2' -ara-F.

In one embodiment, the sense strand and the antisense strand are each independently modified with an unnatural nucleotide, e.g., an acyclic nucleotide, LNA, HNA, ceNA, 2' -methoxyethyl, 2' -O-methyl, 2' -O-allyl, 2' -C-allyl, 2' -deoxy, 2' -fluoro, 2' -O-N-methylacetamido (2 ' -O-NMA), 2' -O-dimethylaminoethoxyethyl (2 ' -O-DMAEOE), 2' -O-aminopropyl (2 ' -O-AP), or 2' -ara-F.

In one embodiment, the sense strand and the antisense strand of the dsRNA agent each contain at least two different modifications.

In some embodiments, the oligonucleotide contains one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve 2' -F modifications. In one example, the oligonucleotide contains nine or ten 2' -F modifications.

In one embodiment, the oligonucleotide does not contain any 2' -F modifications.

The iRNA agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. Phosphorothioate or methylphosphonate internucleotide linkage modifications may occur on any nucleotide in any position of the sense strand or antisense strand or both. For example, internucleotide linkage modifications may occur on each nucleotide on the sense or antisense strand; each internucleotide linkage modification may occur in alternating patterns on either the sense strand or the antisense strand; or the sense or antisense strand may contain an alternating pattern of two internucleotide linkage modifications. The alternating pattern of internucleotide linkage modifications on the sense strand may be the same as or different from the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may be offset relative to the alternating pattern of internucleotide linkage modifications on the antisense strand.

In one embodiment, the iRNA comprises phosphorothioate or methylphosphonate internucleotide linkage modifications in the region of the overhang. For example, an overhang region may contain two nucleotides with phosphorothioate or methylphosphonate internucleotide linkages between the two nucleotides. Internucleotide linkage modifications may also be made to link the overhanging nucleotides to terminal pairing nucleotides within the duplex region. For example, at least 2, 3, 4 or all of the overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkages, and optionally, additional phosphorothioate or methylphosphonate internucleotide linkages can be present, which link the overhang nucleotide to the paired nucleotide of the adjacent overhang nucleotide. For example, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are the overhang nucleotides and the third is the pairing nucleotide adjacent to the overhang nucleotide. Preferably, these terminal three nucleotides may be at the 3' -end of the antisense strand.

In one embodiment, the sense strand and/or antisense strand comprises one or more phosphorothioate or methylphosphonate internucleotide linked blocks. In one example, the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand comprises two blocks of phosphorothioate or methylphosphonate internucleotide linkages. For example, two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16-18 phosphate internucleotide linkages.

In one embodiment, the sense strand and the antisense strand each have 15-30 nucleotides. In one example, the sense strand has 19-22 nucleotides and the antisense strand has 19-25 nucleotides. In another example, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

In one embodiment, the nucleotide at position 1 of the 5' -end of the antisense strand in the duplex is selected from A, dA, dU, U and dT. In one embodiment, at least one of the first, second and third base pairs from the 5' -end of the antisense strand is an AU base pair.

In one embodiment, the antisense strand of the dsRNA agent is 100% complementary to the target RNA to hybridize thereto and inhibit its expression by RNA interference. In another embodiment, the antisense strand of the dsRNA agent is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to the target RNA.

In one aspect, the invention relates to a dsRNA agent as defined herein capable of inhibiting expression of a target gene. dsRNA agents comprise a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand contains at least one thermally destabilizing nucleotide, wherein at least one of the thermally destabilizing nucleotides occurs at or near a site opposite the seed region of the antisense strand (i.e., at positions 2-8 of the 5' -end of the antisense strand).

For example, when the sense strand is 21 nucleotides in length, the thermally destabilizing nucleotide may occur between positions 14-17 of the 5' -end of the sense strand. The antisense strand contains at least two modified nucleic acids that are less than the sterically required 2' -OMe modification. Preferably, two modified nucleic acids that are less than the sterically required 2' -OMe are separated by 11 nucleotides in length. For example, two modified nucleic acids are located at positions 2 and 14 of the 5' -end of the antisense strand.

In one embodiment, the dsRNA agent comprises:

(a) A sense strand having:

(i) 18-23 nucleotides in length;

(ii) Three consecutive 2' -F modifications at positions 7-15; and

(b) An antisense strand having:

(i) 18-23 nucleotides in length;

(ii) At least 2' -F modification at any position on the chain; and

(iii) At least two phosphorothioate internucleotide linkages at the first five nucleotides (counted from the 5' end);

wherein the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand; and either has an overhang of two nucleotides at the 3 '-end of the antisense strand and a blunt end at the 5' -end of the antisense strand; or have blunt ends at both ends of the duplex.

In one embodiment, the dsRNA agent comprises:

(a) A sense strand having:

(i) 18-23 nucleotides in length;

(ii) Less than four 2' -F modifications;

(b) An antisense strand having:

(i) 18-23 nucleotides in length;

(ii) Less than twelve 2' -F modifications; and

In one embodiment, the dsRNA agent comprises:

(a) A sense strand having:

(i) 19-35 nucleotides in length;

(ii) Less than four 2' -F modifications;

(b) An antisense strand having:

(i) 19-35 nucleotides in length;

(ii) Less than twelve 2' -F modifications; and

wherein the duplex region is between 19 and 25 base pairs (preferably 19, 20, 21 or 22); and wherein the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand; and either has an overhang of two nucleotides at the 3 '-end of the antisense strand and a blunt end at the 5' -end of the antisense strand; or have blunt ends at both ends of the duplex.

In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand of 15-30 nucleotides in length; at least two phosphorothioate internucleotide linkages at the first five nucleotides (counted from the 5' end) on the antisense strand; wherein the duplex region is between 19 and 25 base pairs (preferably 19, 20, 21 or 22); wherein the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand; and wherein the dsRNA agent has less than 20%, less than 15%, and less than 10% non-natural nucleotides.

In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand of 15-30 nucleotides in length; at least two phosphorothioate internucleotide linkages at the first five nucleotides (counted from the 5' end) on the antisense strand; wherein the duplex region is between 19 and 25 base pairs (preferably 19, 20, 21 or 22); wherein the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand; and wherein the dsRNA agent has greater than 80%, greater than 85%, and greater than 90% of natural nucleotides, e.g., 2' -OH, 2' -deoxy, and 2' -OMe are natural nucleotides.

In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand of 15-30 nucleotides in length; at least two phosphorothioate internucleotide linkages at the first five nucleotides (counted from the 5' end) on the antisense strand; wherein the duplex region is between 19 and 25 base pairs (preferably 19, 20, 21 or 22); wherein the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand; and wherein the dsRNA agent has 100% natural nucleotides, e.g., 2' -OH, 2' -deoxy, and 2' -OMe are natural nucleotides.

In one embodiment, the dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the sense strand sequence is represented by formula (I):

5’n _p -N _a -(XXX) _i -N _b -YYY-N _b -(ZZZ) _j -N _a -n _q 3’

(I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0 to 6;

N _a each independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two different modified nucleotides;

N _b each independently represents an oligonucleotide sequence comprising 1, 2, 3, 4, 5 or 6 modified nucleotides;

n _p and n _q Each independently represents an overhang nucleotide;

Wherein N is _b And Y does not have the same modification;

wherein XXX, YYY and ZZZ each independently represents one motif of three identical modifications over three consecutive nucleotides;

wherein the dsRNA agent has one or more lipophilic monomers containing one or more lipophilic moieties conjugated to one or more positions on at least one strand; and is also provided with

Wherein the antisense strand of the dsRNA comprises two blocks of one, two or three phosphorothioate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 phosphointernucleotide linkages.

Various publications describe multimeric siRNA and can be used with the iRNA of the invention. Such publications include WO2007/091269, U.S. patent No. 7858769, WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520, which are incorporated herein by reference in their entirety.

In some embodiments, the antisense strand is 100% complementary to the target RNA to hybridize thereto and inhibit its expression by RNA interference. In another embodiment, the antisense strand is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to the target RNA.

Nucleic acid modification

In some embodiments, the oligonucleotide comprises at least one nucleic acid modification described herein. For example, the at least one modification is selected from the group consisting of a modified internucleoside linkage, a modified nucleobase, a modified sugar, and any combination thereof. Without limitation, such modifications may be present anywhere in the oligonucleotide. For example, the modification may be present in one of the RNA molecules.

Nucleic acid modification (nucleobases)

The naturally occurring base portion of a nucleoside is typically a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. For those nucleosides that include ribofuranosyl groups, the phosphate group can be attached to the 2', 3', or 5' hydroxyl moiety of the sugar. In forming the oligonucleotide, those phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In oligonucleotides, phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The naturally occurring linkage or backbone of RNA and DNA is a 3 'to 5' phosphodiester linkage.

In addition to "unmodified" or "natural" nucleobases such as the purine nucleobases adenine (a) and guanine (G) and the pyrimidine nucleobases thymine (T), cytosine (c) and uracil (U), many modified nucleobases or nucleobase mimics known to those skilled in the art are also suitable for use in the oligonucleotides described herein. Unmodified or natural nucleobases can be modified or substituted to provide iRNA with improved properties. For example, nuclease-resistant oligonucleotides can be prepared with these bases or modified with synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanosine, or tubercidine) and any of the oligomers described herein. Alternatively, substituted or modified analogues of any of the bases and "universal bases" described above may be used. When a natural base is replaced with a non-natural and/or universal base, a nucleotide is referred to herein as comprising a modified nucleobase and/or nucleobase modification. Modified nucleobases and/or nucleobase modifications also include natural, unnatural and universal bases comprising a conjugate moiety, e.g., a ligand as described herein. Preferred conjugation moieties for conjugation to a nucleobase include cationic amino groups, which can be conjugated to the nucleobase through a suitable alkyl, alkenyl or linker having an amide bond.

Oligonucleotides described herein may also include nucleobase (commonly referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (a) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (c) and uracil (U). Exemplary modified nucleobases include, but are not limited to, other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularine, isoguanine, tubucine, 2- (halo) adenine, 2- (alkyl) adenine, 2- (propyl) adenine, 2- (amino) adenine, 2- (aminoalkyl) adenine, 2- (aminopropyl) adenine, 2- (methylthio) -N ⁶ - (isopentenyl) adenine, 6- (alkyl) adenine, 6- (methyl) adenine, 7- (deazated) adenine, 8- (alkenyl) adenine, 8- (alkyl) adenine, 8- (alkynyl) adenine, 8- (amino) adenine, 8- (halo) adenine, 8- (hydroxy) adenine, 8- (thioalkyl) adenine, 8- (thiol) adenine, N ⁶ - (isopentyl) adenine, N ⁶ - (methyl) adenine, N ⁶ ,N ⁶ - (dimethyl) adenine, 2- (alkyl) guanine, 2- (propyl) guanine, 6- (alkyl) guanineIn, 6- (methyl) guanine, 7- (alkyl) guanine, 7- (methyl) guanine, 7- (deaza) guanine, 8- (alkyl) guanine, 8- (alkenyl) guanine, 8- (alkynyl) guanine, 8- (amino) guanine, 8- (halo) guanine, 8- (hydroxy) guanine, 8- (thioalkyl) guanine, 8- (thiol) guanine, N- (methyl) guanine 2- (thio) cytosine, 3- (deaza) -5- (aza) cytosine, 3- (alkyl) cytosine, 3- (methyl) cytosine, 5- (alkyl) cytosine, 5- (alkynyl) cytosine, 5- (halo) cytosine, 5- (methyl) cytosine, 5- (propynyl) cytosine, 5- (trifluoromethyl) cytosine, 6- (azo) cytosine, N ⁴ - (acetyl) cytosine, 3- (3-amino-3-carboxypropyl) uracil, 2- (thio) uracil, 5- (methyl) -2- (thio) uracil, 5- (methylaminomethyl) -2- (thio) uracil, 4- (thio) uracil, 5- (methyl) -4- (thio) uracil, 5- (methylaminomethyl) -4- (thio) uracil, 5- (methyl) -2,4- (dithio) uracil, 5- (methylaminomethyl) -2,4- (dithio) uracil, 5- (2-aminopropyl) uracil, 5- (alkyl) uracil, 5- (alkynyl) uracil, 5- (allylamino) uracil, 5- (aminoallyl) uracil, 5- (aminoalkyl) uracil, 5- (guanidinoalkyl) uracil, 5- (1, 3-diazol-1-alkyl) uracil, 5- (cyanoalkyl) uracil, 5- (dialkylaminoalkyl) uracil, 5- (dimethylaminoalkyl) uracil, 5- (halo) uracil, 5- (methoxy) uracil, 5-hydroxy-acetic acid, 5- (methoxycarbonylmethyl) -2- (thio) uracil, 5- (methoxycarbonyl-methyl) uracil, 5- (propynyl) uracil, 5- (trifluoromethyl) uracil, 6- (azo) uracil, dihydro-uracil, N ³ - (methyl) uracil, 5-uracil (i.e., pseudouracil), 2- (thio) pseudouracil, 4- (thio) pseudouracil, 2,4- (dithio) pseudouracil, 5- (alkyl) pseudouracil, 5- (methyl) pseudouracil, 5- (alkyl) -2- (thio) pseudouracil, 5- (methyl) -2- (thio) pseudouracil, 5- (alkyl) -4- (thio) pseudouracil, 5- (methyl) -4- (thio) pseudouracil, 5- (alkyl) -2,4- (dithio) pseudouracil, 5- (methyl) -2,4- (dithio) pseudouracil, 1-substituted 2- (thio) -pseudouracil, 1-substituted pseudouracil 4- (thio) pseudouracil, 1-substituted 2,4- (dithio) pseudouracil, 1- (aminocarbonylvinyl) -2- (thio) -pseudouracil, 1- (aminocarbonylvinyl) -4- (thio) pseudouracil, 1- (aminocarbonylvinyl) -2,4- (dithio) pseudouracil, 1- (aminoalkylaminocarbonylvinyl) -pseudouracil, 1- (aminoalkylamino-carbonylvinyl) -2- (thio) pseudouracil, 1- (aminoalkylaminocarbonylvinyl) -4- (thio) pseudouracil, 1- (aminoalkylaminocarbonylvinyl) -2,4- (dithio) pseudouracil, 1,3- (diaza) -2- (oxo) -phenoxazin-1-yl, 1- (aza) -2- (thio) -3- (aza) -phenoxazin-1-yl, 1,3- (diaza) -2- (oxo) -phenothiazin-1-yl, 1- (aza) -2- (thio) -3- (aza) -phenothiazin-1-yl, 1- (aza) -3- (aza) -1-phenothiazin-1-yl, 7-substituted 1,3- (diaza) -2- (oxo) -phenoxazin-1-yl, 7-substituted 1- (aza) -2- (thio) -3- (aza) -phenoxazin-1-yl, 7-substituted 1,3- (diaza) -2- (oxo) -phenothiazin-1-yl, 7-substituted 1- (aza) -2- (thio) -3- (aza) -phenothiazin-1-yl, 7- (aminoalkylhydroxy) -1,3- (diaza) -2- (oxo) -phenoxazin-1-yl, 7- (aminoalkylhydroxy) -1- (aza) -2- (thio) -3- (aza) -phenoxazin-1-yl, 7- (aminoalkylhydroxy) -1,3- (diaza) -2- (oxo) -phenothiazin-1-yl, 7- (aminoalkylhydroxy) -1- (aza) -2- (thio) -3- (aza) -phenothiazin-1-yl, 7- (alkylhydroxy) -1,3- (diaza) -2- (oxo) -phenoxazin-1-yl, 7- (alkylalkylhydroxy) -1,3- (diaza) -2- (oxo) -phenoxazin-1-yl, 7- (guanidylhydroxy) -1- (aza) -2- (aza) -3- (aza) -phenoxazin-1-yl, 7- (guanidylhydroxy) -1,3- (diaza) -2- (oxo) -phenothiazin-1-yl, 7- (guanidylhydroxy) -1- (aza) -2- (thio) -3- (aza) -phenothiazin-1-yl, 1,3,5- (triaza) -2,6- (dioxa) -naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanosine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitroimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3- (meth) isoquinolyl (isocarbostyril), 5- (meth) -7- (propynyl) isoquinolyl, 7- (aza) indolyl, 6- (meth) -7- (aza) -indolyl, imidazopyridinyl, pyrrolyl, pyrrolopyrazinyl, pyrrolyl, pyrrolidoquinolinyl, pyrrolidoyl, pyrrolidoquinolinyl, or the like Alkenyl) isoquinolinyl, propynyl-7- (aza) indolyl, 2,4,5- (trimethyl) phenyl, 4- (methyl) indolyl, 4,6- (dimethyl) indolyl, phenyl, naphthyl, anthryl, phenanthryl, pyrenyl, stilbene, tetracenyl, pentacenyl, difluoromethylphenyl, 4- (fluoro) -6- (methyl) benzimidazole, 4- (methyl) benzimidazole, 6- (azo) thymine, 2-pyridone, 5-nitroindole, 3-nitropyrrole, 6- (aza) pyrimidine, 2- (amino) purine, 2,6- (diamino) purine, 5-substituted pyrimidine, N ² -substituted purines, N ⁶ -substituted purines, O ⁶ -substituted purines, substituted 1,2, 4-triazoles, pyrrolopyrimidin-2-one-3-yl, 6-phenyl-pyrrolopyrimidin-2-one-3-yl, para-substituted-6-phenyl-pyrrolopyrimidin-2-one-3-yl, ortho-substituted-6-phenyl-pyrrolopyrimidin-2-one-3-yl, di-ortho-substituted-6-phenyl-pyrrolopyrimidin-2-one-3-yl, para- (aminoalkylhydroxy) -6-phenyl-pyrrolopyrimidin-2-one-3-yl, ortho- (aminoalkylhydroxy) -6-phenyl-pyrrolopyrimidin-2-one-3-yl, di-ortho- (aminoalkylhydroxy) -6-phenyl-pyrrolopyrimidin-2-one-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl or any O-alkylated or N-alkylated derivatives thereof. Alternatively, substituted or modified analogues of any of the above bases and "universal bases" may be employed.

As used herein, a universal nucleobase is any nucleobase that can base pair with all four naturally occurring nucleobases without substantially affecting melting behavior, recognition by intracellular enzymes, or iRNA duplex activity. Some exemplary general nucleobases include, but are not limited to, 2, 4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisoquinolinyl, 5-methylisoquinolinyl, 3-methyl-7-propynylisoquinolinyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidazopyridinyl, 9-methyl-imidazopyridinyl, pyrrolopyrazinyl, isoquinolyl, 7-propynylisoquinolyl, propynyl-7-azaindolyl, 2,4, 5-trimethylphenyl, 4-methylindolyl, 4, 6-dimethylindolyl, phenyl, naphthyl, anthracenyl, phenanthrenyl, pyrenyl, stilbene, and structural derivatives thereof (see, e.g., loes, 2001,Nucleic Acids Research,29,2437-2447).

Additional nucleobases include those disclosed in U.S. Pat. No. 3,687,808; those disclosed in international application number PCT/US09/038425 submitted 26 days 3 months 2009; on pages Concise Encyclopedia Of Polymer Science And Engineering, 858-859, kroschwitz, j.i. editions of John Wiley & Sons, 1990; those disclosed by englist et al Angewandte Chemie, international Edition,1991,30,613; those disclosed in Modified Nucleosides in Biochemistry, biotechnology and Medicine, herdiewijin, p.ed.wiley-VCH, 2008; and those disclosed by Sanghvi, y.s., chapter 15, dsRNAResearch and Applications, pages 289-302, rooke, s.t., and lebleeu, b. editions, CRC press, 1993. All of the foregoing is incorporated herein by reference.

In certain embodiments, the modified nucleobase is a nucleobase that is substantially similar in structure to the parent nucleobase, such as 7-deazapurine, 5-methylcytosine, or G-clamp. In certain embodiments, nucleobase mimics include more complex structures, such as tricyclic phenoxazine nucleobase mimics. Methods for preparing the modified nucleobases described above are well known to those skilled in the art.

Nucleic acid modification (sugar)

The oligonucleotides provided herein can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) monomers, including nucleosides or nucleotides having modified sugar moieties. For example, the furanosyl sugar ring of a nucleoside can be modified in a variety of ways, including but not limited to adding substituents, bridging two non-geminal ring atoms to form a locked or bicyclic nucleic acid. In certain embodiments, the oligonucleotide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) monomers that are LNAs.

In some embodiments of the locked nucleic acid, the 2 'position of the furanosyl is linked to the 4' position by a linker independently selected from the group consisting of- [ C (R1) (R2) ] _n -、-[C(R1)(R2)] _n -O-、-[C(R1)(R2)] _n -N(R1)-、-[C(R1)(R2)] _n -N(R1)-O-、-[C(R1R2)] _n -O-N(R1)-、-C(R1)＝C(R2)-O-、-C(R1)＝N-、-C(R1)＝N-O-、-C(═NR1)-、-C(═NR1)-O-、-C(═O)-、-C(═O)O-、-C(═S)-、-C(═S)O-、-C(═S)S-、-O-、-Si(R1)2-、-S(═O) _X -and-N (R1) -;

wherein:

x is 0, 1 or 2;

n is 1, 2, 3 or 4;

r1 and R2 are each independently H, a protecting group, hydroxy, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocyclyl, substituted heterocyclyl, heteroaryl, substituted heteroaryl, C5-C7 alicyclic, substituted C5-C7 alicyclic, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C (=O) -H), substituted acyl, CN, sulfonyl (S (=O) 2-J1) or thioxy (S (=O) -J1); and is also provided with

J1 and J2 are each independently H, C C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C (=O) -H), substituted acyl, heterocyclyl, substituted heterocyclyl, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl or a protecting group.

In some embodiments, the linkers of the LNA compounds are each independently- [ C (R1) (R2)]n-、-[C(R1)(R2)]N-O-, -C (R1R 2) -N (R1) -O-or-C (R1R 2) -O-N (R1) -. In another embodiment, the linkers are each independently 4' -CH ₂ -2’、4’-(CH ₂ ) ₂ -2’、4’-(CH ₂ ) ₃ -2’、4’-CH ₂ -O-2’、4’-(CH ₂ ) ₂ -O-2’、4’-CH ₂ -O-N (R1) -2 'and 4' -CH ₂ -N (R1) -O-2' -, wherein R1 are each independently H, a protecting group or C1-C12 alkyl.

Some LNAs have been prepared and disclosed in the patent literature and scientific literature (Singh et al, chem. Commun.,1998,4,455-456; koshkin et al, tetrahedron,1998,54,3607-3630; wahlstedt et al, proc. Natl. Acad. Sci. U.S.A.,2000,97,5633-5638; kumar et al, bioorg. Med. Chem. Lett.,1998,8,2219-2222; WO 94/14226; WO 2005/021570; singh et al, J. Org. Chem.,1998,63,10035-10039; examples of issued U.S. Pat. Nos. 7,053,207;6,268,490;6,770,748;6,794,499;7,034,133; and 6,525,191; and prior disclosures 2004-0170; 2004-0219565; 2004-001784959; 3541; 0143114; 3541; and 20030082807).

Also provided herein is an LNA in which the 2' -hydroxy group of the ribosyl sugar ring is attached to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy group (4 ' -CH ₂ O-2') to form a bicyclic sugar moiety (reviewed in Elayadi et al, curr. Opinion Invens. Drugs,2001,2,558-561; braasch et al chem.biol.,2001,8, 1-7; and Orum et al, curr. Opiion mol. Ter., 2001,3,239-243; see also U.S. Pat. nos. 6,268,490 and 6,670,461). The linkage may be methylene (-CH) bridging the 2 'oxygen atom and the 4' carbon atom ₂ The (-) group, for which the term methyleneoxy (4' -CH) ₂ -O-2') LNA for a dual loop part; in the case where the position is ethylene, the term ethyleneoxy (4' -CH ₂ CH ₂ -O-2') LNA (Singh et al, chem. Commun.,1998,4,455-456: morita et al, bioorganic Medicinal Chemistry,2003,11,2211-2226). Methyleneoxy (4' -CH) ₂ -O-2 ') LNA and other bicyclic sugar analogues show very high duplex thermal stability (tm= +3 to +10 ℃) with complementary DNA and RNA, stability to 3' -exonucleolytic degradation and good solubility properties. Efficient and non-toxic antisense oligonucleotides comprising BNA have been described (Wahlestedt et al, proc.Natl. Acad.Sci.U.S. A.,2000,97,5633-5638).

The methyleneoxy group (4' -CH) ₂ The isomer of-O-2 ') LNA is α -L-methyleneoxy (4' -CH) ₂ -O-2 ') LNA which has been shown to have excellent stability against 3' -exonucleases. alpha-L-methyleneoxy (4' -CH) ₂ Incorporation of-O-2') LNA into antisense gapmers and chimeras, which show potent antisense activity (Frieden et alHuman, nucleic Acids Research,2003,21,6365-6372).

The methyleneoxy group (4' -CH has been described ₂ -O-2') synthesis and preparation of LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, oligomerization and nucleic acid recognition properties (Koshkin et al Tetrahedron,1998,54,3607-3630). BNA and its preparation are also described in WO98/39352 and WO 99/14226.

Also prepared is methyleneoxy (4' -CH ₂ -O-2 ') LNA, phosphorothioate-methyleneoxy (4' -CH) ₂ -O-2 ') LNA and analogues of 2' -thio-LNA (Kumar et al, biorg. Med. Chem. Lett.,1998,8,2219-2222). The preparation of locked nucleotide analogs comprising oligodeoxyribonucleotides duplex as substrates for nucleic acid polymerases is also described (Wengel et al, WO 99/14226). Furthermore, the synthesis of 2' -amino-LNA, a novel conformationally constrained high affinity oligonucleotide analogue, has been described in the art (Singh et al, J.Org.chem.,1998,63,10035-10039). In addition, 2 '-amino-LNAs and 2' -methylamino-LNAs have been prepared and their thermal stability with duplex of complementary RNA and DNA strands has been previously reported.

Modified sugar moieties are well known and can be used to alter, typically increase the affinity of antisense compounds for their targets and/or increase nuclease resistance. Representative lists of preferred modified sugars include, but are not limited to, bicyclic modified sugars, including methyleneoxy (4' -CH ₂ -O-2 ') LNA and ethyleneoxy (4' - (CH) ₂ ) 2-O-2' bridge) ENA; substituted sugars, in particular with 2'-F, 2' -OCH ₃ Or 2' -O (CH) ₂ ) ₂ -OCH ₃ 2' -substituted sugar of substituent; and 4' -thio modified sugars. Sugar may also be replaced with sugar mimetic groups. Methods for preparing modified sugars are well known to those skilled in the art. Some representative patents and publications teaching the preparation of such modified sugars include, but are not limited to, U.S. patent No. 4,981,957;5,118,800;5,319,080;5,359,044;5,393,878;5,446,137;5,466,786;5,514,785;5,519,134;5,567,811;5,576,427;5,591,722;5,597,909;5,610,300;5,627,053;5,639,873;5,646,265;5,658,873;5,670,633;5,792 747;5,700,920;6,531,584; and 6,600,032; and WO 2005/121371.

Examples of "oxy" -2' hydroxyl modifications include alkoxy OR aryloxy (OR, e.g., r=h, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, OR sugar); polyethylene glycol (PEG), O (CH) ₂ CH ₂ O) _n CH ₂ CH ₂ OR, n=1-50; a "locked" nucleic acid (LNA) in which the furanose portion of the nucleoside comprises a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system; O-AMINE or O- (CH) ₂ ) _n AMINE(n＝1-10，AMINE＝NH ₂ The method comprises the steps of carrying out a first treatment on the surface of the Alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine, or polyamino groups); and O-CH ₂ CH ₂ (NCH ₂ CH ₂ NMe ₂ ) ₂ 。

"deoxy" modifications include hydrogen (i.e., deoxyribose, which is particularly relevant for single-stranded protrusions); halogen (e.g., fluorine); amino (e.g. NH ₂ The method comprises the steps of carrying out a first treatment on the surface of the Alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH (CH) ₂ CH ₂ NH) _n CH ₂ CH ₂ -AMINE(AMINE＝NH ₂ The method comprises the steps of carrying out a first treatment on the surface of the Alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino); -NHC (O) R (r=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); cyano group; a thio group; alkyl-thio-alkyl; thioalkoxy; a thioalkyl group; an alkyl group; cycloalkyl; an aryl group; alkenyl and alkynyl groups, which may be optionally substituted with, for example, amino functional groups.

Other suitable 2' -modifications, for example, modified MOEs, are described in U.S. patent application publication No. 20130130378, the contents of which are incorporated herein by reference.

Modification at the 2' position may be present in the arabinose configuration. The term "arabinose configuration" means that the substituents are placed on the C2 'of ribose in the same configuration as the 2' -OH in arabinose.

The sugar may comprise two different modifications on the same carbon of the sugar, for example, a gem modification. The glycosyl group may also contain one or more carbons having a stereochemical configuration opposite to the corresponding carbon in ribose. Thus, an oligonucleotide may comprise one or more monomers containing, for example, arabinose as a sugar. The monomer may have an alpha linkage, such as an alpha-nucleoside, at the 1' position of the sugar. The monomers may also have the opposite configuration at the 4' -position, for example C5' and H4' or substituents replacing them are interchanged with each other. When C5' and H4' or substituents replacing them are exchanged for each other, the sugar is said to be modified at the 4' position.

The oligonucleotides disclosed herein may also include abasic sugars, i.e., sugars lacking a nucleobase at C-1 'or having other chemical groups replacing nucleobases at C1'. See, for example, U.S. patent No. 5,998,203, the contents of which are incorporated herein by reference in their entirety. These abasic sugars may further contain modifications at one or more of the constituent sugar atoms. The oligonucleotide may also contain one or more saccharides that are L isomers, e.g., L-nucleosides. Modification of the glycosyl group may also include replacement of 4' -O with sulfur, optionally substituted nitrogen or CH ₂ A group. In some embodiments, the linkage between C1' and nucleobase is in the α configuration.

Sugar modifications may also include "acyclic nucleotides," which refer to any nucleotide having an acyclic ribose, e.g., where the c—c bond between ribose carbons (e.g., C1' -C2', C2' -C3', C3' -C4', C4' -O4', C1' -O4 ') is absent and/or at least one of ribose carbons or oxygen (e.g., C1', C2', C3', C4', or O4 ') is absent in the nucleotide, either independently or in combination. In some embodiments, the acyclic nucleotide is Wherein B is a modified or unmodified nucleobase, R ₁ And R is ₂ Independently H, halogen, OR ₃ Or alkyl; and R is ₃ Is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar).

In some embodiments, the sugar modification is selected from the group consisting of 2' -H, 2' -O-Me (2 ' -O-methyl), 2' -O-MOE (2 ' -O-methoxyethyl), 2' -F, 2' -O- [2- (methylamino) -2-oxoethyl](2 ' -O-NMA), 2' -S-methyl, 2' -O-CH ₂ -(4’-C)(LNA)、2’-O-CH ₂ CH ₂ - (4 '-C) (ENA), 2' -O-aminopropyl (2 '-O-AP), 2' -O-dimethylaminoethyl (2 '-O-DMAOE), 2' -O-dimethylaminopropyl (2 '-O-DMAP), 2' -O-dimethylaminoethoxyethyl (2 '-O-DMAEOEE) and gem 2' -OMe/2'F with arabinose configuration 2' -O-Me.

It will be appreciated that when a particular nucleotide is linked to the next nucleotide by its 2' -position, the sugar modifications described herein may be placed at the 3' -position of the sugar of the particular nucleotide, e.g., the nucleotide linked by its 2' -position. Modifications at the 3' position may exist in the xylose configuration. The term "xylose configuration" refers to the placement of substituents on the C3 'of ribose in the same configuration as the 3' -OH in xylose.

The hydrogen attached to C4 'and/or C1' may be replaced by a linear or branched optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, wherein the backbone of alkyl, alkenyl and alkynyl may contain O, S, S (O), SO ₂ N (R '), C (O), N (R') C (O) O, OC (O) N (R '), CH (Z'), a phosphorus-containing linkage, an optionally substituted aryl, an optionally substituted heteroaryl, an optionally substituted heterocycle OR an optionally substituted cycloalkyl, wherein R 'is hydrogen, acyl OR an optionally substituted aliphatic, and Z' is selected from OR ₁₁ 、COR ₁₁ 、CO ₂ R ₁₁ 、 NR ₂₁ R ₃₁ 、CONR ₂₁ R ₃₁ 、CON(H)NR ₂₁ R ₃₁ 、ONR ₂₁ R ₃₁ 、CON(H)N＝CR ₄₁ R ₅₁ 、N(R ₂₁ )C(＝NR ₃₁ )NR ₂₁ R ₃₁ 、N(R ₂₁ )C(O)NR ₂₁ R ₃₁ 、N(R ₂₁ )C(S)NR ₂₁ R ₃₁ 、OC(O)NR ₂₁ R ₃₁ 、SC(O)NR ₂₁ R ₃₁ 、N(R ₂₁ )C(S)OR ₁₁ 、N(R ₂₁ )C(O)OR ₁₁ 、N(R ₂₁ )C(O)SR ₁₁ 、N(R ₂₁ )N＝CR ₄₁ R ₅₁ 、ON＝CR ₄₁ R ₅₁ 、SO ₂ R ₁₁ 、SOR ₁₁ 、SR ₁₁ A substituted or unsubstituted heterocycle; r is R ₂₁ And R is ₃₁ Independently at each occurrence is hydrogen, acyl, unsubstituted OR substituted aliphatic, aryl, heteroaryl, heterocycle, OR ₁₁ 、COR ₁₁ 、CO ₂ R ₁₁ Or NR (NR) ₁₁ R ₁₁ 'A'; or R is ₂₁ And R is ₃₁ Together with the atoms to which they are attached form a heterocyclic ring; r is R ₄₁ And R is ₅₁ Independently at each occurrence is hydrogen, acyl, unsubstituted OR substituted aliphatic, aryl, heteroaryl, heterocyclyl, OR ₁₁ 、COR ₁₁ Or CO ₂ R ₁₁ Or NR (NR) ₁₁ R ₁₁ 'A'; and R is ₁₁ And R is ₁₁ ' is independently hydrogen, an aliphatic group, a substituted aliphatic group, an aryl group, a heteroaryl group, or a heterocyclic group. In some embodiments, the hydrogen attached to the C4 'of the 5' -terminal nucleotide is replaced.

In some embodiments, C4 'and C5' together form an optionally substituted heterocycle, preferably comprising at least one —px (Y) -, wherein X is H, OH, OM, SH, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted alkylamino, or optionally substituted dialkylamino, wherein M is independently at each occurrence an alkali metal or transition metal having a total charge of +1; and Y is O, S or NR ', wherein R' is hydrogen, an optionally substituted aliphatic. Preferably, the modification is at the 5' -end of the iRNA.

In certain embodiments, the oligonucleotide comprises at least two regions of at least two consecutive monomers of the above formula. In certain embodiments, the oligonucleotide comprises a gapped motif. In certain embodiments, the oligonucleotide comprises from about 8 to about 14 contiguous at least one region of β -D-2' -deoxyribonucleosides. In certain embodiments, the oligonucleotide comprises from about 9 to about 12 contiguous at least one region of β -D-2' -deoxyribonucleosides.

In certain embodiments, the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) comprising at least one (S) -cEt monomer of the formula:

wherein Bx is a heterocyclic base moiety.

In certain embodiments, the monomers comprise glycomimetics. In certain such embodiments, a combination of mimetics in place of sugar or sugar-internucleoside linkages is used, and the nucleobases are maintained for hybridization with the selected targets. Representative examples of glycomimetics include, but are not limited to, cyclohexenyl or morpholinyl. Representative examples of mimetics of sugar-internucleoside linkage combinations include, but are not limited to, peptide Nucleic Acids (PNAs) and morpholino groups linked by uncharged achiral bonds. In some cases, a mimetic is used instead of a nucleobase. Representative nucleobase mimics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al, nuc Acid res.2000,28:2911-14, incorporated herein by reference). Methods of synthesis of sugar, nucleoside and nucleobase mimetics are well known to those skilled in the art.

Nucleic acid modification (sugar-sugar linkage)

Described herein are linking groups that link monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) together to form oligonucleotides. Such linking groups are also known as intersugar linkages. The two main classes of linking groups are defined by the presence or absence of phosphorus atoms. Representative phosphorus-containing linkages include, but are not limited to, phosphodiester (p=o), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (p=s). Representative non-phosphorus containing linking groups include, but are not limited to In methylene methylimino (-CH) ₂ -N(CH ₃ )-O-CH ₂ (-), thiodiester (-O-C (O) -S-), thiocarbamate (-O-C (O) (NH) -S-); siloxane (-O-Si (H) ₂ -O-); and N, N' -dimethylhydrazine (-CH) ₂ -N(CH ₃ )-N(CH ₃ )-)。

As described above, described herein are one or more phosphorus-containing internucleotide linkages introduced as temporary protecting groups to an oligonucleotideThe remaining phosphorus-containing internucleotide linkages can also be modified using the following method.

Modified linkages can be used to alter, typically increase, nuclease resistance of the oligonucleotide as compared to the native phosphodiester linkage. In certain embodiments, the linkages having chiral atoms may be prepared as a racemic mixture, as individual enantiomers. Representative chiral linkages include, but are not limited to, alkyl phosphonates and phosphorothioates. Methods of making phosphorus-containing and non-phosphorus-containing linkages are well known to those skilled in the art.

The phosphate group in the linking group may be modified by replacing one of the oxygens with a different substituent. One result of this modification is that the resistance of the oligonucleotide to cleavage by the nucleic acid can be increased. Examples of modified phosphate groups include phosphorothioates, phosphoselenates, boranyl phosphates, hydrogen phosphonates, amide phosphates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the linkage may be replaced by any one of the following: s, se, BR ₃ (R is hydrogen, alkyl, aryl), C (i.e., alkyl, aryl, etc.), H, NR ₂ (R is hydrogen, optionally substituted alkyl, aryl) or (R is optionally substituted alkyl or aryl). The phosphorus atom in the unmodified phosphate group is achiral. However, substitution of one of the above atoms or groups of atoms for one of the non-bridging oxygens renders the phosphorus atom chiral; in other words, the phosphorus atom in the phosphate group modified in this way is a stereocenter. Stereoisomerism phosphorus atom can haveThere is either an "R" configuration (Rp herein) or an "S" configuration (Sp herein).

Dithiophosphate has both non-bridging oxygens replaced with sulfur. The phosphorus center in dithiophosphate is achiral, which prevents the formation of the oligonucleotide diastereomers. Thus, while not wanting to be bound by theory, modification of both non-bridging oxygens that eliminate chiral centers, which eliminates chiral center, e.g., dithiophosphate formation, may be desirable because it does not produce diastereomeric mixtures. Thus, the non-bridging oxygens may independently be either O, S, se, B, C, H, N OR (R is alkyl OR aryl).

The phosphate linker may also be modified by replacing the bridged oxygen (i.e., the oxygen linking the phosphate to the sugar of the monomer) with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate) and carbon (bridged methylphosphonate). Substitution may occur at either or both of the linking oxygens. When the bridged oxygen is the 3' -oxygen of a nucleoside, substitution with carbon is preferred. When the bridged oxygen is the 5' -oxygen of the nucleoside, substitution with nitrogen is preferred.

Modified phosphate linkages (in which at least one oxygen attached to the phosphate is replaced or the phosphate group is replaced with a non-phosphorus group) are also referred to as "non-phosphodiester intersugar linkages" or "non-phosphodiester linkers".

In certain embodiments, the phosphate group may be replaced with a non-phosphorus containing linker, such as a dephosphorylation linker. The dephosphorylated linker is also referred to herein as a non-phosphodiester linker. While not wishing to be bound by theory, it is believed that substitution of a neutral structural mimetic should confer enhanced nuclease stability because the charged phosphodiester group is the reaction center for cleavage degradation of the nucleic acid. Also, while not wanting to be bound by theory, in some embodiments it may be desirable to introduce a change in which the charged phosphate groups are replaced with neutral moieties.

Examples of moieties that may replace the phosphate group include, but are not limited to, amides (e.g., amide-3 (3' -CH) ₂ -C (=o) -N (H) -5 ') and amide-4 (3' -CH) ₂ -N (H) -C (=o) -5')), hydroxyamino, siliconOxa-alkanes (dialkylsiloxanes), carboxamides, carbonates, carboxymethyl, carbamates, carboxylic esters, thioethers, oxirane linkers, sulfides, sulfonic acids, sulfonamide, sulfonate esters, thiomethylal (3' -S-CH) ₂ -O-5 '), methylal (3' -O-CH) ₂ -O-5 '), oxime, methyleneimino, methylenecarbonylamino, methylenemethylimino (MMI, 3' -CH) ₂ -N(CH ₃ ) -O-5 '), methylenehydrazinol, methylenedimethylhydrazinol, methylenemethyloimino, ether (C3' -O-C5 '), thioether (C3' -S-C5 '), thioacetamido (C3' -N (H) -C (=o) -CH) ₂ -S-C5’、C3’-O-P(O)-O-SS-C5’、C3’-CH ₂ -NH-NH-C5’、3’-NHP(O)(OCH ₃ ) -O-5 'and 3' -NHP (O) (OCH ₃ ) -O-5' and containing a mixture N, O, S and CH ₂ Nonionic bonds of the constituent parts. See, e.g., carbohydrate Modifications in Antisense Research; y.s. sanghvi and p.d. cook edit ACS Symposium Series 580; chapter 3 and 4, (pages 40-65). Preferred embodiments include methylenemethylimino (MMI), methylenecarbonylamino, amide, carbamate, and ethylene oxide linkers.

It is well known to those skilled in the art that in some cases, substitution of non-bridging oxygens may result in enhanced cleavage of the linkages between adjacent 2'-OH groups to saccharides, and thus in many cases modification of non-bridging oxygens may require modification of 2' -OH groups, for example, modifications that do not participate in cleavage of linkages between adjacent saccharides, for example, arabinose, 2 '-O-alkyl, 2' -F, LNA and ENA.

Preferred non-phosphodiester inter-sugar linkages include phosphorothioates, phosphorothioates having at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more enantiomeric excess of the Sp isomer, phosphorothioates having at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more enantiomeric excess of the Rp isomer, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkylphosphonates (e.g., methylphosphonates), selenophosphate, phosphoramidates (e.g., N-alkylaminophosphates), and boranephosphonates.

In some embodiments, the oligonucleotide further comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more and up to all including) modified or non-phosphodiester linkage. In some embodiments, the oligonucleotide further comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more and up to all including) phosphorothioate linkage.

Oligonucleotides can also be constructed in which the phosphate linker and sugar are replaced with nuclease resistant nucleosides or nucleotide surrogates. While not wishing to be bound by theory, it is believed that the lack of a repeatedly charged backbone reduces binding to proteins that recognize polyanions (e.g., nucleases). Again, while not wanting to be bound by theory, it may be desirable in some embodiments to introduce a change in which the bases are tethered by a neutral proxy backbone. Examples include morpholino, cyclobutyl, pyrrolidine, peptide Nucleic Acid (PNA), aminoethylglycinyl PNA (aegPNA) and backbone extended pyrrolidine PNA (bepPNA) nucleoside agents. The preferred agent is a PNA agent.

The oligonucleotides described herein may contain one or more asymmetric centers and thus produce enantiomers, diastereomers, and other stereoisomeric configurations, which may be defined as (R) or (S) depending on absolute stereochemistry, e.g., for sugar anomers, or as (D) or (L), e.g., for amino acids, etc. Included among the oligonucleotides are all such possible isomers, as well as their racemic and optically pure forms.

Nucleic acid modification (terminal modification)

In some embodiments, the oligonucleotide further comprises a phosphate or phosphate mimetic at the 5' -end of the antisense strand. In one embodiment, the phosphate ester mimic is a 5' -Vinyl Phosphonate (VP).

In some embodiments, the 5 '-end of the antisense strand is free of 5' -Vinylphosphonate (VP).

The ends of the iRNA agent can be modified. Such modifications may be at one or both ends. For example, the 3 'and/or 5' ends of the iRNA can be conjugated to other functional molecular entities, such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, cy3 or Cy5 dyes) or protecting groups (e.g., based on sulfur, silicon, boron, or esters). The functional molecular entity may be linked to the sugar by a phosphate group and/or a linker. The terminal atom of the linker may be attached to or replace the linking atom of the phosphate group or the C-3 'or C-5' O, N, S or C group of the sugar. Alternatively, the linker may be attached to or replace the terminal atom of a nucleotide surrogate (e.g., PNA).

When an array of adaptor/phosphate-functional molecular entities-adaptor/phosphate is inserted between the two strands of a double stranded oligonucleotide, the array can replace the hairpin loop in the hairpin oligonucleotide.

Terminal modifications that can be used to modulate activity include modifications of the 5' end of iRNA with phosphate or phosphate analogs. In certain embodiments, the 5' -end of the iRNA is phosphorylated or comprises a phosphoryl analog. Exemplary 5' -phosphate modifications include those compatible with RISC-mediated gene silencing. Modifications at the 5' -end may also be used to stimulate or inhibit the immune system of a subject. In some embodiments, the 5' -end of the oligonucleotide comprises a modificationWherein W, X and Y are each independently selected from O, OR (R is hydrogen, alkyl, aryl), S, se, BR ₃ (R is hydrogen, alkyl, aryl), BH ₃ ^- C (i.e., alkyl, aryl, etc.), H, NR ₂ (R is hydrogen, alkyl, aryl) OR OR (R is hydrogen, alkyl OR aryl); a and Z are each independently at each occurrence absent O, S, CH ₂ NR (R is hydrogen, alkyl, aryl) or optionally substituted alkylene wherein the backbone of the alkylene may comprise one or more of O, S, SS and NR (R is hydrogen, alkyl, aryl) internally and/or terminally; and n is 0-2. In some embodiments, n is 1 or 2. It is understood that a replaces the oxygen attached to the 5' carbon of the sugar. When n is 0, W and Y, together with the P to which they are attached, may form an optionally substituted 5-8 membered heterocyclic ring, wherein W and Y are each independently O, S, NR' or alkylene. Preferably, the heterocycle is substituted with aryl or heteroaryl. In some embodiments, one or both hydrogens on C5 'of the 5' -terminal nucleotide are replaced with a halogen, e.g., F.

Exemplary 5 '-modifications include, but are not limited to, 5' -monophosphate ((HO) ₂ (O) P-O-5'); 5' -diphosphate ((HO) ₂ (O) P-O-P (HO) (O) -O-5'); 5' -triphosphate ((HO) ₂ (O) P-O- (HO) (O) P-O-P (HO) (O) -O-5'); 5 '-monothiophosphoric acid (phosphorothioate), (HO) (HS) (S) P-O-5'); 5 '-mono-dithiophosphoric acid (dithiophosphate) (HO) (S) P-O-5'), 5 '-thiophosphoric acid ((HO) 2 (O) P-S-5'); 5' - α -thiotriphosphate; 5' - β -thiotriphosphate; 5' -gamma-thiophosphoric acid; 5' -phosphoramidate ((HO) ₂ (O)P-NH-5’、(HO)(NH ₂ ) (O) P-O-5'). Other 5' -modifications include 5' -alkylphosphonates (R (OH) (O) P-O-5', r=alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5' -alkyletherphosphonates (R (OH) (O) P-O-5', r=alkyl ethers, e.g., methoxymethyl (CH) ₂ OMe), ethoxymethyl, etc.); 5' -guanosine caps (7-methylated or unmethylated) (7 m-G-O-5' - (HO) (O) P-O-P (HO) (O) -O-5 '); 5' -adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5 ' - (HO) (O) P-O-P (HO) (O) -O-5 '). Other exemplary 5' -modifications include, wherein Z is optionally substituted alkyl at least once, e.g., ((HO) ₂ (X)P-O[-(CH ₂ ) _a -O-P(X)(OH)-O] _b -5’、((HO) ₂ (X)P-O[-(CH ₂ ) _a -P(X)(OH)-O] _b -5’、((HO)2(X)P-[-(CH ₂ ) _a -O-P(X)(OH)-O] _b -5'; dialkyl terminal phosphates and phosphate mimics: HO [ - (CH) ₂ ) _a -O-P(X)(OH)-O] _b -5’、H ₂ N[-(CH ₂ ) _a -O-P(X)(OH)-O] _b -5’、H[-(CH ₂ ) _a -O-P(X)(OH)-O] _b -5’、Me ₂ N[-(CH ₂ ) _a -O-P(X)(OH)-O] _b -5’、HO[-(CH ₂ ) _a -P(X)(OH)-O] _b -5’、H ₂ N[-(CH ₂ ) _a -P(X)(OH)-O] _b -5’、H[-(CH ₂ ) _a -P(X)(OH)-O] _b -5’、Me ₂ N[-(CH ₂ ) _a -P(X)(OH)-O] _b -5' wherein a and b are each independently 1-10. Other embodiments include using BH ₃ 、BH ₃ ^- And/or Se replaces oxygen and/or sulfur.

Terminal modifications can also be used to monitor the distribution, and in such cases, the preferred groups to be added include fluorophores, e.g., fluorescein or Alexa dyes, e.g., alexa 488. Terminal modifications can also be used to enhance uptake, useful modifications for which include targeting ligands. Terminal modifications can also be used to crosslink an oligonucleotide with another moiety; modifications useful for this include mitomycin C, psoralen, and derivatives thereof.

Thermal destabilization modification

Oligonucleotides, such as iRNA or dsRNA agents, may be optimized for RNA interference by introducing a thermal destabilizing modification in the sense strand at a site opposite the seed region of the antisense strand (i.e., at positions 2-8 of the 5' -end of the antisense strand) to increase the propensity of the iRNA duplex to dissociate or melt (reduce the free energy of duplex association). This modification may increase the propensity of the duplex to dissociate or melt in the seed region of the antisense strand.

The thermostable modification may include abasic modification; mismatches with the opposite nucleotide in the opposite strand; and sugar modifications such as 2' -deoxy modifications or acyclic nucleotides, e.g., unlocked Nucleic Acids (UNA) or Glycerol Nucleic Acids (GNA).

Exemplary abasic modifications are:

exemplary sugar modifications are:

r=h, OH, O-alkyl

The term "UNA" refers to an unlocked acyclic nucleic acid in which any bonds of the sugar have been removed, thereby forming an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the bond between C1'-C4' is removed (i.e., a covalent carbon-oxygen-carbon bond between C1 'and C4' carbons). In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2 'and C3' carbons) is removed (see Mikhailov et al, tetrahedron Letters,26 (17): 2059 (1985); and fluidir et al, mol. Biosystem., 10:1039 (2009), which is incorporated herein by reference in its entirety). Acyclic derivatives provide greater backbone flexibility without affecting Watson-Crick pairing. The acyclic nucleotides may be linked by a 2'-5' or 3'-5' linkage.

The term "GNA" refers to a diol nucleic acid which is a polymer similar to DNA or RNA, but which differs in the composition of the "backbone" which consists of repeating glycerol units linked by phosphodiester linkages:

the thermal destabilizing modification may be a mismatch (i.e., a non-complementary base pair) between the thermal destabilizing nucleotide and the opposing nucleotide in the opposing strand in the dsRNA duplex. Exemplary mismatched base pairs include G: G, G: A, G: U, G: T, A: A, A: C, C: C, C: U, C: T, U: U, T: T, U: T or a combination thereof. Other mismatched base pairing known in the art are also suitable for use in the present invention. Mismatches may occur between naturally occurring nucleotides or modified nucleotides, i.e., mismatched base pairing may occur between nucleobases of the corresponding nucleotides, regardless of the modification on the ribose of the nucleotide. In certain embodiments, an oligonucleotide, such as an siRNA or iRNA agent, contains at least one nucleobase in mismatch pairing that is a 2' -deoxynucleobase; for example, the 2' -deoxynucleobase is in the sense strand.

Further examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA) and mismatch modifications are described in detail in WO 2011/133876, which is incorporated herein by reference in its entirety.

Thermal destabilizing modifications may also include universal base and phosphate modifications with the ability to reduce or eliminate hydrogen bonding with the opposite base.

Nucleobase modifications with impaired or completely eliminated ability to form hydrogen bonds with bases in the opposite strand have been evaluated for destabilization of the central region of dsRNA duplex as described in WO 2010/0011895, which is incorporated herein by reference in its entirety. Exemplary nucleobase modifications are:

exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplex compared to the natural phosphodiester linkage are:

in some embodiments, the oligonucleotides may comprise 2' -5' linkages (to 2' -H, 2' -OH, and 2' -OMe and to p=o or p=s). For example, 2' -5' linkage modifications may be used to promote nuclease resistance or inhibit binding of the sense strand to the antisense strand, or may be used at the 5' end of the sense strand to avoid activation of the sense strand by RISC.

In another embodiment, the oligonucleotide may comprise an L-sugar (e.g., L-ribose, L-arabinose, with 2' -H, 2' -OH, and 2' -OMe). For example, these L sugar modifications may be used to promote nuclease resistance or inhibit binding of the sense strand to the antisense strand, or may be used at the 5' end of the sense strand to avoid activation of the sense strand by RISC.

In some embodiments, one or more targeting ligands are passed throughR of (2) ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ 、R ₇ 、R ₈ And R is ₉ Optionally linked to the modified phosphate prodrug compound via one or more linkers/tethers.

Targeting ligand passageThe introduction of an oligonucleotide on the sense strand or the antisense strand or both the sense and antisense strands is illustrated in scheme 16 of example 10 below. These targeting ligands can be conjugated to +.>Excision.

In some embodiments, the targeting ligand is selected from the group consisting of antibodies, ligand binding portions of receptors, ligands of receptors, aptamers, carbohydrate-based ligands, fatty acids, lipoproteins, folic acid, thyroid stimulating hormone, melanocyte stimulating hormone, surface active protein a, mucin, glycosylated polyamino acids, transferrin, biphosphoric acid, polyglutamic acid, polyaspartic acid, lipophilic portions that enhance plasma protein binding, cholesterol, steroids, bile acids, vitamin B12, biotin, fluorophores, and peptides.

In one embodiment, the lipophilic moiety contains saturated or unsaturated C ₄ -C ₃₀ A hydrocarbon chain and optionally a functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. For example, the lipophilic moiety contains saturated or unsaturated C ₆ -C ₁₈ A hydrocarbon chain.

Additional lipophilic moieties and additional details regarding the hydrophobicity of the lipophilic and oligonucleotide of the lipophilic moiety can be found in PCT application No. PCT/US20/59399 entitled "extrahepatic delivery," filed on month 11, 6 of 2020, the contents of which are incorporated herein by reference in their entirety.

In certain embodiments, at least one ligand targets a receptor that mediates delivery to CNS tissue. In one embodiment, the targeting ligand is selected from the group consisting of Angiopep-2, lipoprotein receptor-related protein (LRP) ligand, bend.3 cell binding ligand, transferrin receptor (TfR) ligand, mannose receptor ligand, glucose transporter, and LDL receptor ligand.

In certain embodiments, at least one ligand targets a receptor that mediates delivery to ocular tissue. In one embodiment, the targeting ligand is selected from the group consisting of trans retinol, RGD peptide, LDL receptor ligand and carbohydrate based ligand.

The targeting ligand may also be directed (independently) (i.e., not through) Introduced into the oligonucleotide.

In some embodiments, the oligonucleotide contains at least one targeting ligand at the 5 '-end, 3' -end, and/or internal position of the antisense strand.

In some embodiments, the oligonucleotide contains at least one targeting ligand at the 5 '-end, 3' -end, and/or internal position of the sense strand.

In some embodiments, the oligonucleotide contains at least one at the 5 '-end, 3' -end and/or internal position of the antisense strandAnd at least one targeting ligand at the 5 '-end, 3' -end and/or internal position of the sense strand.

In one embodiment, the oligonucleotide contains at least one at the 5' -end of the antisense strand And contains at least one targeting ligand at the 3' -end of the sense strand.

In some embodiments, as described below, one or more targeting ligands are linked to the modified phosphate prodrug compound via one or more linkers/tethers (via )。

In some embodiments, as described below, one or more targeting ligands are directly (i.e., not through) via one or more linkers/tethers (i.e., not through) Ligating to the oligonucleotides.

Joint/tether

The linker/tether is attached to the modified phosphate prodrug compound at a "Tether Attachment Point (TAP)". The linker/tether may include any C ₁ -C ₁₀₀ A carbon-containing moiety (e.g. C) ₁ -C ₇₅ 、C ₁ -C ₅₀ 、C ₁ -C ₂₀ 、C ₁ -C ₁₀ ；C ₁ 、C ₂ 、C ₃ 、C ₄ 、C ₅ 、C ₆ 、C ₇ 、C ₈ 、C ₉ Or C ₁₀ ) And may have at least one nitrogen atom. In certain embodiments, the nitrogen atom forms part of a terminal amino or amido (NHC (O) -) group on the linker/tether that can serve as the point of attachment for the modified phosphate prodrug compound. Non-limiting examples of linkers/tethers (underlined) include TAPs ₂ _n -(CH)NH-；TAP- ₂ _n C(O)(CH)NH-；TAP- ₂ NR””(CH) _n NH-、TAP- ₂ _n C(O)-(CH)-C(O)-；TAP- ₂ _n C(O)-(CH)-C(O)O-；TAP-C(O)-O-；TAP- ₂ _n C(O)-(CH)- NH-C(O)-；TAP- ₂ _n C(O)-(CH)-；TAP-C(O)-NH-；TAP-C(O)-；TAP- ₂ _n (CH)-C(O)-；TAP- ₂ _n (CH)-C (O)O-；TAP- ₂ _n (CH)-The method comprises the steps of carrying out a first treatment on the surface of the Or TAP- ₂ _n (CH)-NH-C(O)-The method comprises the steps of carrying out a first treatment on the surface of the Wherein n is 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) and R "" is C ₁ -C ₆ An alkyl group. Preferably, n is 5, 6 or 11. In other embodiments, the nitrogen may form part of a terminal oxyamino group, e.g., -ONH ₂ Or hydrazino, -NHNH ₂ . The linker/tether may be optionally substituted with, for example, hydroxy, alkoxy, perhaloalkyl and/or optionally interrupted with one or more additional heteroatoms, for example N, O or S. Preferred tether ligands may include, for example, TAP ₂ _n - (CH) NH (ligand)；TAP- ₂ _n C (O) (CH) NH (ligand)；TAP-NR”” ₂ _n (CH) NH (ligand)；TAP ₂ _n - (CH) ONH (ligand)；TAP- ₂ _n C (O) (CH) ONH (ligand)；TAP- ₂ _n NR "" (CH) ONH (ligand) Body type；TAP- ₂ _n ₂ (CH) NHNH (ligand)、TAP- ₂ _n ₂ C (O) (CH) NHNH (ligand)；TAP- ₂ _n ₂ NR "" (CH) NHNH (ligand)；TAP- ₂ _n C (O) - (CH) -C (O) (ligand)；TAP- ₂ _n C (O) - (CH) -C (O) O (ligand);TAP-c (O) -O (ligand)；TAP-C ₂ _n (O) - (CH) -NH-C (O) (ligand)；TAP- ₂ _n C (O) - (CH) (ligand)；TAP-C (O) -NH (ligand)；TAP-C (O) (complex) Body type；TAP- ₂ _n (CH) -C (O) (ligand)；TAP- ₂ _n (CH) -C (O) O (ligand)；TAP- ₂ _n (CH) (ligand)The method comprises the steps of carrying out a first treatment on the surface of the Or TAP- ₂ _n (CH)- NH-C (O) (ligand). In some embodiments, the amino-terminated linker/tether (e.g., NH ₂ 、ONH ₂ 、NH ₂ NH ₂ ) An imino bond (i.e., c=n) may be formed with the ligand. In some embodiments, the amino-terminated linker/tether (e.g., NH ₂ 、ONH ₂ 、NH ₂ NH ₂ ) Can be used, for example, with a C (O) CF ₃ And (3) acylation.

In some embodiments, the linker/tether may be capped with a sulfhydryl group (i.e., SH) or an alkene (e.g., ch=ch2). For example, the tether may be TAP ₂ _n -(CH)-SH、TAP- ₂ _n C(O)(CH)SH、TAP ₂ _n ₂ -(CH)-(CH＝CH)Or TAP-C(O) ₂ _n ₂ (CH)(CH＝CH)Wherein n may be as described elsewhere. The tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally interrupted with one or more additional heteroatoms, e.g., N, O or S. The double bond may be cis or trans or E or Z.

In other embodiments, the linker/tether may include an electrophilic moiety, preferably at a terminal position of the linker/tether. Exemplary electrophilic moieties include, for example, aldehydes, alkyl halides, methanesulfonates, toluenesulfonates, nitrobenzenesulfonates or p-bromophenylsulfonates, or activated carboxylic esters, such as NHS esters or pentafluorophenyl esters. Preferred linkers/tethers (underlined) include TAPs - ₂ _n (CH)CHO；TAP- ₂ _n C(O)(CH)CHOThe method comprises the steps of carrying out a first treatment on the surface of the Or TAP- ₂ _n NR””(CH)CHOWherein n is 1-6 and R "" is C ₁ -C ₆ An alkyl group; or TAP ₂ _n -(CH)C(O)ONHS；TAP- ₂ _n C(O)(CH)C(O)ONHSThe method comprises the steps of carrying out a first treatment on the surface of the Or TAP- ₂ _n NR””(CH)C(O)ONHSWherein n is 1-6 and R "" is C ₁ -C ₆ An alkyl group; TAP- ₂ _n ₆ ₅ (CH)C(O)OCF；TAP- ₂ _n ₆ ₅ C(O)(CH)C(O)OCFThe method comprises the steps of carrying out a first treatment on the surface of the Or TAP- ₂ _n NR””(CH)C(O) ₆ ₅ OCFWherein n is 1-11 and R "" is C ₁ -C ₆ An alkyl group; or (b) ₂ _n ₂ -(CH)CHLG；TAP- ₂ _n ₂ C (O) (CH) CHLG; or (b)TAP-NR”” ₂ _n ₂ (CH)CHLG，Wherein n may be as described elsewhere and R "" is C ₁ -C ₆ Alkyl (LG may be a leaving group, e.g., halide, mesylate, tosylate, p-nitrobenzenesulfonate, p-bromophenylsulfonate). The tethering may be performed by coupling a nucleophilic group of the ligand, e.g., a thiol or amino group, to an electrophilic group on the tether.

In other embodiments, it may be desirable for the monomer to include a phthalimido group (K) at the terminal position of the linker/tether.

In other embodiments, other protected amino groups may be at terminal positions of the linker/tether, such as alloc, monomethoxy trityl (MMT), trifluoroacetyl, fmoc, or arylsulfonyl (e.g., the aryl moiety may be o-nitrophenyl or o, p-dinitrophenyl).

Any of the linkers/tethers described herein may further comprise one or more additional linking groups, e.g., -O- (CH) ₂ ) _n -、-(CH ₂ ) _n -SS-、-(CH ₂ ) _n -or- (ch=ch) -.

Cleavable linker/tether

In some embodiments, the at least one linker/tether may be a redox cleavable linker, an acid cleavable linker, an esterase cleavable linker, a phosphatase cleavable linker, or a peptidase cleavable linker.

In one embodiment, at least one of the linkers/tethers may be a reductively cleavable linker (e.g., a disulfide group).

In one embodiment, at least one of the linkers/tethers may be an acid cleavable linker (e.g., hydrazone group, ester group, acetal group, or ketal group).

In one embodiment, at least one of the linkers/tethers may be an esterase cleavable linker (e.g., an ester group).

In one embodiment, at least one of the linkers/tethers may be a phosphatase cleavable linker (e.g., a phosphate group).

In one embodiment, at least one of the linkers/tethers may be a peptidase cleavable linker (e.g., a peptide bond).

Cleavable linking groups are sensitive to the cleavage agent, e.g., pH, redox potential, or the presence of a degrading molecule. In general, lysing agents are found more commonly or at higher levels or activities within cells than in serum or blood. Examples of such degradation agents include: redox agents selected for a particular substrate or not having substrate specificity, including, for example, an oxidation or reduction enzyme or reducing agent present in the cell, such as a thiol, which can redox-cleave the linking group by reductive degradation; an esterase; endosomes or agents that can create an acidic environment, for example, those that result in a pH of five or less; enzymes, peptidases (which may be substrate specific) and phosphatases that can hydrolyze or degrade acid cleavable linkers by functioning as universal acids.

Cleavable linking groups, such as disulfide bonds, may be pH sensitive. The pH of human serum was 7.4, while the average intracellular pH was slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5-6.0, and lysosomes have an even more acidic pH, around 5.0. Some tethers will have a linking group that cleaves at a preferred pH, thereby releasing the iRNA agent from the ligand (e.g., a targeted or cell permeable ligand such as cholesterol) or into a desired compartment of the cell within the cell.

The chemical juncture (e.g., a linking group) that links the ligand to the iRNA agent can include a disulfide bond. When the iRNA agent/ligand complex is taken up into the cell by endocytosis, the acidic environment of the endosome will cause disulfide cleavage, thereby releasing the iRNA agent from the ligand (Quintana et al, pharm res.19:1310-1316,2002; patri et al, curr. Opin. Curr. Biol.6:466-471, 2002). The ligand may be a targeting ligand or a second therapeutic agent that may complement the therapeutic effect of the iRNA agent.

The tether may include a linking group that can be cleaved by a particular enzyme. The type of linking group incorporated into the tether can depend on the cell to which the iRNA agent is targeted. For example, an iRNA agent that targets mRNA in hepatocytes can be conjugated to a tether that includes an ester group. Hepatocytes are rich in esterases and therefore the tether is more effectively cleaved in hepatocytes than in cell types that are not rich in esterases. Cleavage of the tether releases the iRNA agent from the ligand attached to the distal end of the tether, potentially enhancing the silencing activity of the iRNA agent. Other esterase-enriched cell types include cells of the lung, kidney cortex and testis.

The tether containing the peptide bond can be conjugated to an iRNA agent that targets peptidase-rich cell types (e.g., hepatocytes and synoviocytes). For example, an iRNA agent that targets synovial cells, e.g., for the treatment of inflammatory diseases (e.g., rheumatoid arthritis), may be conjugated to a tether containing a peptide bond.

In general, the suitability of a candidate cleavable linking group can be assessed by testing the ability of a degradant (or condition) to cleave the candidate linking group. It is also desirable to test candidate cleavable linkers for their ability to resist cleavage in blood or when contacted with other non-target tissues (e.g., tissues to which an iRNA agent is to be administered to a subject). Thus, the relative sensitivity to lysis between a first condition selected to indicate lysis in a target cell and a second condition selected to indicate lysis in other tissue or biological fluids (e.g., blood or serum) can be determined. The evaluation can be performed in a cell-free system, cells, cell cultures, organ or tissue cultures, or whole animals. Initial evaluation was performed under cell-free or culture conditions and confirmed by further evaluation throughout the animal. In preferred embodiments, useful candidate compounds lyse at least 2, 4, 10, or 100 times faster in cells (or in vitro conditions selected to mimic intracellular conditions) than blood or serum (or in vitro conditions selected to mimic extracellular conditions).

Redox cleavable linking groups

One type of cleavable linking group is a redox cleavable linking group that cleaves upon reduction or oxidation. An example of a reducing cleavable linking group is a disulfide linking group (-S-). To determine whether a candidate cleavable linking group is a suitable "reduction cleavable linking group," or is suitable for use with a particular iRNA moiety and a particular targeting agent, for example, the methods described herein can be considered. For example, candidates can be evaluated by incubation with Dithiothreitol (DTT) or other reducing agents using agents known in the art that mimic the rate of lysis observed in cells, e.g., target cells. Candidates may also be evaluated under conditions selected to mimic blood or serum conditions. In a preferred embodiment, the candidate compound is cleaved in blood by up to 10%. In preferred embodiments, useful candidate compounds degrade at least 2, 4, 10, or 100 times faster in cells (or in vitro conditions selected to mimic intracellular conditions) than blood (or in vitro conditions selected to mimic extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzymatic kinetic assays under conditions selected to mimic intracellular media and compared to conditions selected to mimic extracellular media.

Phosphate-based cleavable linking groups

The phosphate-based linking group is cleaved by an agent that degrades or hydrolyzes the phosphate group. Examples of agents that cleave phosphate groups in cells are enzymes, such as phosphatase in cells. -O-P (S) (SRk) -O-, O-and S-groups-S-P (O) (ORk) -O-, -O-P (S) (SRk) -O-, -S-P (O) (ORk) -O-, and-O-P (O) (ORk) -S-, -S-P (O) (ORk) -S-, S-and S-groups-O-P (S) (ORk) -S-, -S-P (S) (ORk) -O-, -O-P (O) (Rk) -O-, -O-P (S) (Rk) -O-, -S-P (O) (Rk) -O-, -S-P (S) (Rk) -O-, -S-P (O) (Rk) -S-, -O-P (S) (Rk) -S-. -S-P (O) (OH) -O- -O-P (O) (OH) -S-, -S-P (O) (OH) -O-, -O-P (O) (OH) -S-, and-S-P (O) (OH) -S-, -O-P (S) (OH) -S-, -S-P (S) (OH) -O-, -O-P (O) (H) -O-, -O-P (S) (H) -O-, -S-P (O) (H) -O-, -S-P (S) (H) -O-, -S-P (O) (H) -S-, -O-P (S) (H) -S-. A preferred embodiment is-O-P (O) (OH) -O-. These candidates can be evaluated using methods similar to those described above.

Acid cleavable linking groups

An acid cleavable linking group is a linking group that cleaves under acidic conditions. In preferred embodiments, the acid-cleavable linking group is cleaved in an acidic environment at a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less), or by a reagent such as an enzyme that can be used as a generalized acid. In cells, specific low pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for acid-cleavable linkers. Examples of acid cleavable linking groups include, but are not limited to, hydrazones, ketals, acetals, esters, and esters of amino acids. The acid cleavable group may have the general formula-c=nn-, C (O) O or-OC (O). A preferred embodiment is when the carbon (alkoxy) attached to the oxygen of the ester is aryl, substituted alkyl or tertiary alkyl such as dimethylpentyl or tertiary butyl. These candidates can be evaluated using methods similar to those described above.

Ester-based linking groups

The ester-based linking group is cleaved in the cell by enzymes such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, alkylene, alkenylene, and alkynylene esters. The ester cleavable linking group has the general formula-C (O) O-or-OC (O) -. These candidates can be evaluated using methods similar to those described above.

Peptide-based cleavage groups

The peptide-based linking group is cleaved in the cell by enzymes such as peptidases and proteases. Peptide-based cleavable linkers are peptide bonds formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not include an amide group (-C (O) NH-). The amide groups may be formed between any alkylene, alkenylene or alkynylene groups. Peptide bonds are formed between amino acidsCreating a specific type of amide bond for peptides and proteins. Peptide-based cleavage groups are generally limited to peptide bonds (i.e., amide bonds) formed between the amino acids that produce the peptide and protein, and do not include the entire amide functionality. The peptide cleavable linking group has the general formula-NHCHR ¹ C(O)NHCHR ² C (O) -, wherein R ¹ And R is ² Is the R group of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

Biodegradable linker/tether

The linker may also include a bio-cleavable linker that is a linker that links two portions of a molecule, e.g., one or both strands of two separate siRNA molecules to create a nucleotide and non-nucleotide linker of a double (siRNA), or a combination thereof. In some embodiments, the simple electrostatic or stacked interaction between two separate siRNAs can represent a linker. Non-nucleotide linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides and derivatives thereof, aliphatic, alicyclic, heterocyclic groups, and combinations thereof.

In some embodiments, at least one linker (tether) is a bio-cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, and mannose, and combinations thereof.

In one embodiment, the bio-cleavable carbohydrate linker may have 1 to 10 sugar units with at least one heterohead linkage capable of linking two siRNA units. When two or more saccharides are present, these units may be linked by 1-3, 1-4 or 1-6 saccharides or by alkyl chains.

Exemplary bio-cleavable linkers include:

Further discussion of bio-cleavable linkers can be found in PCT application No. PCT/US18/14213 entitled "Endosomal Cleavable Linkers," filed on 1 month 18 in 2018, the contents of which are incorporated herein by reference in their entirety.

Carrier body

In some embodiments, one or more targeting ligands are linked (via) to one or more carriers as described herein and optionally via one or more linkers/tethers as described herein ) To modified phosphate prodrug compounds.

In some embodiments, one or more targeting ligands are directed (i.e., not passed through ) Is linked to the oligonucleotide by one or more vectors as described herein and optionally by one or more linkers/tethers as described above.

The carrier may be a cyclic group or an acyclic group. In one embodiment, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl. In one embodiment, the acyclic group is based on a portion of a serinol backbone or a diethanolamine backbone.

The vector may replace one or more nucleotides of the iRNA agent.

In some embodiments, the vector replaces one or more nucleotides at an internal position of the iRNA agent.

In other embodiments, the vector replaces the nucleotides at the end of the sense strand or the antisense strand. In one embodiment, the vector replaces the terminal nucleotide on the 3 'end of the sense strand, thereby acting as a terminal cap protecting the 3' end of the sense strand. In one embodiment, the carrier is a cyclic group having an amine, for example, the carrier may be pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl.

Ribonucleotide subunits in which the ribose of the subunit has been so replaced are referred to herein as ribose substitution modified subunits (RRMS). The carrier may be a cyclic or acyclic moiety and includes two "backbone attachment points" (e.g., hydroxyl groups) and a ligand. As described above, the targeting ligand may be directly attached to the carrier or indirectly attached to the carrier via an inserted linker/tether.

The ligand conjugated monomer subunit can be the 5 'or 3' -terminal subunit of the iRNA molecule, i.e., one of the two "W" groups can be a hydroxyl group and the other "W" group can be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the ligand conjugated monomer subunits may occupy internal positions, and the two "W" groups may be one or more unmodified or modified ribonucleotides. More than one ligand conjugated monomer subunit may be present in an iRNA agent. A step of

Monomers based on sugar substitution, e.g. ligand conjugated monomers (cyclic)

Cyclic saccharide-substituted based monomers, e.g., saccharide-substituted ligand conjugated based monomers, also referred to herein asRRMS monomer compound. The carrier may have the general formula (LCM-2) provided below (in this structure, the preferred backbone attachment point may be selected from R ¹ Or R is ² ；R ³ Or R is ⁴ The method comprises the steps of carrying out a first treatment on the surface of the Or R is ⁹ And R is ¹⁰ If Y is CR ⁹ R ¹⁰ (two positions are selected to provide two backbone attachment points, e.g., R ¹ And R is ⁴ Or R is ⁴ And R is ⁹ )). Preferred tether attachment points include R ⁷ The method comprises the steps of carrying out a first treatment on the surface of the When X is CH ₂ When R is ⁵ Or R is ⁶ . The vector is described below as an entity that can be incorporated into a chain. Thus, it should be understood that the structure also encompasses the points of attachment therein (e.g., R ¹ Or R is ² ；R ³ Or R is ⁴ The method comprises the steps of carrying out a first treatment on the surface of the Or R is ⁹ Or R is ¹⁰ (when Y is CR) ⁹ R ¹⁰ When) one (in the case of terminal positions) or both (in the case of internal positions) are attached to a phosphate or modified phosphate (e.g., sulfur-containing) backbone. For example: one of the R groups may be-CH ₂ One of the bonds is attached to the support and one of the bonds is attached to a backbone atom, for example, to an oxygen or central phosphorus atom.

Wherein:

x is N (CO) R ⁷ 、NR ⁷ Or CH (CH) ₂ ；

Y is NR ⁸ 、O、S、CR ⁹ R ¹⁰ ；

Z is CR ¹¹ R ¹² Or is absent;

R ¹ 、R ² 、R ³ 、R ⁴ 、R ⁹ and R is ¹⁰ Each independently H, OR ^a Or (CH) ₂ ) _n OR ^b Provided that R ¹ 、R ² 、R ³ 、R ⁴ 、R ⁹ And R is ¹⁰ At least two of which are OR ^a And/or (CH) ₂ ) _n OR ^b ；

R ⁵ 、R ⁶ 、R ¹¹ And R is ¹² Each independently is a ligand, H, optionally substituted with 1-3R ¹³ Substituted C ₁ -C ₆ Alkyl or C (O) NHR ⁷ The method comprises the steps of carrying out a first treatment on the surface of the Or R is ⁵ And R is ¹¹ Taken together is optionally R ¹⁴ Substituted C ₃ -C ₈ Cycloalkyl;

R ⁷ may be a ligand, e.g. R ⁷ May be R ^d Or R is ⁷ Ligands which may be indirectly tethered to the carrier, e.g. via a tether portion, e.g. by NR ^c R ^d Substituted C ₁ -C ₂₀ An alkyl group; or by NHC (O) R ^d Substituted C ₁ -C ₂₀ An alkyl group;

R ⁸ is H or C ₁ -C ₆ An alkyl group;

R ¹³ is hydroxy, C ₁ -C ₄ Alkoxy or halogen;

R ¹⁴ is NR (NR) ^c R ⁷ ；

R ¹⁵ For C optionally substituted by cyano ₁ -C ₆ Alkyl, or C ₂ -C ₆ Alkenyl groups;

R ¹⁶ is C ₁ -C ₁₀ An alkyl group;

R ¹⁷ is a liquid phase or solid phase supporting reagent;

l is-C (O) (CH ₂ ) _q C (O) -or-C (O) (CH ₂ ) _q S-；

R ^a For protecting groups, e.g. CAr ₃ The method comprises the steps of carrying out a first treatment on the surface of the (e.g., dimethoxytrityl) or Si (X) ^5’ )(X ^5” )(X ^5”‘ ) Wherein (X) ^5’ )、(X ^5” ) And (X) ^5”‘ ) As described elsewhere.

R ^b Is P (O) (O ^- )H、P(OR ¹⁵ )N(R ¹⁶ ) ₂ Or L-R ¹⁷ ；

R ^c Is H or C ₁ -C ₆ An alkyl group;

R ^d is H or a ligand;

ar is each independently optionally C ₁ -C ₄ Alkoxy substituted C ₆ -C ₁₀ An aryl group;

n is 1-4; and q is 0 to 4.

Exemplary carriers include, for example, wherein X is N (CO) R ⁷ Or NR (NR) ⁷ Y is CR ⁹ R ¹⁰ And Z is absent; or X is N (CO) R ⁷ Or NR (NR) ⁷ Y is CR ⁹ R ¹⁰ And Z is CR ¹¹ R ¹² The method comprises the steps of carrying out a first treatment on the surface of the Or X is N (CO) R ⁷ Or NR (NR) ⁷ Y is NR ⁸ And Z is CR ¹¹ R ¹² The method comprises the steps of carrying out a first treatment on the surface of the Or X is N (CO) R ⁷ Or NR (NR) ⁷ Y or O and Z are CR ¹¹ R ¹² The method comprises the steps of carrying out a first treatment on the surface of the Or X is CH ₂ The method comprises the steps of carrying out a first treatment on the surface of the Y is CR ⁹ R ¹⁰ The method comprises the steps of carrying out a first treatment on the surface of the Z is CR ¹¹ R ¹² And R is ⁵ And R is ¹¹ Together form C ₆ Those of cycloalkyl (H, z=2), or indane ring systems, e.g. X is CH ₂ The method comprises the steps of carrying out a first treatment on the surface of the Y is CR ⁹ R ¹⁰ The method comprises the steps of carrying out a first treatment on the surface of the Z is CR ¹¹ R ¹² And R is ⁵ And R is ¹¹ Together form C ₅ Cycloalkyl (H, z=1).

In certain embodiments, the carrier may be based on a pyrroline ring system or a 4-hydroxyproline ring system, e.g., X is N (CO) R ⁷ Or NR (NR) ⁷ Y is CR ⁹ R ¹⁰ And Z is absent (D).OFG ¹ Preferably to a primary carbon, e.g. an exocyclic alkylene group, such as methylene, which is attached to one of the carbons in the five-membered ring (-CH in D ₂ OFG ¹ )。OFG ² Preferably directly to one of the carbons in the five-membered ring (OFG in D) ² ). For pyrroline-based carriers, -CH ₂ OFG ¹ Can be connected to C-2 and OFG ² Can be connected to C-3; or-CH ₂ OFG ¹ Can be connected to C-3 and OFG ² Can be connected to C-4. In certain embodiments, CH ₂ OFG ¹ And OFG ² May be geminally substituted to one of the carbons mentioned above. For 3-hydroxyproline based vectors, -CH ₂ OFG ¹ Can be connected to C-2 and OFG ² Can be connected to C-4. Base groupMonomers at pyrroline-and 4-hydroxyproline may thus contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is limited around that particular linkage, e.g., due to the presence of a ring. Thus, in any of the above-mentioned pairings, CH ₂ OFG ¹ And OFG ² May be cis or trans with respect to each other. Thus, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus exist as racemates and racemic mixtures, single enantiomers, single diastereomers and diastereomeric mixtures. Explicitly including all such isomeric forms of the monomers (e.g., carrying CH ₂ OFG ¹ And OFG ² May have an R configuration in the center; or both have an S configuration; or one center may have an R configuration and the other center may have an S configuration, and vice versa). The tether attachment point is preferably nitrogen. Preferred examples of the carrier D include the following: / >

In certain embodiments, the carrier may be based on a piperidine ring system (E), e.g., X is N (CO) R ⁷ Or NR (NR) ⁷ Y is CR ⁹ R ¹⁰ And Z is CR ¹¹ R ¹² 。OFG ¹ Preferably to a primary carbon, e.g., an exocyclic alkylene group such as methylene (n=1) or ethylene (n=2) attached to one of the carbons in the six-membered ring [ E- (CH) ₂ ) _n OFG ¹ ]。OFG ² Preferably directly to one of the carbons in the six-membered ring (OFG in E) ² )。-(CH ₂ )nOFG ¹ And OFG ² May be arranged on the ring in a geminal fashion, i.e. both groups may be attached to the same carbon, for example at C-2, C-3 or C-4. Alternatively, - (CH) ₂ ) _n OFG ¹ And OFG ² May be arranged in ortho-position on the ring, i.e. two groups may be attached to adjacent ring carbon atoms, e.g. - (C)H ₂ ) _n OFG ¹ Can be connected to C-2 and OFG ² May be attached to C-3; - (CH) ₂ ) _n OFG ¹ Can be connected to C-3 and OFG ² May be attached to C-2; - (CH) ₂ ) _n OFG ¹ Can be connected to C-3 and OFG ² May be attached to C-4; or- (CH) ₂ ) _n OFG ¹ Can be connected to C-4 and OFG ² May be attached to C-3. Thus, the piperidine-based monomer may contain a linkage (e.g., a carbon-carbon bond) where bond rotation is limited around that particular linkage, such as a limitation due to the presence of a ring. Thus, in any of the above pairs, - (CH) ₂ )nOFG ¹ And OFG ² May be cis or trans with respect to each other. Thus, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus exist as racemates and racemic mixtures, single enantiomers, single diastereomers and diastereomeric mixtures. Explicitly including all such isomeric forms of the monomers (e.g., carrying CH ₂ OFG ¹ And OFG ² May have an R configuration in the center; or both have an S configuration; or one center may have an R configuration and the other center may have an S configuration, and vice versa). The tether attachment point is preferably nitrogen.

In certain embodiments, the carrier may be based on a piperazine ring system (F), e.g., X is N (CO) R ⁷ Or NR (NR) ⁷ Y is NR ⁸ And Z is CR ¹¹ R ¹² Or morpholine ring systems (G), e.g. X is N (CO) R ⁷ Or NR (NR) ⁷ Y is O and Z is CR ¹¹ R ¹² 。

OFG1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene group, such as methylene, which is attached to one of the carbons in the six-membered ring (F or-CH in G) ₂ OFG ¹ )。OFG ² Preferably directly to one of the carbons in the six-membered ring (F or G-OFG ² ). For F and G, -CH ₂ OFG ¹ Can be connected to C-2 andand OFG ² May be attached to C-3; or vice versa. In certain embodiments, CH ₂ OFG ¹ And OFG ² May be geminally substituted to one of the carbons mentioned above. Piperazine and morpholine based monomers may thus contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is limited around that particular linkage, e.g., limitations due to the presence of a ring. Thus, in any of the above-mentioned pairings, CH ₂ OFG ¹ And OFG ² May be cis or trans with respect to each other. Thus, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus exist as racemates and racemic mixtures, single enantiomers, single diastereomers and diastereomeric mixtures. Explicitly including all such isomeric forms of the monomers (e.g., carrying CH ₂ OFG ¹ And OFG ² May have an R configuration in the center; or both have an S configuration; or one center may have an R configuration and the other center may have an S configuration, and vice versa). R' "can be, for example, C ₁ -C ₆ Alkyl, preferably CH ₃ . The tether attachment points are preferably nitrogen in F and G.

In certain embodiments, the carrier may be based on a decalin ring system, e.g., X is CH ₂ The method comprises the steps of carrying out a first treatment on the surface of the Y is CR ⁹ R ¹⁰ The method comprises the steps of carrying out a first treatment on the surface of the Z is CR ¹¹ R ¹² R is as follows ⁵ And R is ¹¹ Together form C ₆ Cycloalkyl (H, z=2), or indane ring systems, e.g. X is CH ₂ The method comprises the steps of carrying out a first treatment on the surface of the Y is CR ⁹ R ¹⁰ The method comprises the steps of carrying out a first treatment on the surface of the Z is CR ¹¹ R ¹² R is as follows ⁵ And R is ¹¹ Together form C ₅ Cycloalkyl (H, z=1).

OFG ¹ Preferably to a primary carbon, e.g. an exocyclic methylene group (n=1) or ethylene group (n=2) linked to one of C-2, C-3, C-4 or C-5 [ in H- (CH 2) _n OFG ¹ ]。OFG ² Preferably directly to one of C-2, C-3, C-4 or C-5 (OFG in H) ² )。-(CH ₂ ) _n OFG ¹ And OFG ² May be arranged on the ring in a geminal fashion, i.e. both groups may be attached to the same carbon, for example at C-2, C-3, C-4 or C-5. Alternatively, - (CH) ₂ ) _n OFG ¹ And OFG ² May be arranged in ortho-position on the ring, i.e. two groups may be attached to adjacent ring carbon atoms, e.g. - (CH) ₂ ) _n OFG ¹ Can be connected to C-2 and OFG ² May be attached to C-3; - (CH) ₂ ) _n OFG ¹ Can be attached to C-3 and OFG ² May be attached to C-2; - (CH) ₂ ) _n OFG ¹ Can be connected to C-3 and OFG ² May be attached to C-4; or- (CH) ₂ ) _n OFG ¹ Can be connected to C-4 and OFG ² May be attached to C-3; - (CH) ₂ ) _n OFG ¹ Can be connected to C-4 and OFG ² May be attached to C-5; or- (CH) ₂ ) _n OFG ¹ Can be connected to C-5 and OFG ² May be attached to C-4. Thus, decalin or indane based monomers may contain linkages (e.g., carbon-carbon bonds) in which bond rotation is limited around the particular linkage, such as by the presence of a ring. Thus, in any of the above pairs, - (CH) ₂ ) _n OFG ¹ And OFG ² May be cis or trans with respect to each other. Thus, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus exist as racemates and racemic mixtures, single enantiomers, single diastereomers and diastereomeric mixtures. Explicitly including all such isomeric forms of the monomers (e.g., carrying CH ₂ OFG ¹ And OFG ² May have an R configuration in the center; or both have an S configuration; or one center may have an R configuration and the other center may have an S configuration, and vice versa). In a preferred embodiment, the substituents at C-1 and C-6 are trans relative to each other. The tether attachment point is preferably C-6 or C-7.

Other carriers may include those based on 3-hydroxyproline (J).

Thus, - (CH) ₂ ) _n OFG ¹ And OFG ² May be cis or trans with respect to each other. Thus, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus exist as racemates and racemic mixtures, single enantiomers, single diastereomers and diastereomeric mixtures. Explicitly including all such isomeric forms of the monomers (e.g., carrying CH ₂ OFG ¹ And OFG ² May have an R configuration in the center; or both have an S configuration; or one center may have an R configuration and the other center may have an S configuration, and vice versa). The tether attachment point is preferably nitrogen.

Details about more representative cyclic, sugar-substituted-based vectors can be found in U.S. patent nos. 7,745,608 and 8,017,762, which are incorporated herein by reference in their entirety.

Sugar-substituted monomer (acyclic)

Acyclic sugar-substituted-based monomers, such as sugar-substituted ligand conjugated-based monomers, are also referred to herein as ribose-substituted monomer subunit (RRMS) monomer compounds. Preferred acyclic carriers may have the formula LCM-3 or LCM-4:

in some embodiments, x, y, and z may each be, independently of one another, 0, 1, 2, or 3. In formula LCM-3, when y and z are different, then the tertiary carbon may have the R or S configuration. In a preferred embodiment, x is 0, and in formula LCM-3 (e.g., based on serinol), y and z are each 1, and in formula LCM-3, y and z are each 1. Each of the following formulas LCM-3 or LCM-4 may be optionally substituted with, for example, hydroxy, alkoxy, perhaloalkyl.

Details about more representative acyclic sugar-substituted-based carriers can be found in U.S. patent nos. 7,745,608 and 8,017,762, which are incorporated herein by reference in their entirety.

In some embodiments, the oligonucleotide comprises one or more targeting ligands conjugated to the 5 'end of the sense strand or the 5' end of the antisense strand, optionally through a carrier and/or linker/tether.

In some embodiments, the oligonucleotide comprises one or more targeting ligands conjugated to the 3 'end of the sense strand or the 3' end of the antisense strand, optionally through a carrier and/or linker/tether.

In some embodiments, the oligonucleotide comprises one or more targeting ligands conjugated to both ends of the sense strand, optionally through a carrier and/or linker/tether.

In some embodiments, the oligonucleotide comprises one or more targeting ligands conjugated to both ends of the antisense strand, optionally through a carrier and/or linker/tether.

In some embodiments, the oligonucleotide comprises one or more targeting ligands conjugated to an internal position of the sense strand or antisense strand, optionally through a carrier and/or linker/tether.

In some embodiments, one or more targeting ligands are conjugated to ribose, nucleobase, and/or internucleotide linkages. In some embodiments, one or more targeting ligands are conjugated to the ribose at the 2 'position, 3' position, 4 'position, and/or 5' position of the ribose. In some embodiments, one or more targeting ligands are conjugated at a natural (e.g., A, T, G, C or U) or modified nucleobase as defined herein. In some embodiments, one or more targeting ligands are conjugated at a phosphate or modified phosphate group as defined herein.

In some embodiments, the oligonucleotide comprises one or more targeting ligands conjugated to the 5 'or 3' end of the sense strand, and one or more identical or different targeting ligands conjugated to the 5 'or 3' end of the antisense strand,

in some embodiments, at least one targeting ligand is located at one or more terminal positions of the sense strand or the antisense strand. In one embodiment, at least one targeting ligand is located on the 3 'or 5' end of the sense strand. In one embodiment, at least one targeting ligand is located on the 3 'or 5' end of the antisense strand.

In some embodiments, at least one targeting ligand is conjugated to one or more internal positions on at least one strand. The internal position of a strand refers to a nucleotide at any position of the strand except for the terminal positions of the 3 '-end and the 5' -end of the strand (e.g., excluding 2 positions: position 1 counted from the 3 '-end and position 1 counted from the 5' -end).

In one embodiment, at least one targeting ligand is located at one or more internal positions on at least one strand, including all positions except for the terminal two positions at each end of the strand (e.g., excluding 4 positions: positions 1 and 2 counted from the 3 'end and positions 1 and 2 counted from the 5' end). In one embodiment, the targeting ligand is located at one or more internal positions on at least one strand, including all but three positions at the end of each end of the strand (e.g., excluding 6 positions: positions 1, 2 and 3 counted from the 3 'end and positions 1, 2 and 3 counted from the 5' end).

In one embodiment, at least one targeting ligand is located at one or more positions on at least one end of the duplex region, including all positions within the duplex region, but not including the overhang region or vector replacing the terminal nucleotide on the 3' end of the sense strand.

In one embodiment, at least one targeting ligand is located on the sense strand within the first five, four, three, two, or the first base pair of the 5' end of the antisense strand of the duplex region.

In one embodiment, at least one targeting ligand (e.g., lipophilic moiety) is located at one or more internal positions on at least one strand, except for the cleavage site region of the sense strand, e.g., the targeting ligand (e.g., lipophilic moiety) is not located at positions 9-12 counted from the 5 '-end of the sense strand, e.g., the targeting ligand (e.g., lipophilic moiety) is not located at positions 9-11 counted from the 5' -end of the sense strand. Alternatively, the internal positions exclude positions 11-13 counted from the 3' -end of the sense strand.

In one embodiment, at least one targeting ligand (e.g., a lipophilic moiety) is located at one or more internal positions on at least one strand that excludes the cleavage site region of the antisense strand. For example, the internal positions exclude positions 12-14 counted from the 5' -end of the antisense strand.

In one embodiment, at least one targeting ligand (e.g., a lipophilic moiety) is located at one or more internal positions on at least one strand that excludes positions 11-13 on the sense strand where the 3 '-end begins to count and positions 12-14 on the antisense strand where the 5' -end begins to count.

In one embodiment, one or more targeting ligands (e.g., lipophilic moieties) are located at one or more of the following internal positions: positions 4-8 and 13-18 of the sense strand and positions 6-10 and 15-18 of the antisense strand are counted from the 5' end of each strand.

In one embodiment, one or more targeting ligands (e.g., lipophilic moieties) are located at one or more of the following internal positions: positions 5, 6, 7, 15 and 17 of the sense strand and positions 15 and 17 of the antisense strand are counted from the 5' end of each strand.

Target gene

Without limitation, the target genes of the siRNA include, but are not limited to, genes that promote unwanted cell proliferation, growth factor genes, growth factor receptor genes, genes that express kinases, adapter protein genes, genes encoding G protein superfamily molecules, genes encoding transcription factors, genes that mediate angiogenesis, viral genes, genes required for viral replication, genes that mediate viral functions, genes of bacterial pathogens, genes of amoeba pathogens, genes of parasitic pathogens, genes of fungal pathogens, genes that mediate unwanted immune responses, genes that mediate processing of pain, genes that mediate neurological diseases, alleles (allenes) found in cells characterized by loss of heterozygosity, or one allele of a polymorphic gene.

specific exemplary target genes for siRNA include, but are not limited to, PCSK-9, apoC3, AT3, AGT, ALAS1, TMPR, HAO1, AGT, C5, CCR-5, PDGF beta genes; the Erb-B gene, the Src gene; CRK gene; the GRB2 gene; RAS gene; MEKK gene; JNK gene; RAF gene; erk1/2 gene; PCNA (p 21) gene; MYB genes; c-MYC gene; the JUN gene; FOS gene; BCL-2 gene; cyclin D gene; a VEGF gene; an EGFR gene; cyclin a gene; a cyclin E gene; WNT-1 gene; a beta-catenin gene; c-MET gene; PKC genes; NFKB gene; STAT3 gene; survivin gene; her2/Neu genes; a topoisomerase I gene; topoisomerase II alpha gene; a p73 gene; the p21 (WAF 1/CIP 1) gene, the p27 (KIP 1) gene; PPM1D gene; a litter protein I gene; MIB I gene; MTAI gene; m68 gene; a tumor suppressor gene; a p53 gene; DN-p63 gene; pRb tumor suppressor gene; APC1 tumor suppressor gene; BRCA1 tumor suppressor gene; PTEN tumor suppressor gene; MLL fusion genes, e.g., MLL-AF9, BCR/ABL fusion genes; TEL/AML1 fusion gene; EWS/FLI1 fusion gene; TLS/FUS1 fusion gene; PAX3/FKHR fusion gene; AML1/ETO fusion gene; an αv-integrin gene; flt-1 receptor gene; a tubulin gene; human papillomavirus gene, gene required for human papillomavirus replication, human immunodeficiency virus gene, gene required for human immunodeficiency virus replication, hepatitis A virus gene, gene required for hepatitis A virus replication hepatitis B virus gene, gene required for replication of hepatitis B virus, hepatitis C virus gene, gene required for replication of hepatitis C virus, hepatitis D virus gene genes required for replication of hepatitis D virus, hepatitis E virus gene, genes required for replication of hepatitis E virus, hepatitis G virus gene, genes required for replication of hepatitis G virus, hepatitis octyl virus gene, genes required for replication of hepatitis octyl virus, genes required for replication of respiratory syncytial virus, and herpes simplex virus gene, gene required for replication of herpes simplex virus, herpes megacell virus gene, gene required for replication of herpes megacell virus, herpes Epstein Barr virus gene, gene required for replication of herpes Epstein Barr virus, kaposi sarcoma-associated herpes virus gene, gene required for replication of Kaposi sarcoma-associated herpes virus, JC virus gene, human gene required for replication of JC virus, myxovirus gene genes required for myxovirus gene replication, rhinovirus genes, genes required for rhinovirus replication, coronavirus genes, genes required for coronavirus replication, west Nile virus genes, genes required for West Nile virus replication, st.Louis encephalitis genes, genes required for St.Louis encephalitis replication, tick-borne encephalitis genes, genes required for tick-borne encephalitis virus replication, murray Valley encephalitis virus genes, genes required for replication of murray valley encephalopathy virus, dengue virus genes, genes required for replication of dengue virus genes, simian virus 40 genes, genes required for replication of simian virus 40, human T-lymphophilic virus genes, genes required for replication of human T-lymphophilic virus, moloney murine leukemia virus genes, genes required for replication of Moloney murine leukemia virus, encephalomyocarditis virus genes, genes required for replication of encephalomyocarditis virus, measles virus genes, genes required for replication of measles virus, varicella zoster virus genes, genes required for replication of varicella zoster virus, adenovirus genes, genes required for replication of adenovirus, yellow fever virus genes, genes required for replication of yellow fever virus, polio virus genes, genes required for replication of polio virus, poxvirus genes, genes required for replication of poxvirus plasmodium genes, genes required for plasmodium gene replication, mycobacterium ulcerans genes, genes required for Mycobacterium ulcerans replication, mycobacterium tuberculosis genes, genes required for Mycobacterium tuberculosis replication, mycobacterium leprae genes, genes required for Mycobacterium leprae replication, staphylococcus aureus genes, genes required for Staphylococcus aureus replication, streptococcus pneumoniae genes, genes required for Streptococcus pneumoniae replication, streptococcus pyogenes genes, chlamydia pneumoniae genes, genes required for Chlamydia pneumoniae replication, mycoplasma pneumoniae genes, genes required for Mycoplasma pneumoniae replication, integrin genes, selectin genes, complement system genes, chemokine receptor genes, GCSF genes, gro1 genes, gro2 genes, gro3 genes, PF4 genes, streptococcus pyogenes genes, MIG gene, pre-platelet alkaline protein gene, MIP-1I gene, MIP-1J gene, RANTES gene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene, CMBKR3 gene, CMBKR5v, AIF-1 gene, I-309 gene, ion channel component gene, neurotransmitter receptor gene, neurotransmitter ligand gene, amyloid family gene, presenilin gene, HD gene, DRPLA gene, SCA1 gene, SCA2 gene, mxd 1 gene, CACNL1A4 gene, SCA7 gene, SCA8 gene, allele found in heterozygous deletion (LOH) cells, one allele of the polymorphic gene, and combinations thereof.

Loss of heterozygosity (LOH) can result in hemizygosity of a sequence (e.g., a gene) in the LOH region. This can result in significant genetic differences between normal cells and disease state cells (e.g., cancer cells) and provide useful differences between normal cells and disease state cells (e.g., cancer cells). This difference may be due to the fact that the gene or other sequence is heterozygous in diploid cells and hemizygous in cells with LOH. The region of LOH will typically include genes whose loss promotes unwanted proliferation, such as tumor suppressor genes, and other sequences including, for example, other genes, in some cases genes that are essential for normal function, such as growth. The methods of the invention rely in part on the specific modulation of one allele of an essential gene with the compositions of the invention.

In certain embodiments, the invention provides oligonucleotides that modulate micrornas.

Targeting CNS

In some embodiments, the invention provides oligonucleotides of C9orf72 that target APP, spinocerebellar ataxia 2, and ALS of early onset familial alzheimer's disease, and amyotrophic lateral sclerosis and frontotemporal dementia.

In some embodiments, the invention provides oligonucleotides that target TARDBP of ALS, MAPT (Tau) of frontotemporal dementia, and HTT of huntington's disease.

In some embodiments, the invention provides SNCA targeting parkinson's disease, FUS of ALS, ATXN3 of spinocerebellar ataxia 3, ATXN1 of SCA1, genes of SCA7 and SCA8, ATN1 of DRPLA, meCP2 of XLMR, PRNP of prion disease, recessive CNS disorders: lafora disease, DMPK of DM1 (CNS and skeletal muscle), and TTR of hATTR (CNS, eye and systemic).

Spinocerebellar ataxia is a hereditary brain dysfunction. Dominant inherited forms of spinocerebellar ataxia, such as SCA1-8, are destructive disorders without disease-modifying therapies. Exemplary targets include SCA2, SCA3, and SCA1.

A more detailed description of these CNS-targeted receptors and related diseases can be found in PCT application No. PCT/US20/59399 entitled "extrahepatic delivery," filed on 11/6/2020, which is incorporated herein by reference in its entirety.

In some embodiments, the invention provides oligonucleotides targeting genes of disease including, but not limited to, age-related macular degeneration (AMD) (dry and wet), avian elastic chorioretinopathy, dominant retinitis 4, fuch dystrophy, hATTR amyloidosis, hereditary and sporadic glaucoma, and stargardt disease.

In some embodiments, the oligonucleotide targets VEGF for wet (or exudative) AMD.

In some embodiments, the oligonucleotide targets C3 of dry (or non-exudative) AMD.

In some embodiments, the oligonucleotide targets CFB of dry (or non-exudative) AMD.

In some embodiments, the oligonucleotide targets MYOC of glaucoma.

In some embodiments, the oligonucleotide targets ROCK2 of glaucoma.

In some embodiments, the oligonucleotide targets ADRB2 for glaucoma.

In some embodiments, the oligonucleotide targets CA2 of glaucoma.

In some embodiments, the oligonucleotide targets CRYGC for the cataract.

In some embodiments, the oligonucleotide targets PPP3CB of dry eye syndrome.

Ligand

In certain embodiments, the oligonucleotide is further modified by covalent attachment of one or more conjugate groups. Typically, the conjugate group alters one or more properties of the attached compound of the invention, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are commonly used in the chemical arts and are attached to a parent compound, such as an oligonucleotide, either directly or through an optional linking moiety or linking group. Preferred lists of conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterols, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes.

In some embodiments, the oligonucleotide further comprises a targeting ligand that targets a receptor that mediates delivery to a particular CNS tissue. These targeting ligands can also be conjugated in combination with lipophilic moieties to achieve specific intrathecal and systemic delivery.

Exemplary targeting ligands for receptor mediated delivery to CNS tissues are peptide ligands, e.g., angiopep-2, lipoprotein receptor-related protein (LRP) ligand, bend.3 cell binding ligand; transferrin receptor (TfR) ligands (which can utilize the iron transport system in the brain and transport cargo into the brain parenchyma); mannose receptor ligands (which target olfactory ensheathing cells, glial cells), glucose transporters and LDL receptor ligands.

In some embodiments, the oligonucleotide further comprises a targeting ligand that targets a receptor that mediates delivery to a particular ocular tissue. These targeting ligands can also be conjugated in combination with lipophilic moieties to achieve specific ocular delivery (e.g., intravitreal delivery) and systemic delivery. Exemplary targeting ligands for targeting receptor-mediated delivery to ocular tissue are lipophilic ligands such as all-trans retinol (which targets retinoic acid receptors); RGD peptides (which target retinal pigment epithelial cells), such as H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH or cyclo (-Arg-Gly-Asp-D-Phe-Cys); LDL receptor ligands; and carbohydrate-based ligands (which target endothelial cells in the posterior eye).

Preferred conjugate groups suitable for use in the present invention include lipid moieties such as cholesterol moieties (Letsinger et al, proc. Natl. Acad. Sci. USA,1989,86,6553); cholic acid (Manoharan et al, biorg. Med. Chem. Lett.,1994,4,1053); thioethers, for example, hexyl-S-tritylthiol (Manoharan et al, ann.N. Y. Acad. Sci.,1992,660,306; manoharan et al, bioorg. Med. Chem. Let.,1993,3,2765); thiocholesterol (obelhauser et al, nucleic acids res.,1992,20,533); aliphatic chains, for example, dodecanediol or undecyl residues (Saison-Behmoaras et al, EMBO J.,1991,10,111; kabanov et al, FEBS Lett.,1990,259,327; svinarchuk et al, biochimie,1993,75,49); phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium-1, 2-di-O-hexadecyl-rac-glycerol-3-H-phosphonate (Manoharan et al, tetrahedron lett.,1995,36,3651; shea et al, nucleic acids res.,1990,18,3777); polyamine or polyethylene glycol chains (Manoharan et al, nucleosides & Nucleotides,1995,14,969); adamantane acetic acid (Manoharan et al, tetrahedron lett.,1995,36,3651); palmitoyl moieties (Mishra et al, biochim.Biophys. Acta,1995,1264,229); or octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety (Crooke et al, J.Pharmacol.exp.Ther.,1996,277,923).

In general, a wide variety of entities, such as ligands, can be coupled to the oligonucleotides described herein. The ligand may comprise a naturally occurring molecule or a recombinant or synthetic molecule. Exemplary ligands include, but are not limited to, polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly (L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N- (2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [ MPEG ]] ₂ Polyvinyl alcohol (PVA), polyurethane, poly (2-ethyl acrylic acid), N-isopropyl acrylamide polymer, polyphosphazine, polyethylenimine, cationic groups, spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic-polyamineDendritic polyamines, arginine, amidines, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, thyrotropins, melanotropins, lectins, glycoproteins, surfactant protein a, mucins, glycosylated polyamino acids, transferrin, biphosphoric acid, polyglutamic acid, polyaspartic acid, aptamers, desialylated fetuin, hyaluronic acid, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, sugar-albumin conjugates, intercalators (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (e.g., TPPC4, texaphyrin), sha Feilin (sapphirin)), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., steroids, bile acids, cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1, 3-bis-O (hexadecyl) glycerol, geranoxyhexyl, hexadecyl glycerol, borneol, menthol, 1, 3-propanediol, heptadecyl, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) cholic acid, dimethoxytrityl or phenoxazine), peptides (e.g., alpha helical peptides, amphiphilic peptides, RGD peptides, cell penetrating peptides, endosomal cleavage/fusion peptides), alkylating agents, phosphates, amino groups, mercapto groups, polyamino groups, alkyl groups, substituted alkyl groups, radiolabelled labels, enzymes, haptens (e.g., biotin), transport/absorption enhancers (e.g., naproxen, aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, eu3+ complexes of the tetraazamacrocycle), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, multivalent carbohydrates, vitamins (e.g., vitamin a, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin, and pyridoxal), vitamin cofactors, lipopolysaccharide, activators of p38 MAP kinase, activators of NF- κb, taxol (tamon), vincristine, vinblastine, cytochalasin, nocodazole, florolactone, lankurine a, phalloidin, swinholide a, indanocine, myoservin, tumor necrosis factor α (tnfα), interleukins- 1 beta, gamma interferon, natural or recombinant Low Density Lipoprotein (LDL), natural or recombinant High Density Lipoprotein (HDL), and cell permeabilizers (e.g., helical cell permeabilizers).

Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; an alpha, beta or gamma peptide; an N-methyl peptide; an aza peptide; peptides having one or more amide (i.e., peptide) linkages substituted with one or more urea, thiourea, carbamate or sulfonylurea linkages; or a cyclic peptide. Peptide mimetics (also referred to herein as oligopeptide mimetics) are molecules capable of folding into a defined three-dimensional structure similar to a natural peptide. The peptide or peptidomimetic ligand can be about 5-50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

Exemplary amphiphilic peptides include, but are not limited to, cecropin, lycotoxins, paradaxins, bufoin, CPF, bombesin-like peptide (BLP), antibacterial peptide (cathelicidins), ceratoxins, handle tumor sea squirt (S.clava) peptide, cecropin (HFIAP), xenopus antibacterial peptide (magainines), brevinins-2, dermatopontin, melittin, pleurocidin, H ₂ Peptide A, xenopus peptide, esculontitis-1 and caens.

As used herein, the term "endosomolytic ligand" refers to a molecule having endosomolytic properties. Endosomolytic ligands facilitate the lysis and/or transport of the compositions of the invention or components thereof from a cellular compartment (e.g., endosome, lysosome, endoplasmic Reticulum (ER), golgi apparatus, microtubule, peroxisome, or other vesicle within a cell) to the cytoplasm of the cell. Some exemplary endosomolytic ligands include, but are not limited to, imidazoles, polyimidazoles or oligomeric imidazoles, linear or branched Polyethylenimines (PEI), linear and branched polyamines, for example, spermines, cationic linear and branched polyamines, polycarboxylates, polycations, masked oligo-or polycations or anions, acetals, polyacetals, ketals/polyketals, orthoesters, linear or branched polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptide mimics, pH-sensitive peptides, natural and synthetic fusogenic lipids, natural and synthetic cationic lipids.

Exemplary endosomolytic/fusogenic peptides include, but are not limited to

AALEALAEALEALAEALEALAEAAAAGGC(GALA)；

AALAEALAEALAEALAEALAEALAAAAGGC(EALA)；

ALEALAEALEALAEA；GLFEAIEGFIENGWEGMIWDYG(INF-7)；

GLFGAIAGFIENGWEGMIDGWYG(Inf HA-2)；

GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMID

GWYGC(diINF-7)；

GLFEAIEGFIENGWEGMIDGGCGLFEAIEGFIENGWEGMIDGGC

(diINF-3)；GLFGALAEALAEALAEHLAEALAEALEALAAGGSC

(GLF)；GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC(GALA-

INF3)；GLF EAI EGFI ENGW EGnIDG K GLF EAI EGFI ENGW EGnI

DG (INF-5, n is norleucine); LFEALLELLESLWELLLEA (JTS-1);

GLFKALLKLLKSLWKLLLKA(ppTG1)；

GLFRALLRLLRSLWRLLLRA(ppTG20)；

WEAKLAKALAKALAKHLAKALAKALKACEA(KALA)；

GLFFEAIAEFIEGGWEGLIEGC(HA)；

GIGAVLKVLTTGLPALISWIKRKRQQ (melittin); h ₅ WYG; and

CHK ₆ HC。

without wishing to be bound by theory, fusogenic lipids fuse with and thus destabilize the membrane. Fusogenic lipids generally have a small head group and an unsaturated acyl chain. Exemplary fusogenic lipids include, but are not limited to, 1, 2-dioleoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyl Oleoyl Phosphatidylcholine (POPC), (6Z, 9Z,28Z, 31Z) -heptadecan-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl (2, 2-Di ((9Z, 12Z) -octadeca-9, 12-dienyl) -1, 3-dioxolan-4-yl) methylamine (DLiN-k-DMA), and N-methyl-2- (2, 2-Di ((9Z, 12Z) -octadeca-9, 12-dienyl) -1, 3-dioxolan-4-yl) ethylamine (also referred to herein as XTC).

Synthetic polymers having endosomolytic activity suitable for the present invention are described in U.S. patent application publication No. 2009/0048410;2009/0023890;2008/0287630;2008/0287628;2008/0281044;2008/0281041;2008/0269450;2007/0105804;20070036865; and 2004/0198687, the contents of which are incorporated herein by reference in their entirety.

Exemplary cell penetrating peptides include, but are not limited to, RQIKIWFQNRRMKWKK (transmembrane peptide); GRKKRRQRRRPPQC (Tat fragment 48-60);

GALFLGWLGAAGSTMGAWSQPKKKRKV (peptide based signal sequence);

LLIILRRRIRKQAHAHSK(PVEC)；

GWTLNSAGYLLKINLKALAALAKKIL (transshipment);

KLALKLALKALKAALKLA (amphiphilic model peptide); RRRRRRRRR (Arg 9);

KFFKFFKFFK (bacterial cell wall penetrating peptide);

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES(LL-37)；

SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin P1);

ACYCRIPACIAGERRYGTCIYQGRLWAFCC (α -defensin);

DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (β -defensin);

RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2(PR-39)；ILPWKWPWWPWRR-NH ₂ (endolicidin); AAVALLPAVLLALLAP (RFGF); AALLPVLLAAP (RFGF analog); and RKCRIVVIRVCR (bacitracin).

Exemplary cationic groups include, but are not limited to, those derived from, for example, O-AMINE (amine=nh) ₂ The method comprises the steps of carrying out a first treatment on the surface of the Alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino) are described. Aminoalkoxy groups, e.g. O (CH) ₂ ) _n AMINE, (e.g., amine=nh ₂ The method comprises the steps of carrying out a first treatment on the surface of the Alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino); amino (e.g. NH ₂ The method comprises the steps of carrying out a first treatment on the surface of the Alkylamino, dialkylamino, heterocycleA group, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); and NH (CH) ₂ CH ₂ NH) _n CH ₂ CH ₂ -AMINE(AMINE＝NH ₂ The method comprises the steps of carrying out a first treatment on the surface of the Alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino).

As used herein, the term "targeting ligand" refers to any molecule that provides enhanced affinity for a selected target (e.g., a cell, cell type, tissue, organ, body region or compartment (e.g., a cell, tissue or organ compartment)). Some exemplary targeting ligands include, but are not limited to, antibodies, antigens, folic acid, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands.

Carbohydrate-based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, e.g., galNAc ₂ And GalNAc ₃ (GalNAc and multivalent GalNAc are collectively referred to herein as GalNAc conjugates); d-mannose, multivalent lactose, N-acetylglucosamine, glucose, multivalent fucose, glycosylated polyamino acids, and lectins. The term multivalent means that more than one monosaccharide unit is present. Such monosaccharide subunits may be linked to each other or to the scaffold molecule by glycosidic linkages.

Many folic acid and folic acid analogs suitable for use in the present invention as ligands are described in U.S. patent No. 2,816,110;5,552,545;6,335,434 and 7,128,893, the contents of which are incorporated herein by reference in their entirety.

As used herein, the terms "PK modulating ligand" and "PK modulator" refer to molecules that can modulate the pharmacokinetics of the compositions of the present invention. Some exemplary PK modulators include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogs, peptides, protein binding agents, vitamins, fatty acids, phenoxazine, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S) - (+) -pranoprofen, carprofen, PEG, biotin, and transthyretin binding ligands (e.g., tetraiodothyroacetic acid, 2,4, 6-triiodophenol, and flufenamic acid). Oligonucleotides comprising a number of phosphorothioate sugar linkages are also known to bind to serum proteins, so short oligonucleotides, such as oligonucleotides comprising about 5 to 30 nucleotides (e.g., 5 to 25 nucleotides, preferably 5 to 20 nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides), and oligonucleotides comprising a number of phosphorothioate linkages in the backbone are also suitable for use in the present invention as ligands (e.g., as PK modulating ligands). The PK modulating oligonucleotide may comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more phosphorothioate and/or phosphorodithioate linkages. In some embodiments, all internucleotide linkages in the PK-modulating oligonucleotide are phosphorothioate and/or phosphorodithioate linkages. In addition, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use in the present invention as PK modulating ligands. Binding to serum components (e.g., serum proteins) can be predicted from albumin binding assays, such as those described in Oravcova, et al, journal of Chromatography B (1996), 677:1-27.

When two or more ligands are present, the ligands may all have the same properties, all have different properties, or some ligands have the same properties while others have different properties. For example, the ligand may have targeting properties, have endosomolytic activity, or have PK modulating properties. In a preferred embodiment, all ligands have different properties.

When a monomer is incorporated into a component of a compound of the invention (e.g., a compound or linker of the invention), a ligand or tether ligand may be present on the monomer. In some embodiments, after a "precursor" monomer has been incorporated into a component of a compound of the invention (e.g., a compound or linker of the invention), a ligand may be incorporated into the "precursor" monomer by coupling. For example, monomers having, for example, an amino-terminated tether (i.e., having no associated ligand), e.g., monomer-linker-NH ₂ The components of the compounds of the present invention may be incorporated (examplesSuch as a compound or linker of the invention). In a subsequent operation, i.e., after incorporation of the precursor monomer into a component of the compounds of the invention (e.g., the compounds or linkers of the invention), the ligand having an electrophilic group (e.g., pentafluorophenyl ester or aldehyde group) may then be attached to the precursor monomer by coupling the electrophilic group of the ligand to the terminal nucleophilic group of the tether of the precursor monomer.

In another example, monomers having chemical groups suitable for participating in click chemistry reactions, such as azide or alkyne terminated tethers/linkers, may be incorporated. In a subsequent operation, i.e. after incorporation of the precursor monomer into the chain, a ligand having complementary chemical groups, such as an alkyne or azide, can be attached to the precursor monomer by coupling the alkyne and azide together.

In some embodiments, the ligand may be conjugated to a nucleobase, sugar moiety, or internucleoside linkage of the oligonucleotide. Conjugation to the purine nucleobase or derivative thereof may occur at any position, including in-and out-of-loop atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is attached to the conjugate moiety. Conjugation to pyrimidine nucleobases or derivatives thereof may also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of the pyrimidine nucleobases may be substituted with conjugated moieties. When the ligand binds to a nucleobase, the preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions required for base pairing.

Conjugation to the sugar moiety of the nucleoside can occur at any carbon atom. Exemplary carbon atoms that may be attached to the sugar moiety of the conjugate moiety include 2', 3', and 5' carbon atoms. The 1' position may also be linked to a conjugate moiety, for example in an abasic residue. Internucleoside linkages may also carry a conjugate moiety. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the conjugate moiety may be directly linked to a phosphorus atom or to a O, N or S atom bound to a phosphorus atom. For amine-containing or amide-containing internucleoside linkages (e.g., PNAs), the conjugate moiety may be attached to the nitrogen atom or an adjacent carbon atom of the amine or amide.

There are many methods of preparing conjugates of oligonucleotides. Typically, the oligonucleotide is attached to the conjugate moiety by contacting a reactive group (e.g., OH, SH, amine, carboxyl, aldehyde, etc.) on the oligonucleotide with a reactive group on the conjugate moiety. In some embodiments, one reactive group is electrophilic, while the other is nucleophilic.

For example, the electrophilic group may be a carbonyl-containing functional group, and the nucleophilic group may be an amine or a thiol. Methods for conjugating nucleic acids and related oligonucleotides with and without a linking group are well described in the literature, e.g., manoharan in Antisense Research and Applications, rooke and LeBleu editions, CRC press, boca Raton, fli, 1993, chapter 17, which is incorporated herein by reference in its entirety.

The ligand may be attached to the oligonucleotide by a linker or carrier monomer, e.g., a ligand carrier. The carrier comprises (i) at least one "backbone attachment point", preferably two "backbone attachment points", and (ii) at least one "tether attachment point". "backbone attachment point" as used herein refers to a functional group, such as a hydroxyl group or typically a bond, such as a phosphate or modified phosphate (e.g., sulfur-containing) backbone, that can be used and is suitable for incorporating a carrier monomer into an oligonucleotide backbone. "tethered attachment point" (TAP) refers to an atom, e.g., a carbon atom or a heteroatom (other than the atom providing the backbone attachment point), of a carrier monomer linking the selected moiety. The selected moiety may be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, and polysaccharide. Optionally, the selected portion is attached to the carrier monomer by an intervening tether. Thus, the support will typically comprise a functional group, such as an amino group, or will typically provide a bond suitable for incorporating or tethering another chemical entity, such as a ligand, to a constituent atom.

Representative U.S. patents that teach the preparation of conjugates of nucleic acids include, but are not limited to, U.S. patent No. 4,828,979;4,948,882;5,218,105;5,525,465;5,541,313;5,545,730;5,552,538;5,578,717,5,580,731;5,580,731;5,591,584;5,109,124;5,118,802;5,138,045;5,414,077;5,486,603;5,512,439;5,578,718;5,608,046;4,587,044;4,605,735;4,667,025;4,762,779;4,789,737;4,824,941;4,835,263;4,876,335;4,904,582;4,958,013;5,082,830;5,112,963;5,214,136;5,082,830;5,112,963;5,149,782;5,214,136;5,245,022;5,254,469;5,258,506;5,262,536;5,272,250;5,292,873;5,317,098;5,371,241,5,391,723;5,416,203,5,451,463;5,510,475;5,512,667;5,514,785;5,565,552;5,567,810;5,574,142;5,585,481;5,587,371;5,595,726;5,597,696;5,599,923;5,599,928;5,672,662;5,688,941;5,714,166;6,153,737;6,172,208;6,300,319;6,335,434;6,335,437;6,395,437;6,444,806;6,486,308;6,525,031;6,528,631;6,559,279; the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the oligonucleotide further comprises a targeting ligand that targets liver tissue. In some embodiments, the targeting ligand is a carbohydrate-based ligand. In one embodiment, the targeting ligand is a GalNAc conjugate.

Because the ligand may be conjugated to the iRNA agent through a linker or carrier, and because the linker or carrier may contain a branched linker, the iRNA agent may then contain multiple ligands through the same or different backbone attachment point as the carrier or through a branched linker. For example, the branching point of the branched linker may be a divalent, trivalent, tetravalent, pentavalent, or hexavalent atom, or a group exhibiting such polyvalent. In some embodiments of the present invention, in some embodiments, branch points are-N, -N (Q) -C, -O-C-S-C, -SS-C, -C (O) N (Q) -C-OC (O) N (Q) -C, -N (Q) C (O) -C or-N (Q) C (O) O-C; wherein Q is independently at each occurrence H or optionally substituted alkyl. In other embodiments, the branching point is glycerol or a glycerol derivative.

Evaluation of candidate iRNA

Candidate iRNA agents, e.g., modified RNAs, can be evaluated for their selected properties by exposing the iRNA agent or modified molecule and the control molecule to appropriate conditions and evaluating for the presence of the selected properties. For example, resistance to degradation agents can be assessed as follows. Candidate modified RNAs (and control molecules, typically in unmodified form) may be exposed to degradation conditions, e.g., to environments that include degradation agents, e.g., nucleases. For example, biological samples, e.g., similar to the environment that may be encountered in therapeutic applications, e.g., blood or cell fractions, e.g., cell-free homogenates or disrupted cells, may be used. The degradation resistance of the candidate and control can then be assessed by any of a variety of methods. For example, the candidate and control may be labeled with, for example, a radioactive or enzymatic label or a fluorescent label such as Cy3 or Cy5 prior to exposure. The control and modified RNAs can be incubated with a degradant, and optionally a control, e.g., an inactivated (e.g., heat-inactivated) degradant. Physical parameters, such as size, of the modified and control molecules are then determined. They can be determined by physical methods, such as by polyacrylamide gel electrophoresis or a fractionation column, to assess whether the molecule retains its original length, or functional assessment. Alternatively, northern blot analysis can be used to determine the length of unlabeled modified molecules.

Functional assays may also be used to evaluate candidate agents. Functional assays may be applied initially or after an earlier nonfunctional assay (e.g., an assay for resistance to degradation) to determine whether a modification alters the ability of a molecule to silence gene expression. For example, cells, e.g., mammalian cells, such as mouse or human cells, may be co-transfected with a plasmid expressing a fluorescent protein, e.g., GFP, and a candidate RNA agent homologous to a transcript encoding the fluorescent protein (see, e.g., WO 00/44914). For example, the ability of modified dsiRNA homologous to GFP mRNA to inhibit GFP expression can be assayed by monitoring a decrease in cellular fluorescence as compared to control cells in which the transfection does not include the candidate dsiRNA (e.g., a control without the addition of reagent and/or a control with the addition of unmodified RNA). The efficacy of a candidate agent on gene expression can be assessed by comparing the fluorescence of cells in the presence of modified and unmodified dssiRNAs.

In an alternative functional assay, candidate dssiRNAs homologous to an endogenous mouse gene (e.g., a maternally expressed gene such as c-mos) can be injected into an immature mouse oocyte to assess the ability of the agent to inhibit gene expression in vivo (see, e.g., WO 01/36646). The phenotype of the oocyte, e.g., the ability to maintain metaphase II arrest, may be monitored as an indication of agent inhibition expression. For example, cleavage of c-mos mRNA by dssiRNA will cause the oocyte to exit metaphase arrest and initiate parthenogenesis (Colledge et al, nature 370:65-68,1994; hashimoto et al, nature,370:68-71,1994). The effect of the modified agent on target RNA levels can be confirmed by measuring the decrease in target mRNA levels by Northern blotting, or by measuring the decrease in target protein levels by Western blotting, as compared to negative controls. Controls may include cells to which no reagent is added and/or cells to which unmodified RNA is added.

Physiological effects

The sirnas described herein can be designed to make it easier to determine therapeutic toxicity by the complementarity of the siRNA to human and non-human animal sequences. By these methods, the siRNA can consist of a sequence that is fully complementary to a nucleic acid sequence from a human and a nucleic acid sequence from at least one non-human animal (e.g., a non-human mammal such as a rodent, ruminant, or primate). For example, the non-human mammal may be a mouse, rat, dog, pig, goat, sheep, cow, monkey, bonobo, chimpanzee, macaque or cynomolgus monkey. The sequence of the siRNA can be complementary to sequences within homologous genes of non-human mammals and humans, for example, oncogenes or tumor suppressor genes. By determining the toxicity of the siRNA in a non-human mammal, the toxicity of the siRNA in humans can be extrapolated. For more vigorous toxicity assays, the siRNA can be complementary to humans and more than one, e.g., two or three or more non-human animals.

The methods described herein can be used to correlate any physiological effect of siRNA on a human, e.g., any unwanted effect, such as a toxic effect or any positive or desired effect.

Increasing cellular uptake of siRNA

Described herein are various siRNA compositions containing covalently linked conjugates that increase cellular uptake and/or intracellular targeting of siRNA.

Further provided are methods of the invention comprising administering an siRNA and a drug that affects the uptake of the siRNA into a cell. The drug may be administered before, after, or simultaneously with the administration of the siRNA. The drug may be covalently or non-covalently linked to the siRNA. The drug may be, for example, lipopolysaccharide, an activator of p38MAP kinase or an activator of NF- κB. The drug may have a transient effect on the cell. The agent may increase the uptake of the siRNA into the cell, e.g., by disrupting the cytoskeleton of the cell, e.g., by disrupting microtubules, microfilaments, and/or intermediate filaments of the cell. The drug may be, for example, taxane, vincristine, vinblastine, cytochalasin, nocodazole, jasmonate, lankurine A, phalloidin, swinholide A, indanocine indancin, or myoervin. For example, drugs can also increase siRNA uptake into a given cell or tissue by activating an inflammatory response. Exemplary agents having such effects include tumor necrosis factor alpha (tnfα), interleukin-1 beta, cpG motifs, gamma interferon or more generally agents that activate toll-like receptors.

SiRNA production

siRNA can be produced by a variety of methods, such as batch production. An exemplary method includes: organic synthesis and RNA cleavage, e.g., in vitro cleavage.

And (5) organic synthesis. siRNA can be prepared by synthesizing each respective strand of a single-stranded RNA molecule or a double-stranded RNA molecule separately, and then the constituent strands can be annealed.

Large bioreactors, such as OligoPilot II from Pharmacia Biotec AB (Uppsala Sweden), may be used to produce large amounts of a given RNA strand of a given siRNA. The OligoPilot II reactor can efficiently couple nucleotides using only a 1.5 molar excess of phosphoramidite nucleotides. For the preparation of RNA strands, ribonucleotide imides (amidites) are used. Standard cycles of monomer addition can be used to synthesize a 21 to 23 nucleotide strand of siRNA. Typically, the two complementary strands are generated separately and then annealed, e.g., after release from the solid support and deprotection.

Organic synthesis can be used to generate discrete siRNA species. The complementarity of the species to a particular target gene can be precisely specified. For example, the species may be complementary to a region that includes a polymorphism, e.g., a single nucleotide polymorphism. Furthermore, the location of the polymorphism can be precisely defined. In some embodiments, the polymorphism is located in an internal region, e.g., at least 4, 5, 7, or 9 nucleotides from one or both ends.

dsiRNA cleavage. siRNA can also be prepared by cleaving larger siRNA. Cleavage may be mediated in vitro or in vivo. For example, to produce iRNA by in vitro cleavage, the following method can be used:

and (5) in vitro transcription. dsiRNA is produced by bi-directional transcription of nucleic acid (DNA) fragments. For example, hiScribe ^TM RNAi transcription kit (New England Biolabs) provides a vector and a method for producing dsiRNA of a nucleic acid fragment cloned into a position with T7 promoter on either side of the vector. Separate templates were generated for T7 transcription of the two complementary strands of dsiRNA. Templates were transcribed in vitro by addition of T7 RNA polymerase and dsiRNA was produced. Similar methods using PCR and/or other RNA polymerases (e.g., T3 or SP6 polymerase) can also be dotoxin that can contaminate the preparation of the recombinase.

And (5) in vitro cracking. In one embodiment, the RNA produced by this method is carefully purified to remove terminal siRNA. For example, siRNA is cleaved in vitro into siRNA using Dicer or equivalent RNAse III-based activity. For example, dsiRNA may be incubated in an in vitro extract from drosophila or using purified components, e.g., purified RNAse or RISC complex (RNA-induced silencing complex). See, e.g., ketting et al Genes Dev2001Oct 15;15 (20) 2654-9; and Hammond Science 2001 Aug 10;293 (5532):1146-50.

dsiRNA cleavage typically produces multiple siRNA species, each of which is a specific 21 to 23nt fragment of the source dsiRNA molecule. For example, there may be an siRNA comprising sequences complementary to overlapping and adjacent regions of the source dsiRNA molecule.

Regardless of the synthetic method, the siRNA formulation can be prepared in a solution (e.g., aqueous and/or organic solution) suitable for formulation. For example, the siRNA formulation may be precipitated and redissolved in pure double distilled water and lyophilized. The dried siRNA can then be resuspended in a solution suitable for the intended formulation process.

Conjugation of iRNA agents to targeting ligands

In some embodiments, the targeting ligand is conjugated to the iRNA agent via a nucleobase, sugar moiety, or internucleoside linkage.

Conjugation to the purine nucleobase or derivative thereof may occur at any position, including in-and out-of-loop atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is attached to the conjugate moiety. Conjugation to pyrimidine nucleobases or derivatives thereof may also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of the pyrimidine nucleobases may be substituted with conjugate moieties. When the targeting ligand is conjugated to a nucleobase, the preferred position is one that does not interfere with hybridization, i.e., does not interfere with hydrogen bonding interactions required for base pairing. In one embodiment, the targeting ligand can be conjugated to the nucleobase through a linker containing an alkyl, alkenyl or amide linkage.

Conjugation to the sugar moiety of the nucleoside can occur at any carbon atom. Exemplary carbon atoms of the targeting ligand-attachable sugar moiety include 2', 3', and 5' carbon atoms. The targeting ligand may also be attached to the 1' position, for example in an abasic residue. In one embodiment, the targeting ligand may be conjugated to the saccharide moiety through a 2' -O modification, with or without a linker.

The internucleoside linkage may also carry a targeting ligand. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the targeting ligand may be directly linked to the phosphorus atom or to O, N or S atoms bound to the phosphorus atom. For amine-or amide-containing internucleoside linkages (e.g., PNAs), the targeting ligand may be attached to the nitrogen atom or an adjacent carbon atom of the amine or amide.

There are many methods for preparing conjugates of oligonucleotides. Typically, the oligonucleotide is attached to the conjugate moiety by contacting a reactive group (e.g., OH, SH, amine, carboxyl, aldehyde, etc.) on the oligonucleotide with a reactive group on the conjugate moiety. In some embodiments, one reactive group is electrophilic, while the other is nucleophilic.

For example, the electrophilic group may be a carbonyl-containing functional group, while the nucleophilic group may be an amine or a thiol. Methods of conjugation of nucleic acids and related oligonucleotides with and without a linking group are fully described in the literature, e.g., manoharan in Antisense Research and Applications, rooke and leblue editions, CRC press, boca Raton, fli, 1993, chapter 17, which is incorporated herein by reference in its entirety.

In one embodiment, the first (complementary) RNA strand and the second (sense) RNA strand can be synthesized separately, wherein one of the RNA strands comprises a flanking targeting ligand, and the first and second RNA strands can be mixed to form the dsRNA. The step of synthesizing the RNA strand preferably involves solid phase synthesis, wherein the individual nucleotides are joined end-to-end by forming internucleotide 3'-5' phosphodiester bonds in successive synthesis cycles.

In one embodiment, the targeting ligand with a phosphoramidite group is coupled to the 3 '-end or the 5' -end of the first (complementary) or second (sense) RNA strand in the final synthesis cycle. In solid phase synthesis of RNA, the nucleotide is initially in the form of a nucleoside phosphoramidite. In each synthesis cycle, further nucleoside phosphoramidites are attached to the-OH group of the previously incorporated nucleotide. If the targeting ligand has a phosphoramidite group, it can be coupled to the free OH end of RNA previously synthesized in solid phase synthesis in a manner similar to nucleoside phosphoramidites. The synthesis can be carried out in an automated and standardized manner using a conventional RNA synthesizer. Synthesis of targeting ligands with phosphoramidite groups can include phosphitylation of free hydroxyl groups to produce phosphoramidite groups.

In general, oligonucleotides can be synthesized using protocols known in the art, such as, for example, caruthers et al, methods in Enzymology (1992) 211:3-19; WO 99/54459; wincott et al, nucleic acids Res. (1995) 23:2677-2684; wincott et al Methods mol. Bio. (1997) 74:59; brennan et al, biotechnol.Bioeng. (1998) 61:33-45; and U.S. Pat. No. 6,001,311; each of which is incorporated herein by reference in its entirety. In general, oligonucleotide synthesis involves conventional nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5 '-end and phosphoramidite at the 3' -end. In a non-limiting example, small scale synthesis was performed on an expodite 8909RNA synthesizer sold by Applied Biosystems, inc (Weiterstadt, germany) using ribonucleoside phosphoramidites sold by ChemGenes Corporation (Ashland, mass.). Alternatively, the synthesis may be performed on a 96-well plate synthesizer, such as an instrument produced by Protogene (Palo Alto, calif.), or by Usman et al, J.am.chem.Soc. (1987) 109:7845; scaringe et al, nucleic acids Res (1990) 18:5433; wincott et al, nucleic acids Res.

(1990) 23:2677-2684; and Wincott, et al, methods mol. Bio (1997) 74:59, each of which is incorporated herein by reference in its entirety.

The nucleic acid molecules of the invention may be synthesized separately and joined together after synthesis, for example, by ligation (Moore et al, science (1992) 256:9923; WO 93/23569; shabarova et al, nucleic acids Res. (1991) 19:4247; bellon et al, nucleic acids & nucleic oxides (1997) 16:951; bellon et al, bioconjugate chem. (1997) 8:204; or by post-synthesis hybridization and/or deprotection. The nucleic acid molecules may be purified by gel electrophoresis using conventional methods, or may be purified by high pressure liquid chromatography (HPLC; see Wincott et al, supra), the entire contents of which are incorporated herein by reference), and resuspended in water.

Pharmaceutical composition

In one aspect, the invention features a pharmaceutical composition including an iRNA (siRNA), e.g., a double-stranded siRNA or ssiRNA, (e.g., a precursor, such as a larger siRNA that can be processed into a ssiRNA, or a DNA encoding an siRNA, e.g., a double-stranded siRNA or ssiRNA, or a precursor thereof) that includes a nucleotide sequence that is complementary, e.g., substantially and/or fully complementary, to a target RNA. The target RNA may be a transcript of an endogenous human gene. In one embodiment, the siRNA (a) is 19 to 25 nucleotides long, e.g., 21 to 23 nucleotides long, (b) is complementary to the endogenous target RNA, and optionally, (c) comprises at least one 3' overhang that is 1 to 5nt long. In one embodiment, the pharmaceutical composition may be an emulsion, microemulsion, cream, jelly, or liposome.

In one example, the pharmaceutical composition includes an iRNA (siRNA) admixed with a local delivery agent. The local delivery agent may be a plurality of microscopic vesicles. The microvesicles may be liposomes. In some embodiments, the liposome is a cationic liposome.

In another aspect, the pharmaceutical composition includes an iRNA (siRNA), e.g., a double stranded siRNA or ssiRNA (e.g., a precursor, such as a larger siRNA that can be processed into a ssiRNA or a DNA encoding an siRNA, e.g., a double stranded siRNA or ssiRNA, or a precursor thereof) in admixture with a local permeation enhancer. In one embodiment, the topical penetration enhancer is a fatty acid. The fatty acid may be arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, glyceryl monooleate (monolein), glyceryl dilaurate, glyceryl 1-monocaprate, 1-dodecylazepan-2-one, acylcarnitine, acylcholine or C _1-10 Alkyl esters, monoglycerides, diglycerides or pharmaceutically acceptable salts thereof.

In another embodiment, the topical permeation enhancer is a bile salt. The bile salt may be cholic acid, dehydrocholic acid, deoxycholic acid, glycocholic acid (glycocholic acid), glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, taurine-24, 25-dihydro-sodium fusidate, sodium glycodihydrofusidate, polyoxyethylene-9-lauryl ether or pharmaceutically acceptable salts thereof.

In another embodiment, the permeation enhancer is a chelating agent. The chelating agent may be EDTA, citric acid, salicylate, N-acyl derivatives of collagen, laureth-9, N-aminoacyl derivatives of beta-diketones or mixtures thereof.

In another embodiment, the penetration enhancer is a surfactant, e.g., an ionic or nonionic surfactant. The surfactant may be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, perfluorinated chemical emulsion or mixtures thereof.

In another embodiment, the permeation enhancer may be selected from the group consisting of unsaturated cyclic ureas, 1-alkyl-alkanones, 1-alkenyl aza-alkanones, steroidal anti-inflammatory agents, and mixtures thereof. In yet another embodiment, the permeation enhancer may be a glycol, pyrrole, azone, or terpene.

In one aspect, the invention features a pharmaceutical composition that includes an iRNA (siRNA) in a form suitable for oral delivery, e.g., a double stranded siRNA or a ssiRNA, (e.g., a precursor, such as a larger siRNA that can be processed into the ssiRNA, or DNA encoding an siRNA, e.g., a double stranded siRNA or a ssiRNA, or a precursor thereof). In one embodiment, oral delivery can be used to deliver the siRNA composition to cells or regions of the gastrointestinal tract, e.g., the small intestine, colon (e.g., to treat colon cancer), and the like. The oral delivery form may be a tablet, capsule or gel capsule. In one embodiment, the siRNA of the pharmaceutical composition modulates the expression of a cell adhesion protein, modulates a cell proliferation rate, or has biological activity against a eukaryotic pathogen or retrovirus. In another embodiment, the pharmaceutical composition comprises an enteric material that substantially prevents dissolution of the tablet, capsule or gel capsule in the stomach of a mammal. In some embodiments, the enteric material is a coating. The coating may be phthalate acetate, propylene glycol, sorbitan monooleate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate or cellulose acetate phthalate.

In another embodiment, the oral dosage form of the pharmaceutical composition includes a permeation enhancer. The permeation enhancer may be a bile salt or a fatty acid. The bile salt can be ursodeoxycholic acid, chenodeoxycholic acid and their salts. The fatty acid may be capric acid, lauric acid, and salts thereof.

In another embodiment, the oral dosage form of the pharmaceutical composition comprises an excipient. In one example, the excipient is polyethylene glycol. In another example, the excipient is procilol.

In another embodiment, the oral dosage form of the pharmaceutical composition comprises a plasticizer. The plasticizer may be diethyl phthalate, dibutyl triacetate, dibutyl phthalate or triethyl citrate.

In one aspect, the invention features a pharmaceutical composition that includes an iRNA (siRNA) and a delivery vehicle. In one embodiment, the siRNA (a) is 19 to 25 nucleotides long, e.g., 21 to 23 nucleotides long, (b) is complementary to the endogenous target RNA, and optionally, (c) comprises at least one 3' overhang of 1 to 5 nucleotides long.

In one embodiment, the delivery vehicle can deliver an iRNA (siRNA), e.g., a double stranded siRNA or ssiRNA (e.g., a precursor, such as a larger siRNA that can be processed into a ssiRNA, or a DNA encoding an siRNA, e.g., a double stranded siRNA or ssiRNA, or a precursor thereof) to the radial cell by local administration. The delivery vehicle may be a microvesicle. In one example, the microvesicles are liposomes. In some embodiments, the liposome is a cationic liposome. In another example, the microvesicles are micelles. In one aspect, the invention features pharmaceutical compositions that include an siRNA in an injectable dosage form, e.g., a double stranded siRNA or ssiRNA (e.g., a precursor, such as a larger siRNA that can be processed into ssiRNA, or a DNA encoding an siRNA, e.g., a double stranded siRNA or ssiRNA, or a precursor thereof). In one embodiment, the injectable dosage form of the pharmaceutical composition includes a sterile aqueous solution or dispersion and a sterile powder. In some embodiments, the sterile solution may include a diluent such as water; a brine solution; fixed oils, polyethylene glycols, glycerol or propylene glycol.

In one aspect, the invention features pharmaceutical compositions comprising an iRNA (siRNA) in an oral dosage form, e.g., a double-stranded siRNA or ssiRNA (e.g., a precursor, such as a larger siRNA that can be processed into a ssiRNA, or a DNA encoding an siRNA, e.g., a double-stranded siRNA or ssiRNA, or a precursor thereof). In one embodiment, the oral dosage form is selected from the group consisting of a tablet, a capsule, and a gel capsule. In another embodiment, the pharmaceutical composition comprises an enteric material that substantially prevents dissolution of the tablet, capsule or gel capsule in the stomach of a mammal. In some embodiments, the enteric material is a coating. The coating may be phthalate acetate, propylene glycol, sorbitan monooleate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate or cellulose acetate phthalate. In one embodiment, the oral dosage form of the pharmaceutical composition includes a permeation enhancer, e.g., a permeation enhancer as described herein.

In one aspect, the invention features pharmaceutical compositions including an iRNA (siRNA) in a rectal dosage form, e.g., a double-stranded siRNA or ssiRNA (e.g., a precursor, such as a larger siRNA that can be processed into a ssiRNA, or a DNA encoding an siRNA, e.g., a double-stranded siRNA or ssiRNA, or a precursor thereof). In one embodiment, the rectal dosage form is an enema. In another embodiment, the rectal dosage form is a suppository.

In one aspect, the invention features a pharmaceutical composition in vaginal dosage form that includes an iRNA (siRNA), e.g., a double-stranded siRNA or ssiRNA (e.g., a precursor, such as a larger siRNA that can be processed into a ssiRNA, or a DNA encoding an siRNA, e.g., a double-stranded siRNA or ssiRNA, or a precursor thereof). In one embodiment, the vaginal dosage form is a suppository. In another embodiment, the vaginal dosage form is a foam, cream or gel.

In one aspect, the invention features a pharmaceutical composition including an iRNA (siRNA) in a pulmonary or nasal dosage form, e.g., a double stranded siRNA or ssiRNA, e.g., a precursor, such as a larger siRNA that can be processed into a ssiRNA, or a DNA encoding an siRNA, e.g., a double stranded siRNA or ssiRNA, or a precursor thereof). In one embodiment, the siRNA is incorporated into a particle, e.g., a large particle, such as a microsphere. The particles may be prepared by spray drying, lyophilization, evaporation, fluidized bed drying, vacuum drying, or a combination thereof. Microspheres may be formulated as suspensions, powders or implantable solids.

Therapeutic methods and delivery routes

Another aspect of the invention relates to a method of reducing expression of a target gene in a cell comprising contacting the cell with an oligonucleotide. In one embodiment, the cell is an extrahepatic cell.

Another aspect of the invention relates to a method of reducing expression of a target gene in a subject comprising administering an oligonucleotide to the subject.

Another aspect of the invention relates to a method of treating a subject suffering from a CNS disorder comprising administering to the subject a therapeutically effective amount of a double stranded iRNA agent of the invention, thereby treating the subject. Exemplary CNS disorders treatable by the methods of the invention include alzheimer's disease, amyotrophic Lateral Sclerosis (ALS), frontotemporal dementia, huntington's disease, parkinson's disease, spinocerebellar disease, prions, and lafora.

Depending on the type of gene targeted and the type of disorder to be treated, the oligonucleotide may be delivered to the subject by a variety of routes. In some embodiments, the oligonucleotide is administered extrahepatic, such as ocular (e.g., intravitreal) or intrathecal or intraventricular.

In one embodiment, the oligonucleotide is administered intrathecally or intraventricularly. By intrathecal or intraventricular administration of the double stranded iRNA agent, the method can reduce expression of the target gene in brain or spinal tissues, e.g., cortex, cerebellum, cervical, lumbar and thoracic vertebrae.

In some embodiments, exemplary target genes are APP, ATXN2, C9orf72, TARDBP, MAPT (Tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA7, SCA8, meCP2, PRNP, SOD1, DMPK, and TTR. To reduce expression of these target genes in a subject, the oligonucleotides can be administered directly (e.g., intravitreally) to the eye. The method can reduce expression of a target gene in ocular tissue by intravitreal administration of a double stranded iRNA agent.

For ease of illustration, the formulations, compositions and methods in this section are discussed primarily with respect to modified siRNA. However, it is understood that these formulations, compositions and methods can be practiced with other siRNAs, e.g., unmodified siRNAs, and that such implementations are within the present invention. Compositions comprising iRNA can be delivered to a subject by a variety of routes. Exemplary approaches include: intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, and ocular.

The iRNA molecules of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more iRNA species and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional medium or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds may also be incorporated into the compositions.

The pharmaceutical compositions of the present invention may be administered in a variety of ways, depending on the area to be treated and the local or systemic treatment desired. Administration may be topical (including ocular, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular infusion, or intrathecal or intraventricular administration.

The route and site of administration may be selected to enhance targeting. For example, for targeting muscle cells, intramuscular injection into the muscle of interest would be a reasonable choice. Lung cells can be targeted by administration of iRNA in aerosol form. Vascular endothelial cells can be targeted by coating the balloon catheter with iRNA and mechanically introducing DNA.

Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily matrices, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, troches or lozenges. In the case of tablets, carriers that may be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants, for example starches and lubricants, such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are desired for oral use, the nucleic acid composition may be combined with emulsifying and suspending agents. If desired, certain sweeteners and/or flavoring agents may be added.

Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Intraventricular injection may be facilitated by, for example, an intraventricular catheter attached to a reservoir. For intravenous use, the total concentration of solutes can be controlled to render the formulation isotonic.

For ocular administration, ointments or drops may be delivered by ocular delivery systems known in the art, such as an applicator or an eye dropper. Such compositions may include a mucous mimetic such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose, or poly (vinyl alcohol), a preservative such as sorbic acid, EDTA, or benzalkonium chloride (benzychronium chloride), in combination with a common amount of diluents and/or carriers.

In one embodiment, the administration of an iRNA (siRNA), e.g., a double stranded siRNA or ssiRNA composition, is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracerebroventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral, or ocular. Administration may be provided by the subject or by another person (e.g., a health care provider). The medicament may be provided in measured doses or in dispensers delivering metered doses. The selected delivery mode is discussed in more detail below.

Intrathecal administration. In one embodiment, delivery is by intrathecal injection (i.e., injection into spinal fluid that bathes brain and spinal cord tissue). intrathecal injection of the iRNA agent into the spinal fluid may be performed as a bolus or by an implantable subcutaneous micropump, providing periodic and sustained delivery of siRNA into the spinal fluid. Spinal fluid circulates from the choroid plexus, which produces spinal fluid, down around the spinal cord and dorsal root ganglion, and then up through the cerebellum and through the cortex to the arachnoid particles (where fluid may leave the CNS), which depends on the size, stability and solubility of the injected compound, the intrathecally delivered molecules may hit targets throughout the entire CNS.

In some embodiments, intrathecal administration is by pump. The pump may be an osmotic pump that is surgically implanted. In one embodiment, an osmotic pump is implanted into the subarachnoid space of the spinal canal to facilitate intrathecal administration.

In some embodiments, intrathecal administration is by an intrathecal delivery system for a drug comprising a reservoir containing a volume of a medicament and a pump configured to deliver a portion of the medicament contained in the reservoir. Further details regarding this intrathecal delivery system can be found in PCT/US 2015/013153 submitted on day 28, 1, 2015, which is incorporated herein by reference in its entirety.

The amount of the iRNA agent injected intrathecally or intraventricularly can vary from one target gene to another and the appropriate amount that must be administered is determined separately for each target gene. Typically, the amount ranges from 10 μg to 2mg, preferably from 50 μg to 1500 μg, more preferably from 100 μg to 1000 μg.

Rectal administration. The invention also provides methods, compositions, and kits for rectal administration or delivery of the siRNA described herein.

Thus, an iRNA (siRNA) described herein, e.g., a double-stranded siRNA or ssiRNA (e.g., a precursor, such as a larger siRNA that can be processed into a ssiRNA, or a DNA encoding an siRNA, e.g., a double-stranded siRNA or ssiRNA, or a precursor thereof), e.g., a therapeutically effective amount of an siRNA described herein, e.g., an siRNA having a duplex region of less than 40, e.g., less than 30 nucleotides, and having a single-stranded 3' overhang of one or two 1-3 nucleotides, can be administered rectally, e.g., introduced into the lower or upper colon by rectum. The method is particularly useful for treating inflammatory disorders, disorders characterized by unwanted cell proliferation, such as polyps or colon cancer.

By introducing a dispensing device, e.g. a flexible camera-guided device similar to that used for examination of the colon or removal of polyps, comprising a device for delivering a drug, the drug can be delivered to a site in the colon.

Rectal administration of siRNA is by means of enema. The siRNA of the enema can be dissolved in saline or buffer solution. Rectal administration may also be by suppositories, which may contain other ingredients, for example, excipients such as cocoa butter or hydroxypropyl methylcellulose.

Ocular delivery. The iRNA agents described herein can be administered to ocular tissue. For example, the medicament may be applied to tissue at or near the surface of the eye, such as the interior of the eyelid. They can be applied topically, for example, by spraying, in the form of drops, as eye washes or ointments. Administration may be provided by the subject or another person (e.g., a health care provider). The medicament may be provided in a metered dose or in a dispenser delivering a metered dose. The drug may also be administered to the interior of the eye and may be introduced through a needle or other delivery device that may introduce the drug into a selected area or structure. Eye treatment is particularly desirable for treating inflammation of the eye or nearby tissue.

In certain embodiments, the double-stranded iRNA agent may be delivered directly to the eye by ocular tissue injection, such as periocular, conjunctival, sub-tenon's capsule, intra-anterior, intravitreal, intra-ocular, anterior or posterior juxtascleral, subretinal, subconjunctival, retrobulbar, or intratubular injection; direct application to the eye by use of a catheter or other placement device such as retinal pellets, intraocular inserts, suppositories, or implants comprising porous, non-porous, or gel-like materials; by topical eye drops or ointments; or by a slow release device implanted in the conjunctival sac or adjacent to the sclera (transsclera) or in the sclera (intra sclera) or in the eye. Intracameral injection may be passed through the cornea into the anterior chamber to allow the agent to reach the trabecular meshwork. The intratubular injection may be into or within a venous collection channel draining schlemm's canal.

In one embodiment, the double stranded iRNA agent can be administered to an eye, such as in the vitreous cavity of the eye, by intravitreal injection, for example, using a prefilled syringe in the form of an instant injection for use by medical personnel.

For ocular delivery, the double-stranded iRNA agent may be combined with an ophthalmically acceptable preservative, co-solvent, surfactant, viscosity enhancing agent, penetration enhancing agent, buffer, sodium chloride, or water to form an aqueous, sterile ophthalmic suspension or solution. Solution formulations may be prepared by dissolving the conjugate in a physiologically acceptable isotonic aqueous buffer. In addition, the solution may include an acceptable surfactant to aid in dissolving the double stranded iRNA agent. Viscosity enhancing agents, such as hydroxymethyl cellulose, hydroxyethyl cellulose, methyl cellulose, polyvinylpyrrolidone, and the like, may be added to the pharmaceutical compositions to improve retention of the double stranded iRNA agent.

To prepare a sterile ophthalmic ointment formulation, a double stranded iRNA agent is combined with a preservative in a suitable vehicle, such as mineral oil, liquid lanolin, or white petrolatum. Sterile ophthalmic gel formulations can be prepared by suspending a double stranded iRNA agent in a gel prepared by, for example, a method known in the art -940 (BF Goodrich, charlotte, n.c.), etc.

Local delivery. Any of the sirnas described herein can be directly applied to the skin. For example, the drug may be applied topically or delivered in a layer of skin, e.g., by using a microneedle or set of microneedles that penetrate into the skin but, e.g., do not enter the underlying musculature. The administration of the siRNA composition may be topical. Topical application may, for example, deliver the composition to the dermis or epidermis of a subject. Topical administration may be in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids or powders. Compositions for topical administration may be formulated as liposomes, micelles, emulsions or other lipophilic molecular assemblies. Transdermal administration may be applied with at least one penetration enhancer, such as iontophoresis, sonophoresis and sonophoresis.

For ease of illustration, the formulations, compositions, and methods in this section are discussed mostly with respect to modified siRNA. However, it is understood that these formulations, compositions and methods can be practiced with other siRNAs, e.g., unmodified siRNAs, and that such implementations are within the present invention. In some embodiments, the siRNA, e.g., a double stranded siRNA or ssiRNA (e.g., a precursor, such as a larger siRNA that can be processed into ssiRNA, or DNA encoding the siRNA, e.g., a double stranded siRNA or ssiRNA, or a precursor thereof) is delivered to the subject by topical administration. "topical administration" refers to delivery to a subject by bringing the formulation into direct contact with the surface of the subject. The most common form of topical delivery is to the skin, but the compositions disclosed herein may also be applied directly to other surfaces of the body, for example to the eye, mucous membranes, body cavity surfaces or inner surfaces. As mentioned above, the most common topical delivery is to the skin. The term encompasses several routes of administration, including, but not limited to, topical administration and transdermal administration. These modes of application generally include penetration of the skin across the permeation barrier and effective delivery to the target tissue or layer. Topical application can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition. Topical administration may also be used as a means of selectively delivering the oligonucleotide to the epidermis or dermis of a subject, or to a particular layer thereof, or to underlying tissue.

As used herein, the term "skin" refers to the epidermis and/or dermis of an animal. Mammalian skin is composed of two major, distinct layers. The outer layer of the skin is called the epidermis. The epidermis is composed of the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale, which is the deepest portion of the epidermis, at the skin surface. The thickness of the epidermis is between 50 μm and 0.2mm depending on the location on the body.

Below the epidermis is the dermis, which is significantly thicker than the epidermis. The dermis is mainly composed of collagen in the form of fiber bundles. Collagen bundles support, inter alia, blood vessels, lymphatic capillaries, glands, nerve endings and immunocompetent cells.

One of the main functions of the skin as an organ is to regulate the entry of substances into the body. The primary permeability barrier of the skin is provided by the stratum corneum, which is formed by multiple layers of cells in various states of differentiation. The spaces between cells in the stratum corneum are filled with different lipids arranged in a lattice configuration that provides a seal to further enhance the skin penetration barrier.

The permeation barrier provided by the skin makes it largely impermeable to molecules having a molecular weight greater than about 750 Da. In order for larger molecules to cross the permeation barrier of the skin, mechanisms other than normal permeation must be used.

Several factors determine the permeability of the skin to the applied agent. These factors include the characteristics of the skin being treated, the characteristics of the delivery agent, the drug and the delivery agent and the interaction between the drug and the skin, the dosage of the drug administered, the form of treatment, and the post-treatment regimen. To selectively target the epidermis and dermis, compositions may sometimes be formulated that include one or more permeation enhancers that enable the permeation of a drug to a preselected layer.

Transdermal delivery is a valuable route of administration of liposoluble therapeutic agents. The dermis is more permeable than the epidermis and is therefore absorbed much faster by bruises, burns or bare skin. Inflammation and other physiological conditions that increase blood flow to the skin also enhance transdermal absorption. Absorption by this route may be enhanced by the use of an oily vehicle (oiling) or by the use of one or more permeation enhancers. Other effective means of delivering the compositions disclosed herein by the transdermal route include hydration of the skin and the use of controlled release topical patches. The transdermal route provides a potentially effective means of delivering the compositions disclosed herein for systemic and/or local treatment.

In addition, iontophoresis (transfer of ionic solutes through biological membranes under the influence of an electric field) (Lee et al, critical Reviews in Therapeutic Drug Carrier Systems,1991, page 163), sonophoresis or sonophoresis (use of ultrasound to enhance absorption of various therapeutic agents across biological membranes, particularly the skin and cornea) (Lee et al, critical Reviews in Therapeutic Drug Carrier Systems,1991, page 166), and optimization of dose position and retained vehicle characteristics relative to the site of administration (Lee et al, critical Reviews in Therapeutic Drug Carrier Systems,1991, page 168) can be useful methods for enhancing transport of topically applied compositions across skin and mucosal sites.

The provided compositions and methods can also be used to detect the function of various proteins and genes in dermal tissue and in animals cultured or maintained in vitro. Thus, the present invention can be applied to examine the function of any gene. The methods of the invention may also be used therapeutically or prophylactically. For example, for treating animals known or suspected to have a disease such as psoriasis, lichen planus, toxic epidermonecrobiosis, erythema multiforme, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, paget's disease, kaposi's sarcoma, pulmonary fibrosis, lyme disease and viral, fungal and bacterial infections of the skin.

Pulmonary delivery. Any of the sirnas described herein can be administered to the pulmonary system. Pulmonary administration can be achieved by inhalation or by introducing a delivery device into the pulmonary system, for example by introducing a dispensable drug delivery device. Certain embodiments may use methods of pulmonary delivery by inhalation. The medicament may be provided in a dispenser which delivers the medicament in a sufficiently small form, e.g. wet or dry medicament, so that it may be inhaled. The device may deliver a metered dose of the drug. The subject or another person may administer the medicament. Pulmonary delivery is effective not only for conditions that directly affect lung tissue, but also for conditions that affect other tissues. The siRNA can be formulated as a liquid or non-liquid, e.g., powder, crystal, or aerosol, for pulmonary delivery.

For ease of illustration, the formulations, compositions, and methods in this section are discussed in large part with respect to modified siRNA. However, it is understood that these formulations, compositions and methods can be practiced with other siRNAs, e.g., unmodified siRNAs, and that such implementations are within the present invention. Compositions comprising an siRNA, e.g., a double stranded siRNA or a ssiRNA (e.g., a precursor, such as a larger siRNA that can be processed into a ssiRNA, or a DNA encoding an siRNA, e.g., a double stranded siRNA or a ssiRNA, or a precursor thereof) can be administered to a subject by pulmonary delivery. Pulmonary delivery compositions can be delivered by inhalation of the dispersion by a patient such that the composition within the dispersion, e.g., iRNA, can reach the lungs where it can be readily absorbed directly into the blood circulation through the alveolar region. Pulmonary delivery can be effective in treating pulmonary diseases for systemic delivery and local delivery.

Pulmonary delivery can be achieved by different methods, including the use of atomized, aerosolized, micelle, and dry powder-based formulations. Delivery may be achieved by liquid nebulizers, aerosol-based inhalers, and dry powder dispensing devices. A metered dose device may be used. One of the benefits of using a nebulizer or inhaler is that the possibility of contamination can be minimized because the device is self-contained. For example, dry powder dispersion devices deliver drugs that can be readily formulated as dry powders. The iRNA composition may be stably stored as a lyophilized or spray-dried powder by itself or in combination with a suitable powder carrier. Delivery of the composition for inhalation may be mediated by an administration timing element, which may include a timer, a dose counter, a time measurement device or a time indicator, which when incorporated into the device, enables dose tracking, compliance monitoring and/or dose triggering of the patient during administration of the aerosol medicament.

The term "powder" means a composition consisting of finely divided solid particles that are free flowing and can be readily dispersed in an inhalation device and subsequently inhaled by a subject such that the particles reach the lungs to allow penetration into the alveoli. Thus, the powder is said to be "respirable". For example, the average particle size diameter is less than about 10 μm, with a relatively uniform spherical distribution. In some embodiments, the diameter is less than about 7.5 μm, and in some embodiments, less than about 5.0 μm. Typically, the particle size distribution is between about 0.1 μm to about 5 μm in diameter, sometimes between about 0.3 μm to about 5 μm.

The term "dry" means that the moisture content of the composition is less than about 10 weight percent (% w) of water, typically less than about 5% w, and in some cases less than about 3% w. The dry composition may allow the particles to be readily dispersed in the inhalation device to form an aerosol.

The term "therapeutically effective amount" is the amount present in the composition required to provide the desired level of drug to produce the desired physiological response in the subject to be treated.

The term "physiologically effective amount" refers to an amount delivered to a subject to produce a desired reduction or therapeutic effect.

The term "pharmaceutically acceptable carrier" means that the carrier can be taken up into the lungs without significant adverse toxicological effects on the lungs.

Types of pharmaceutical excipients that can be used as carriers include stabilizers such as Human Serum Albumin (HSA), fillers such as carbohydrates, amino acids, and polypeptides; a pH adjustor or a buffering agent; salts such as sodium chloride; etc. These carriers may be in crystalline or amorphous form or may be a mixture of both.

Particularly valuable compatibilizers include compatible carbohydrates, polypeptides, amino acids, or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, etc.; disaccharides such as lactose, trehalose, and the like; cyclodextrins, such as 2-hydroxypropyl-beta-cyclodextrin; and polysaccharides such as raffinose, maltodextrin, dextran, and the like; alditols such as mannitol, xylitol, and the like. One group of carbohydrates may include lactose, trehalose, raffinose, maltodextrin and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine, with glycine being used in some embodiments.

Additives may be included as minor components of the compositions of the invention for conformational stability during spray drying and to improve the dispersibility of the powder. These additives include hydrophobic amino acids such as tryptophan, tyrosine, leucine, phenylalanine, and the like.

Suitable pH adjusting agents or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate may be used in some embodiments.

Pulmonary administration of micellar iRNA formulations can be achieved by a metered dose spray device with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFCs and CFC propellants.

Oral or nasal delivery. Any of the sirnas described herein can be administered orally, e.g., in the form of a tablet, capsule, gel capsule, lozenge, troche, or liquid syrup. Furthermore, the composition may be applied topically to the oral surface.

Any of the sirnas described herein can be administered nasally. Nasal administration may be achieved by introducing a delivery device into the nose, for example by introducing a delivery device that can dispense a drug. Methods of nasal delivery include sprays, aerosols, liquids, such as by drops, or by topical application to the surface of the nasal cavity. The medicament may be in a dispenser and delivered in a form small enough, e.g. wet or dry, so that it may be inhaled. The device may deliver a metered dose of the drug. The subject or another person may administer the medicament.

Nasal delivery is effective not only for conditions that directly affect nasal tissue, but also for conditions that affect other tissues. siRNA can be formulated as a liquid or non-liquid, e.g., powder, crystals, or for nasal delivery. As used herein, the term "crystal" describes a solid having the structure or characteristics of a crystal, i.e., a particle of three-dimensional structure, wherein planes intersect at defined angles and wherein a regular internal structure exists. The compositions of the present invention may have different crystal forms. The crystalline forms may be prepared by a variety of methods including, for example, spray drying.

For ease of illustration, the formulations, compositions, and methods in this section are discussed in large part with respect to modified siRNA. However, it is understood that these formulations, compositions and methods can be practiced with other siRNAs, e.g., unmodified siRNAs, and that such implementations are within the present invention. Oral and nasal membranes offer advantages over other routes of administration. For example, drugs administered through these membranes have a rapid onset, provide therapeutic plasma levels, avoid first pass effects of liver metabolism, and avoid exposure of the drug to adverse Gastrointestinal (GI) environments. Additional advantages include easy access to the membrane site so that the drug can be easily applied, positioned and removed.

In oral delivery, the composition may be targeted to oral surfaces, for example, the film including the ventral surface of the tongue and the sublingual mucosa of the buccal mucosa, or the buccal mucosa that constitutes the inner wall of the cheek. The sublingual mucosa is relatively permeable, thus giving rapid absorption and acceptable bioavailability of many drugs. In addition, sublingual mucosa is convenient, acceptable and easy to access.

The ability of molecules to penetrate the oral mucosa appears to be related to molecular size, lipid solubility and peptide protein ionization. Small molecules smaller than 1000 daltons appear to rapidly cross the mucosa. As the molecular size increases, permeability decreases rapidly. Fat-soluble compounds are more permeable than non-fat-soluble molecules. Maximum absorption occurs when the molecule is not ionized or the charge is neutral. Thus, the charged molecules present the greatest challenge to absorption across the oral mucosa.

The pharmaceutical composition of iRNA can also be administered to the buccal cavity of a human by spraying the mixed micelle pharmaceutical formulation and propellant as described above into the buccal cavity from a metered dose spray dispenser, rather than inhalation. In one embodiment, the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity. For example, the drug may be sprayed into the buccal cavity or applied directly to the surface of the buccal cavity, for example in liquid, solid or gel form. Such administration is particularly desirable for treating inflammation of the buccal cavity, e.g., gums or tongue, e.g., in one embodiment, buccal administration is by spraying from a dispenser, e.g., a metered dose spray dispenser that dispenses a pharmaceutical composition and a propellant, into the buccal cavity, e.g., non-inhalation.

Aspects of the application also relate to methods of delivering oligonucleotides into the CNS by intrathecal or intraventricular delivery, or into ocular tissue by ocular delivery, e.g., intravitreal delivery.

Some embodiments relate to methods of reducing expression of a target gene in a subject comprising administering an oligonucleotide described herein to the subject. In one embodiment, the oligonucleotide is administered intrathecally or intraventricularly (to reduce expression of the target gene in brain or spinal tissue). In one embodiment, the oligonucleotide is administered ocularly, e.g., intravitreally (to reduce expression of the target gene in ocular tissue).

The application is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references, pending application patent applications and published patents cited throughout this application are expressly incorporated herein by reference.

Examples

The application will now be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the application and are not intended to limit the application.

EXAMPLE 1 Synthesis of Cyclic disulfide modified phosphate prodrug derivatives

Scheme 1

Compound 800: trans-1, 2-dithiane-4, 5-diol (3.04 g,20 mmol) was dissolved in anhydrous THF (30 mL) under inert atmosphere and cooled in a water/ice bath. 60% dispersion of sodium hydride in oil (0.84 g,21 mmol) was added and the mixture was stirred for 30 minutes. Methyl iodide (3.7 mL,60 mmol) was added and the mixture was slowly warmed to room temperature overnight. The reaction mixture was concentrated under vacuum to a colorless liquid. The product was isolated by flash chromatography on silica gel of the crude product (3.66 g) with an isocratic 30% ethyl acetate/hexane (1:9 to 1:1 gradient). 1.11g (33%) of 800 was obtained as a pale yellow oil. ¹ H NMR,(400MHz,DMSO-d ₆ )δ5.29(d,J＝4.8Hz,1H)；3.44(septet,J＝4.8Hz,1H)；3.30(dd,J＝3.6,13.2Hz,1H)；3.11-3.03(m,2H)；2.74(dd,J＝10.0,13.6Hz,1H)；2.67(dd,J＝10.0,13.6Hz,1H)。

Compound 801: 2-cyanoethyl N, N-diisopropylchlorophosphamide (1.80 mL,8 mmol) was added to a stirred solution of methanol 800 (1.07 g,6.4 mmol) and DIEA (1.40 mL,8 mmol) in anhydrous ethyl acetate (30 mL) under Ar. The mixture was stirred at room temperature for 1 hour and quenched. The organic phase was separated, washed twice with 5% NaCl, saturated NaCl, dried over anhydrous sodium sulfate and the crude residue was purified on a silica gel column with a solution containing 1% TEAPurification of a hexane solution with an isocratic 25% ethyl acetate gave 1.88g (80%) of pure phosphoramidite 801 as a pale yellow oil. ¹ H NMR(400MHz,CD ₃ CN):δ3.94-3.74(m,2.5H)；3.74-3.55(m,2.5H)；3.39(s,1.5H)；3.38(s,1.5H)；3.36-3.15(m,3H)；2.91(dd,J＝9.2,13.6Hz,1H)；2.85-2.77(m,1H)；2.72-2.59(m,2H)；1.24-1.13(m,12H)。 ¹³ C NMR(101MHz,CD ₃ CN)δ119.64；119.61；59.84；59.67；59.11；58.91；58.38；58.10；57.64；44.10；43.98；25.03；24.96；24.95；24.92；24.90；24.84；24.76；21.09；21.03. ³¹ P NMR(202MHz,CD ₃ CN):δ150.68；150.37。

Scheme 2

Compound 802: trans-1, 2-dithiane-4, 5-diol (5.0 g,32.8 mmol) was dissolved in pyridine (160 mL) under an inert atmosphere and cooled in a water/ice bath. Trimethylacetyl chloride (14.2 mL,98.5 mmol) was added over 5 minutes, the mixture was warmed to room temperature and stirred for 1.5 hours. The mixture was concentrated under vacuum, redissolved in ethyl acetate (100 mL), washed with 5% NaCl (2X 100 mL), saturated NaCl (1X 100 mL), and dried over Na ₂ SO ₄ Dried, filtered and concentrated to an oil. The product was purified by flash chromatography on silica gel (120 g silica gel column) with ethyl acetate: hexane (1:4 to 1:2 gradient). The product containing fractions were concentrated, rinsed with acetonitrile (2×), and dried under high vacuum. Compound 802 was obtained as a white solid in 66% yield (5.12 g). ¹ H NMR,(500MHz,DMSO-d ₆ )δ5.43(d,J＝5.9Hz,1H),4.67-4.60(m,1H),3.64-3.55(m,1H),3.14(dd,J＝13.2,3.9Hz,2H),2.90-2.80(m,2H),1.15(s,9H). ¹³ C NMR(126MHz,DMSO-d ₆ )δ176.54,38.28,26.79。

Compound 803: compound 802 (3.0 g,12.7 mmol) was dissolved in anhydrous ethyl acetate (60 mL) under an inert atmosphere. N, N-diisopropylethylamine (2.9 mL,16.5 mmol) and 2-cyanoethyl N, N-diisopropylchlorophosphamide (3.7 mL,16.5 mmol) were added and the mixture was stirred atStirred at room temperature for 2 hours. The reaction mixture was quenched, washed with 5% NaCl (3X 200 mL), saturated NaCl (1X 100 mL), anhydrous Na ₂ SO ₄ Dried, filtered and concentrated. The product was purified by flash chromatography on silica gel, 80g silica gel column, using isocratic ethyl acetate (+0.5% triethylamine): hexane (1:10). The product containing fractions were concentrated in vacuo, rinsed with acetonitrile (2×), and dried in high vacuum. Compound 803 was isolated as a colorless oil in 77% yield (4.24 g). ¹ H NMR, (500 MHz, acetonitrile-d) ₃ )δ4.88-4.75(m,1H),4.03-3.92(m,1H),3.89-3.68(m,2H),3.67-3.56(m,2H),3.46-3.32(m,1H),3.03-2.84(m,2H),2.71-2.59(m,2H),1.25-1.11(m,21H)。 ¹³ C NMR (101 MHz, acetonitrile-d) ₃ )δ178.02,119.47,59.71,59.51,59.25,59.05,44.29,44.16,44.09,43.96,39.59,27.55,27.52,25.10,25.04,25.03,24.96,24.90,24.82,21.13,21.10,21.06,21.03. ³¹ P NMR (202 MHz, acetonitrile-d) ₃ )δ151.22,148.72。

Scheme 3

Compound 804: trans-1, 2-cyclohexanediol (10.1 g,87.0 mmol) was dissolved in THF (120 mL) under an inert atmosphere and cooled in a water/ice bath. 60% dispersion of sodium hydride in oil (3.96 g,91.4 mmol) was added and the reaction stirred for 1.5 hours. Methyl iodide (16.3 mL,261.1 mmol) was added and the reaction was allowed to slowly warm to room temperature over 17 hours. The reaction mixture was concentrated under vacuum to a colorless liquid. The product was isolated by flash chromatography on silica gel, 220g silica gel column, using ethyl acetate: hexane (1:9 to 1:1 gradient). The product containing fractions were concentrated and rinsed with dichloromethane (2×). Compound 804 was obtained as a colorless oil in 14% yield (1.6 g). ¹ H NMR,(400MHz,DMSO-d ₆ )δ4.57(d,J＝4.2Hz,1H),3.30-3.22(m,4H),2.89-2.79(m,1H),1.93-1.85(m,1H),1.77-1.66(m,1H),1.62-1.48(m,2H),1.17-1.00(m,4H)。 ¹³ C NMR(126MHz,DMSO-d ₆ )δ83.29,71.52,56.35,32.64,28.30,23.14,23.03。

Compound 805: at the position ofCompound 804 (0.51 g,3.9 mmol) was dissolved in anhydrous ethyl acetate (20 mL) under an inert atmosphere. N, N-diisopropylethylamine (1.0 mL,5.9 mmol) and 2-cyanoethyl N, N-diisopropylchlorophosphamide (1.3 mL,5.9 mmol) were added and the mixture was stirred at room temperature for 3 hours. The mixture was quenched and diluted with ethyl acetate (60 mL). The organic phase was separated, washed with 5% NaCl (3X 150 mL), saturated NaCl (1X 150 mL), and dried over anhydrous Na ₂ SO ₄ Dried, filtered, and concentrated in vacuo. The product was purified by flash chromatography on a 24g silica gel column quenched with triethylamine (10 mL) using ethyl acetate: hexane (1:9 to 1:2 gradient). The product containing fractions were concentrated, rinsed with acetonitrile (2×), and dried under high vacuum. Compound 805 was obtained as a colorless oil in 79% yield (1.02 g). ¹ H NMR, (400 MHz, acetonitrile-d) ₃ )δ3.87-3.54(m,5H),3.32(d,J＝2.4Hz,3H),3.14-3.02(m,1H),2.70-2.57(m,2H),2.02-1.83(m,2H),1.66-1.55(m,2H),1.47-1.21(m,4H),1.21-1.13(m,12H)。 ¹³ C NMR (126 MHz, acetonitrile-d) ₃ )δ83.00,82.75,76.03,75.55,59.64,59.50,59.13,58.98,57.46,56.97,44.05,44.03,43.95,43.93,32.77,32.42,29.34,29.17,25.07,25.05,25.01,25.00,24.87,24.83,24.81,24.77,23.89,23.76,23.63,23.60,21.19,21.13,21.08. ³¹ P NMR (162 MHz, acetonitrile-d) ₃ )δ148.85,148.56。

Scheme 4

Compound 807: a suspension of sodium sulfide nonahydrate (4.08 g,17 mmol) and elemental sulfur (1.09 g,34 mmol) in MMA (N-methylacetamide) (35 mL) was stirred overnight at 30deg.C to form a homogeneous pale yellow solution. A solution of dibromoketone 806 (3.45 g,14 mmol) in MMA (10 mL) was added dropwise for about 30 minutes while maintaining a bath temperature of 30 ℃. The mixture was stirred at 30 ℃ for an additional 2 hours, cooled to room temperature, and quenched by the addition of 5% aqueous NaCl (200 mL). The mixture was extracted with ethyl acetate, the organic phase was separated, washed with 5% aqueous NaCl, saturated sodium chloride and dried with anhydrous sodium sulfate. The solvent was evaporated in vacuo to give a crude residue (217 g) was purified on a silica gel column with 5% ethyl acetate/hexane to give 0.97g (47%) of pure dimethyl ketone 807. ¹ H NMR(400MHz,CDCl3):δ3.58(s,2H)；1.52(s,6H)。 ¹³ C NMR(126MHz,CDCl3):δ210.5；55.8；41.7；23.7。

Compound 808: sodium borohydride (122 mg,3.2 mmol) was added to a cooled (-78 ℃) and stirred solution of ketone 807 (0.94 g,6.4 mmol) and acetic acid (0.37 mL,6.4 mmol) in absolute ethanol (15 mL) under Ar. The mixture was stirred at-78 ℃ for 2 hours, the cooling bath was removed, and the mixture was quenched by the addition of saturated ammonium chloride (15 mL) and ethyl acetate (20 mL). The mixture was warmed to room temperature and water (8 mL) was added to dissolve the solid. The organic phase was separated, washed successively with 15% aqueous NaCl solution, saturated sodium bicarbonate, saturated sodium chloride and dried over anhydrous sodium sulfate. The solvent was removed in vacuo to give the crude product (0.93 g) which was purified on a silica gel column with a gradient of 10 to 30% ethyl acetate/hexanes to give 0.66g (70%) 808 as a slowly crystallizing pale yellow oil. ¹ H NMR(500MHz,CD ₃ CN):δ4.10-4.05(m,1H)；3.39(dd,J＝5.5,11.0Hz,1H)；3.17(d,J＝8Hz,1H)；3.03(dd,J＝4.0,11.0Hz,1H)；1.41(s,3H)；1.37(s,3H).C13 NMR(126MHz,CDCl3):δ82.7；65.0；43.4；26.6；21.4。

Scheme 5

Compounds 809 and 810: n-methylacetamide (100 mL) was heated to 30℃under an inert atmosphere. Disodium sulfide nonahydrate (8.38 g,34.8 mmol) and sulfur (2.24 g,69.7 mmol) were added and the suspension stirred at 35 ℃ for 24 hours to dissolve the solids. A solution of 2, 4-dibromo-3-pentanone (8.47 g,34.8 mmol) in N-methylacetamide (10 mL) was slowly added over 20 min. The mixture was stirred at 30 ℃ for 20 hours and quenched by slowly pouring into a stirred solution of 5% NaCl (400 mL). The mixture was diluted with ethyl acetate (400 mL), the organic layer was separated, washed with 5% NaCl (1X 300 mL), saturated NaCl (1X 300 mL), and dried over anhydrous Na ₂ SO ₄ Dried, filtered and concentrated. Oil is added toThe residue was diluted in hexane (200 mL) and stirred for 18 hours. The solids were removed by filtration and the filtrate was concentrated to an oil. The product was purified by flash chromatography on silica gel, 120g silica gel column, using ethyl acetate: hexane (0 to 10% gradient). Early eluting compound 809 was isolated as a yellow liquid in 31% yield (1.31 g). Compound 810 eluted later was isolated as a yellow oil in 9% yield (0.38 g, 4:1 mixture of 810 and 809). Compound 809: ¹ H NMR(400MHz,DMSO-d ₆ ) δ3.84 (q, j=6.9 hz,2 h), 1.35 (d, j=6.9 hz,6 h). Compound 810: ¹ H NMR(400MHz,DMSO-d ₆ )δ3.93(q,J＝7.0Hz,2H),1.37(d,J＝7.0Hz,6H)。

compound 811: ketone 809 (1.02 g,6.75 mmol) was dissolved in ethanol (15 mL) under an inert atmosphere and cooled to-78 ℃. Acetic acid (0.39 mL,6.75 mmol) was added followed by sodium borohydride (130 mg,3.37 mmol). The mixture was stirred for 5 hours and a second portion of sodium borohydride (130 mg,3.37 mmol) was added. The mixture was stirred for a further 2 hours with saturated NH ₄ Cl (10 mL) was quenched and allowed to warm to room temperature. Ethyl acetate (20 mL) and saturated NH were added ₄ Cl (10 mL) and water (10 mL), and the mixture was stirred at room temperature overnight. The organic layer was separated and saturated with 1:1 NH ₄ Cl: water (1X 20 mL), saturated NaHCO ₃ (1X 25 mL) and saturated NaCl (1X 25 mL), washed with Na ₂ SO ₄ Dried, filtered and concentrated. The product was purified by flash chromatography on silica gel, 24g silica gel column, using ethyl acetate: hexane (gradient 1:15 to 1:9) purification. The product containing fractions were concentrated, rinsed with dichloromethane (2×), and dried under high vacuum overnight. Compound 811 was obtained as a colorless oil in 42% yield (427 mg). ¹ H NMR,(400MHz,DMSO-d ₆ )δ5.35(d,J＝5.6Hz,1H),3.93(q,J＝5.1Hz,1H),3.62-3.53(m,1H),3.44-3.35(m,1H),1.28(t,J＝6.9Hz,6H)。

Compound 812: compound 811 (0.40 g,2.66 mmol) was dissolved in anhydrous ethyl acetate (13 mL) under an inert atmosphere. N, N-diisopropylethylamine (0.70 mL,4.0 mmol) and 2-cyanoethyl N, N-diisopropylchlorophosphamide (0.95 mL,4.0 mmol) were added and the mixture was stirred at room temperature for 1.5 h. The mixture was quenched with 5%NaCl (3X 40 mL), saturated NaCl (1X 40 mL), and Na ₂ SO ₄ Dried, filtered and concentrated. The product was purified by flash chromatography on silica gel, 24g silica gel column, using ethyl acetate (+1% triethylamine): hexane (1:15 to 1:9 gradient). The product containing fractions were concentrated in vacuo, rinsed with acetonitrile (2×), and dried under high vacuum. Compound 812 was isolated as a yellow oil in 20% yield (182 mg). ¹ H NMR, (500 MHz, acetonitrile-d) ₃ )δ4.28-4.19(m,1H),3.89-3.57(m,6H),2.73-2.61(m,2H),1.42-1.35(m,6H),1.22-1.16(m,12H). ³¹ P NMR (202 MHz, acetonitrile-d) ₃ )δ150.85,150.47。

Scheme 6

Compound 813: 2-methyl-3-pentanone (23.3 g,233 mmol) was dissolved in diethyl ether (100 mL) under an inert atmosphere. A solution of separately prepared bromine (25.7 mL, 460 mmol) in dichloromethane (50 mL) (12 drops) was added to the ketone solution and stirred for 1 minute to initiate the reaction. The mixture was cooled in an ice/water bath and bromine solution (65 mL) was added dropwise to the cooled and stirred ketone solution over 3 hours. The ice bath was removed and the mixture was stirred at room temperature for 20 minutes. The mixture was diluted with diethyl ether (300 mL) and added in portions to a stirred aqueous 5% NaCl solution (300 mL) and stirred for 10 minutes. The organic layer was treated with 5% NaCl (2X 500 mL), 5% Na ₂ S ₂ O ₅ (1X 450 mL) and saturated NaCl (1X 500 mL), washed with Na ₂ SO ₄ Dried, filtered and concentrated. Compound 813 was isolated as a pale yellow liquid in 95% yield (57.1 g). ¹ H NMR,(500MHz,DMSO-d ₆ )δ5.41(q,J＝6.6Hz,1H),1.98(s,3H),1.87(s,3H),1.74(d,J＝6.6Hz,3H)。 ¹³ C NMR(126MHz,DMSO-d ₆ )δ198.94,64.58,41.06,29.35,28.59,22.10。

Compound 814: n-methylacetamide (300 mL) was heated at 33℃under an inert atmosphere. Disodium sulfide nonahydrate (27.9 g,116 mmol) and sulfur (7.46 g,233 mmol) were added and the suspension was stirred at 35℃for 24 hours to dissolveAnd (5) dissolving solids. The reaction was cooled to 30℃and a solution of compound 813 (30 g,116 mmol) in N-methylacetamide (20 mL) was slowly added over 15 min. The mixture was stirred at 30 ℃ for 22 hours and quenched by slowly pouring into a stirred solution of 5% NaCl (1200 mL). The mixture was diluted with ethyl acetate (1200 mL) and washed with 5% NaCl (3 x 1200 mL) and saturated NaCl (1 x 800 mL). The organic layer was taken up with Na ₂ SO ₄ Dried, filtered and concentrated. The oily residue was diluted in hexane (600 mL) and stirred for 18 hours. The solids were removed by decantation and the supernatant concentrated to an oil. The product was purified by flash chromatography on silica gel, 220g silica gel column, using dichloromethane: hexane (gradient 1:9 to 1:8). The product containing fractions were concentrated and rinsed with dichloromethane (2×). Compound 814 was isolated as a yellow oil in 32% yield (6.1 g). ¹ H NMR,ELN0021-16-7(400MHz,DMSO-d ₆ )δ3.98(q,J＝7.0Hz,1H),1.45(d,J＝9.0Hz,6H),1.37(d,J＝7.0Hz,3H)。 ¹³ C NMR(126MHz,DMSO-d ₆ )δ211.58,56.27,50.15,24.69,24.11,16.00.。

Compounds 815 and 816: compound 814 (2.3 g,14.2 mmol) was dissolved in ethanol (35 mL) under an inert atmosphere and cooled to-78 ℃. Sodium borohydride (531 mg,4.17 mmol) was added and the reaction mixture was stirred for 15 min, warmed to room temperature and stirred for an additional 3 h. The mixture was cooled to-78 ℃ and saturated NH ₄ Cl (10 mL) quench. Ethyl acetate (50 mL) and saturated NH were added ₄ Cl (35 mL) and water (20 mL), and the mixture was stirred at room temperature overnight. The organic layer was separated and quenched with 1:1 saturated NH4Cl: water (1X 50 mL), saturated NaHCO ₃ (1X 50 mL), saturated NaCl (1X 50 mL), and Na ₂ SO ₄ Dried, filtered and concentrated. The product was isolated by flash chromatography on silica gel, 80g silica gel column, using ethyl acetate: hexane (1:20 to 1:9 gradient). The product containing fractions were concentrated and rinsed with dichloromethane (2×). Early eluting compound 815 was isolated as a yellow solid in 40% yield (0.93 g). Compound 816 eluted later was isolated as a yellow oil in 10% yield (0.23 g). Compound 815: ¹ H NMR,(500MHz,DMSO-d ₆ )δ5.13(d,J＝6.9Hz,1H),3.88-3.82(m,1H),3.76(dd,J＝6.9,4.5Hz,1H),1.37(d,J＝6.5Hz,6H),1.28(d,J＝6.8Hz,3H)。 ¹³ C NMR(101MHz,DMSO-d ₆ ) Delta 83.39,63.88,51.88,28.15,22.83,15.24. Compound 816: ¹ H NMR,(400MHz,DMSO-d ₆ )δ5.67(d,J＝6.3Hz,1H),3.36(dd,J＝8.4,6.2Hz,1H),3.27-3.19(m,1H),1.37(d,J＝6.5Hz,3H),1.31(d,J＝3.9Hz,6H)。 ¹³ C NMR(101MHz,DMSO-d ₆ )δ88.45,57.99,48.86,25.74,21.63,19.17。

compound 817: compound 815 (0.2 g,1.2 mmol) was dissolved in anhydrous ethyl acetate (6 mL) under an inert atmosphere. N, N-diisopropylethylamine (0.32 mL,1.8 mmol) and 2-cyanoethyl N, N-diisopropylchlorophosphamide (0.41 mL,1.8 mmol) were added and the mixture was stirred at room temperature for 3 hours. The reaction was quenched, diluted with ethyl acetate (20 mL), washed with 5% NaCl (3X 40 mL), saturated NaCl (1X 40 mL), and dried over Na ₂ SO ₄ Dried, filtered and concentrated. The product was isolated by flash chromatography on a standard 24g silica gel column quenched with triethylamine (10 mL) using a gradient of ethyl acetate/hexane (1:9 to 1:2). The product containing fractions were concentrated, rinsed with acetonitrile (2×), and dried under high vacuum. Compound 817 was obtained as a slowly crystallizing yellow oil in 54% yield (0.24 g). ¹ H NMR (500 MHz, acetonitrile-d) ₃ )δ4.19-4.10(m,1H),4.01-3.64(m,5H),2.75-2.63(m,2H),1.52(s,3H),1.50-1.37(m,6H),1.27-1.20(m,12H). ³¹ P NMR (202 MHz, acetonitrile-d) ₃ )δ152.39,150.75。

Scheme 7

Compound 819: a suspension of sodium sulfide nonahydrate (4.08 g,17 mmol) and elemental sulfur (1.09 g,34 mmol) in MMA (N-methylacetamide) (35 mL) was stirred overnight at 30deg.C to form a homogeneous yellow solution. A solution of dibromoketone 818 (2.4 mL,14 mmol) in MMA (10 mL) was added dropwise for about 20 minutes while maintaining the bath temperature at 30deg.C. The mixture was stirred at 30 ℃ for an additional 3 hours, cooled to room temperature, and quenched by the addition of 5% aqueous NaCl (200 mL). The mixture was extracted with ethyl acetate The organic phase was separated, washed with 5% aqueous NaCl, saturated sodium chloride and dried over anhydrous sodium sulfate. The solvent was evaporated in vacuo to give a crude residue (2.49 g) which was purified on a silica gel column with a 0-10% ethyl acetate/hexanes gradient to give 1.18g (48%) of pure tetramethyl ketone 819. ¹ H NMR(400MHz,CD3CN):δ1.50(s,12H)。 ¹³ C NMR(126MHz,CD3CN):δ215.4；58.6；25.7。

Compound 820: sodium borohydride (420 mg,11 mmol) was added in portions to a cooled (0 ℃) and stirred solution of ketone 819 (0.78 g,4.4 mmol) and acetic acid (0.5 mL,8.7 mmol) in absolute ethanol (15 mL) over 3 hours under Ar. The mixture was stirred at 0 ℃ for an additional 3 hours, the cooling bath was removed, and the mixture was quenched by the addition of saturated ammonium chloride (30 mL) and ethyl acetate (10 mL). The mixture was warmed to room temperature, water (5 mL) was added to dissolve the solids, and the mixture was vigorously stirred in the presence of air for 48 hours. The organic phase was separated, washed with saturated NaCl and dried over anhydrous sodium sulfate. The solvent was removed in vacuo to give the crude product (0.84 g) which was purified on a silica gel column with a 5-20% ethyl acetate/hexanes gradient to give 0.65g (83%) 820 as a slowly crystallizing pale yellow liquid. ¹ H NMR(500MHz,CD ₃ CN):δ3.50(d,J＝6.5Hz,1H)；3.43(d,J＝7.0Hz,1H)；1.44(s,6H)；1.36(s,6H)。

Compound 821: 2-cyanoethyl N, N-diisopropylchlorophosphamide (0.45 mL,2 mmol) was added to a cooled (0 ℃) and stirred solution of tetramethyl methanol 820 (0.27 g,1.5 mmol) and N, N-diisopropylethylamine (0.35 mL,2 mmol) in anhydrous ethyl acetate (7 mL) under Ar. The cooling bath was removed and the mixture was stirred at room temperature for 24 hours, cooled to 0 ℃ and quenched by addition of saturated sodium bicarbonate solution. The organic phase was separated, dried over anhydrous sodium sulfate and the crude residue was purified on a silica gel column with a 35 to 100% dichloromethane/hexane gradient to give 0.37g (65%) of pure phosphoramidite 821 as a pale yellow oil. ¹ H NMR(400MHz,CD ₃ CN):δ3.89-3.80(m,1H)；3.77(d,J＝12.4Hz,1H)；3.73-3.59(m,3H)；2.65(t,J＝6.0Hz,2H)；1.55(s,3H)；1.50(s,3H)；1.44(s,3H)；1.42(s,3H)；1.22(d,J＝6.8Hz,6H)；1.18(d,J＝6.8Hz,6H). ³¹ P NMR(202MHz,CD ₃ CN):δ150.8。

Scheme 8

Compound 825: 3-methyl-1-phenyl-2-butanone, compound 822 (4.0 g,24.7 mmol) was dissolved in dry diethyl ether (20 mL). To the ketone solution was added a solution of 3 drops of bromine (7.9 g,2.5mL,49.3 mmol) and DCM (10 mL) to initiate the reaction. Once the reaction turned from orange to colorless, the remaining bromine solution was added dropwise over one hour. The reaction was stirred for an additional 2 hours, then diluted with diethyl ether (100 mL) and added in portions to a stirred solution of 5% sodium chloride (100 mL). The organic layer was then washed with 5% NaCl (3X 100 mL), 5% Na ₂ S ₂ O ₅ (1X 100 mL) and saturated NaCl (1X 100 mL). Na for organic layer ₂ SO ₄ Dried, filtered and concentrated. The pale brown crude residue was purified on a silica gel column with a gradient of 2% to 7% ethyl acetate/hexanes to give pure compound 825 as a white solid in 96% yield (7.6 g). ¹ H NMR(400MHz,DMSO)δ7.67-7.63(m,2H),7.41-7.33(m,3H),6.60(s,1H),1.99(s,3H),1.79(s,3H)。

Compound 826: 1- (4-methyl) -3-methylbutan-2-one, compound 823 (4.51 g,25.6 mmol) was dissolved in anhydrous diethyl ether (20 mL) under argon. To the ketone solution was added 10 drops of a solution of bromine (8.18 g,2.62mL,51.2 mmol) and DCM (15 mL) to initiate the reaction. Once the reaction changed from orange to pale orange, the remaining bromine solution was added dropwise over 1 hour. The reaction was stirred for an additional 2 hours, then diluted with diethyl ether (100 mL) and added in portions to a stirred solution of 5% sodium chloride (100 mL). Then using 5% NaCl (3X 100 mL) and 5% Na ₂ S ₂ O ₅ The organic layer was washed with (1X 100 mL) and saturated NaCl (1X 100 mL). Na for organic layer ₂ SO ₄ Dried, filtered and concentrated. The pale brown crude residue was purified on a silica gel column with a gradient of 3% to 20% ethyl acetate/hexanes to give pure compound 826 as a white solid, 79% yield (6.77 g). ¹ H NMR(500MHz,DMSO)δ7.53(d,J＝8.2Hz,2H),7.19(d,J＝7.7Hz,2H),6.57(s,1H),2.28(s,3H),1.97(s,3H),1.78(s,3H)。

Compound 827: 1- (4-methoxyphenyl) -3-methylbutan-2-one, compound 824 (4.82 g,25.1 mmol) was dissolved in anhydrous diethyl ether (20 mL) under argon. To the ketone solution was added 3 drops of a solution of bromine (8.01 g,2.6mL,50.1 mmol) and DCM (15 mL) to initiate the reaction. Once the reaction turned from orange to pale orange, the remaining bromine solution was added dropwise over 1 hour. The reaction was stirred for an additional 2 hours, then diluted with diethyl ether (100 mL) and added in portions to a stirred solution of 5% sodium chloride (100 mL). Then using 5% NaCl (3X 100 mL) and 5% Na ₂ S ₂ O ₅ The organic layer was washed with (1X 100 mL) and saturated NaCl (1X 100 mL). Na for organic layer ₂ SO ₄ Dried, filtered and concentrated. The pale brown crude residue was purified on a silica gel column with a gradient of 0% to 15% ethyl acetate/hexanes to give pure compound 827 as a white solid, 91% yield (8.0 g). ¹ H NMR(600MHz,DMSO)δ7.59(d,J＝6.8Hz,2H),6.95(d,J＝6.8Hz,2H),6.61(s,1H),3.76(s,3H),1.97(s,3H),1.79(s,3H)。

Compound 828: a reactor containing N-methylacetamide (40 mL) heated to 33℃was charged with sodium sulfide nonahydrate (6.0 g,25 mmol) and sulfur (1.6 g,50 mmol). The suspension was stirred at 35 ℃ overnight to dissolve the solids. The mixture was cooled to 30℃and then compound 825 (4.0 g,25.0 mmol) was added. The reaction was stirred for 3 hours and then quenched by the addition of a stirred 5% NaCl solution (200 mL). The mixture was extracted with ethyl acetate (100 mL) and washed with 5% NaCl (2X 150 mL) and saturated NaCl (1X 150 mL). Na for organic layer ₂ SO ₄ Dried, filtered and concentrated. The crude product was diluted with hexane (100 mL) and the precipitated residual sulfur was removed by vacuum filtration. The filtrate was concentrated to give a crude residue which was purified on a silica gel column with a 4% to 10% ethyl acetate/hexanes gradient to give pure compound 828 as a yellow solid, 89% yield (2.49 g). ¹ H NMR(500MHz,DMSO)δ7.44-7.25(m,6H),5.28(s,1H),1.62(s,3H),1.52(s,3H)。 ¹³ C NMR(101MHz,DMSO)δ210.06,135.77,129.16,128.75,128.32,58.97,56.66,24.56,24.16。

Compound 829: to a solution containing N-methylacetamide (40 mL) heated to 33 ℃) Sodium sulfide nonahydrate (6.0 g,25 mmol) and sulfur (1.6 g,50 mmol) were charged into the reactor. The suspension was stirred at 35 ℃ overnight to dissolve the solids. The mixture was cooled to 30℃and then compound 826 (4.0 g,25.0 mmol) was added. The reaction was stirred for 3 hours and then quenched by the addition of stirred 5% NaCl solution (200 mL). The mixture was extracted with ethyl acetate (100 mL) and washed with 5% NaCl (2X 150 mL) and saturated NaCl (1X 150 mL). Na for organic layer ₂ SO ₄ Dried, filtered and concentrated. The crude product was diluted with hexane (100 mL) and the precipitated residual sulfur was removed by vacuum filtration. The filtrate was concentrated to give a crude residue which was purified on a silica gel column with a gradient of 4% to 10% ethyl acetate/hexane to give pure compound 829 as a yellow solid in 53% yield (1.51 g). ¹ H NMR(500MHz,DMSO)δ7.17(s,4H),5.23(s,1H),2.28(s,3H),1.62(s,3H),1.50(s,3H)。 ¹³ C NMR(101MHz,DMSO)δ210.21,137.81,132.70,129.32,129.09,58.91,56.59,24.65,24.19,20.71。

Compound 830: a reactor containing N-methylacetamide (30 mL) heated to 33℃was charged with sodium sulfide nonahydrate (4.71 g,19.6 mmol) and sulfur (1.26 g,39.2 mmol). The suspension was stirred at 35 ℃ overnight to dissolve the solids. The mixture was cooled to 30℃and then compound 827 (3.43 g,9.8 mmol) was added. The reaction was stirred for 3 hours and then quenched by addition to a stirred 5% NaCl solution (150 mL). The mixture was extracted with ethyl acetate (100 mL) and washed with 5% NaCl (2X 150 mL) and saturated NaCl (1X 150 mL). Na for organic layer ₂ SO ₄ Dried, filtered and concentrated. The crude product was diluted with hexane (100 mL) and the precipitated residual sulfur was removed by vacuum filtration. The filtrate was concentrated to give a crude residue which was purified on a silica gel column with a gradient of 0% to 15% ethyl acetate/hexane to give pure compound 830 as a yellow solid, 78% yield (1.75 g). ¹ H NMR(600MHz,DMSO)δ7.21(d,J＝6.9Hz,2H),6.94(d,J＝6.9Hz,2H),5.24(s,1H),3.75(s,3H),1.63(s,3H),1.50(s,3H)。 ¹³ C NMR(151MHz,DMSO)δ210.87,159.73,131.03,127.99,114.71,59.26,56.98,55.65,25.23,24.72。

Compounds 831 and 832: compound 82 was placed in an oven-dried flask under argon8 (2.32 g,10.34 mmol) was dissolved in ethanol (25 mL) and then cooled to-78 ℃. Acetic acid (0.62 g,0.60mL,10.34 mmol) was added followed by NaBH ₄ (0.39 g,10.34 mmol). The reaction was stirred at-78 ℃ for 10 minutes, at 0 ℃ for 1 hour, and then at room temperature overnight. The reaction was cooled to 0deg.C and additional aliquots of NaBH were added ₄ (0.10 g,2.59 mmol). The reaction was stirred at 0deg.C for 5 hours, then saturated NH was slowly added ₄ Cl (15 mL) quench. The mixture was treated with ethyl acetate (80 mL), saturated NH ₄ Cl (40 mL) and water (35 mL). The mixture was stirred at room temperature for 18 hours. The organic layer was saturated with 1:1 NH ₄ Cl: water (1X 50 mL), saturated NaHCO ₃ (1X 50 mL) and saturated NaCl (1X 50 mL). Na for organic layer ₂ SO ₄ Dried, filtered and concentrated. The crude yellow residue was purified on a silica gel column with a gradient of 6% to 10% ethyl acetate in hexanes to give early eluting compound 832 (racemic mixture) as a yellow solid (1.45 g,62% yield) and late eluting compound 831 (racemic mixture) as a yellow solid (0.13 g,6% yield). Compound 831: ¹ H NMR(500MHz,DMSO)δ7.51-7.42(m,2H),7.38-7.24(m,3H),5.68(d,J＝6.7Hz,1H),4.29(d,J＝8.7Hz,1H),3.93(dd,J＝8.6,6.6Hz,1H),1.46(s,3H),1.38(s,3H)。 ¹³ c NMR (101 MHz, DMSO). Delta. 140.17,128.44,128.15,127.59,89.28,58.61,57.01,25.32,21.12. Compound 832: ¹ H NMR(500MHz,DMSO)δ7.56-7.46(m,2H),7.33-7.22(m,3H),5.23(d,J＝7.2Hz,1H),5.00(d,J＝3.8Hz,1H),3.94(dd,J＝6.9,3.8Hz,1H),1.52(s,3H),1.44(s,3H)。 ¹³ C NMR(101MHz,DMSO)δ136.49,130.01,127.66,127.40,83.58,65.70,61.53,28.68,23.27。

compounds 833 and 834: compound 829 (1.25 g,5.24 mmol) was dissolved in ethanol (13 mL) under argon in an oven-dried flask and then cooled to-78 ℃. Acetic acid (0.31 g,0.30mL,5.24 mmol) was added followed by NaBH ₄ (0.20 g,5.24 mmol). The reaction was stirred at-78 ℃ for 10 minutes, at 0 ℃ for 1 hour, then at room temperature overnight. The reaction was cooled to 0deg.C and additional aliquots of NaBH were added ₄ (0.05 g,1.31 mmol). The reaction was stirred at 0deg.C for 5 hours and then passed throughToo slowly add saturated NH ₄ Cl (10 mL) quench. The mixture was treated with ethyl acetate (50 mL), saturated NH ₄ Cl (25 mL) and water (20 mL). The mixture was stirred at room temperature for 18 hours. The organic layer was saturated with 1:1 NH ₄ Cl: water (1X 50 mL), saturated NaHCO ₃ (1X 50 mL) and saturated NaCl (1X 50 mL). Na for organic layer ₂ SO ₄ Dried, filtered and concentrated. The crude yellow residue was purified on a silica gel column with a gradient of 5% to 20% ethyl acetate/hexanes to give early eluting compound 834 (racemic mixture) as a yellow solid (0.87 g,69% yield), and late eluting compound 833 (racemic mixture) as a yellow solid (0.052 g,4% yield). Compound 833: ¹ h NMR (600 mhz, dmso) delta 7.37-7.32 (m, 2H), 7.16 (d, j=7.6 hz, 2H), 5.66 (d, j=6.7 hz, 1H), 4.26 (d, j=8.6 hz, 1H), 3.91 (dd, j=8.8, 6.5hz, 1H), 2.29 (s, 3H), 1.45 (s, 3H), 1.38 (s, 3H). Compound 834: ¹ H NMR(600MHz,DMSO)δ7.44-7.36(m,2H),7.13-7.08(m,2H),5.24 -5.16(m,1H),4.97(d,J＝3.1Hz,1H),3.93-3.86(m,1H),2.28(s,3H),1.52(s,3H),1.44(s,3H)。 ¹³ C NMR(151MHz,DMSO)δ137.11,133.81,130.38,128.75,84.03,66.08,61.83,29.28,23.84,21.17。

compounds 835 and 836: compound 830 (1.9 g,7.5 mmol) was suspended in ethanol (20 mL) under argon in a dry flask and then cooled to-78 ℃. Acetic acid (0.45 g,0.43mL,7.5 mmol) was added followed by NaBH ₄ (0.28 g,7.5 mmol). The reaction was stirred at-78 ℃ for 10 minutes, at 0 ℃ for 1 hour, and then at room temperature overnight. The reaction was cooled to 0deg.C and additional aliquots of NaBH were added ₄ (0.05 g,1.31 mmol). The reaction was stirred at 0deg.C for 5 hours, then saturated NH was slowly added ₄ Cl (40 mL) and water (35 mL). The mixture was stirred for 48 hours. The organic layer was saturated with 1:1 NH ₄ Cl: water (1X 50 mL), saturated NaHCO ₃ (1X 50 mL) and saturated NaCl (1X 50 mL). Na for organic layer ₂ SO ₄ Dried, filtered and concentrated. The crude yellow residue was purified on a silica gel column with 3:48.5:48.5 diethyl ether: DCM: hexane to give early eluting compound 836 (racemic mixture) as a yellow solid (1.0 g,52% yield), and late eluting compound 835(racemic mixture) as a yellow solid (0.07 g,4% yield). Compound 835: ¹ H NMR(600MHz,DMSO)δ7.37(d,J＝8.7Hz,2H),6.91(d,J＝8.7Hz,2H),5.66(d,J＝6.7Hz,1H),4.27(d,J＝8.8Hz,1H),3.89(dd,J＝8.8,6.6Hz,1H),3.74(s,3H),1.43(s,3H),1.38(s,3H)。 ¹³ c NMR (151 MHz, DMSO). Delta. 159.21,131.67,128.54,113.60,83.93,66.04,61.41,55.54,29.36,23.88. Compound 836: ¹ H NMR(600MHz,DMSO)δ7.45(d,J＝8.7Hz,2H),6.86(d,J＝8.8Hz,2H),5.27(d,J＝7.2Hz,1H),4.97(d,J＝3.7Hz,1H),3.85(d,J＝3.3Hz,1H),3.73(s,3H),1.51(s,3H),1.43(s,3H)。 ¹³ C NMR(151MHz,DMSO)δ159.29,132.02,129.81,114.37,89.34,58.74,57.19,55.62,26.10,21.89。

compound 837: compound 831 (0.1 g,0.44 mmol) was dissolved in anhydrous ethyl acetate (1 mL) under an inert atmosphere. N, N-diisopropylethylamine (0.15 mL,0.88 mmol) and 2-cyanoethyl N, N-diisopropylchlorophosphamide (0.15 mL,0.66 mmol) were added and the mixture was stirred at room temperature for 18 hours. The reaction was quenched, diluted with ethyl acetate (20 mL), washed with 5% NaCl (3X 20 mL) and saturated NaCl (1X 40 mL), and dried over Na ₂ SO ₄ Dried, filtered and concentrated. The crude residue was purified on a silica gel column with a gradient of 5% to 30% ethyl acetate/hexane to give pure compound 837 as a yellow oil in 77% yield (0.15 g). ¹ H NMR(600MHz,CD ₃ CN)δ7.55-7.50(m,2H),7.41-7.30(m,3H),4.57-4.43(m,2H),3.68-3.49(m,3H),3.27-3.13(m,1H),2.560-2.56(m,1H),2.26-2.20(m,1H),1.62-1.56(m,6H),1.16(d,J＝6.8Hz,3H),1.10(d,J＝6.8Hz,3H),1.05(d,J＝6.8Hz,3H),0.97(d,J＝6.8Hz,3H)。 ¹³ C NMR(151MHz,CD ₃ CN)δ140.19,139.67,129.22,129.17,129.14,129.07,128.60,128.46,92.49,92.40,92.22,92.15,59.68,59.65,59.62,59.20,59.16,58.38,58.25,58.19,58.05,43.55,43.47,43.40,43.31,26.16,25.94,25.91,24.37,24.32,24.29,24.26,24.20,24.14,22.59,22.00,20.39,20.34,20.14,20.09. ³¹ P NMR(243MHz,CD ₃ CN)δ150.08,148.64。

Compound 839: compound 833 (0.05 g,0.21 mmol) is dissolved in anhydrous ethyl acetate (0.5 mL) under an inert atmosphere. N, N-diisopropylethylamine (0.07 mL,0.42 mmol) and were added2-cyanoethyl N, N-diisopropylchlorophosphamide (0.07 mL,0.31 mmol) and the mixture was stirred at room temperature for 18 hours. The reaction was quenched, diluted with ethyl acetate (10 mL), washed with 5% NaCl (3X 15 mL) and saturated NaCl (1X 15 mL), and washed with Na ₂ SO ₄ Dried, filtered and concentrated. The crude residue was purified on a silica gel column with a gradient of 5% to 30% ethyl acetate/hexanes to give pure compound 839 as a yellow oil in 46% yield (0.042 g). ¹ H NMR(600MHz,CD ₃ CN)δ7.42-7.37(m,2H),7.24-7.14(m,2H),4.54-4.40(m,2H),3.68-3.48(m,3H),3.25-3.11(m,1H),2.59-2.57(m,1H),2.34(d,J＝12.2Hz,3H),2.26-2.20(m,1H),1.61-1.56(m,5H),1.16(d,J＝6.8Hz,3H),1.10(d,J＝6.8Hz,3H),1.05(d,J＝6.8Hz,3H),0.97(d,J＝6.8Hz,3H)。 ¹³ C NMR(151MHz,CD ₃ CN)δ138.49,138.43,136.85,136.42,129.70,129.67,129.09,129.08,92.42,92.31,92.12,92.04,59.74,59.71,59.43,59.40,59.09,58.99,58.44,58.30,58.17,58.03,43.59,43.51,43.41,43.32,26.31,26.05,26.02,24.38,24.33,24.30,24.29,24.26,24.15,24.09,22.71,22.12,20.71,20.70,20.39,20.34,20.08,20.03. ³¹ P NMR(243MHz,CD ₃ CN)δ150.32,148.64。

Compound 841: compound 835 (0.042 g,0.16 mmol) is dissolved in anhydrous ethyl acetate (0.5 mL) under an inert atmosphere. N, N-diisopropylethylamine (0.04 mL,0.25 mmol) and 2-cyanoethyl N, N-diisopropylchlorophosphamide (0.05 mL,0.25 mmol) were added and the mixture was stirred at room temperature for 18 hours. The reaction was quenched, diluted with ethyl acetate (10 mL), washed with 5% NaCl (3X 15 mL) and saturated NaCl (1X 15 mL), and dried over Na ₂ SO ₄ Dried, filtered and concentrated. The crude residue was purified on a silica gel column with a gradient of 5% to 30% ethyl acetate/hexanes to give pure compound 841 as a yellow oil in 44% yield (0.033 g). ¹ H NMR(600MHz,CD ₃ CN)δ7.46-7.40(m,2H),6.97-6.87(m,2H),4.52-4.37(m,2H),3.80(d,J＝11.7Hz,3H),3.70-3.19(m,4H),2.59(t,J＝5.9Hz,1H),2.31-2.27(m,1H),1.63-1.55(m,6H),1.16(d,J＝6.8Hz,3H),1.11(d,J＝6.8Hz,3H),1.06(d,J＝6.8Hz,3H),0.98(d,J＝6.8Hz,3H). ³¹ P NMR(243MHz,CD ₃ CN)δ150.16,148.78。

Compound 838: compound 832 (0.40 g,1.77 mmol) was dissolved in anhydrous ethyl acetate (9 mL) under an inert atmosphere. N, N-diisopropylethylamine (0.4 mL,2.65 mmol) and 2-cyanoethyl N, N-diisopropylchlorophosphamide (0.59 mL,2.65 mmol) were added and the mixture was stirred at room temperature for 18 hours. The reaction was quenched, diluted with ethyl acetate (40 mL), washed with 5% NaCl (3X 80 mL) and saturated NaCl (1X 80 mL), and dried over Na ₂ SO ₄ Dried, filtered and concentrated. The crude residue was purified on a silica gel column with a gradient of 5% to 30% ethyl acetate/hexane to give pure compound 838 as a yellow oil in 80% yield (0.60 g). ¹ H NMR(400MHz,CD ₃ CN)δ7.48-7.42(m,2H),7.34-7.21(m,3H),5.01(d,J＝5.6Hz,1H),4.36-4.31(m,1H),3.76-3.66(m,1H),3.62-3.43(m,3H),2.64-2.55(m,2H),1.66(s,3H),1.61(s,3H),1.04(d,J＝6.8Hz,6H),0.94(d,J＝6.8Hz,6H). ³¹ P NMR(162MHz,CD ₃ CN)δ150.93。

Compound 840: compound 834 (0.61 g,2.53 mmol) was dissolved in anhydrous ethyl acetate (10 mL) under an inert atmosphere. N, N-diisopropylethylamine (0.66 mL,3.8 mmol) and 2-cyanoethyl N, N-diisopropylchlorophosphamide (0.85 mL,3.8 mmol) were added and the mixture was stirred at room temperature for 18 hours. The reaction was quenched, diluted with ethyl acetate (50 mL), washed with 5% NaCl (3X 50 mL) and saturated NaCl (1X 50 mL), and dried over Na ₂ SO ₄ Dried, filtered and concentrated. The crude residue was purified on a silica gel column with a gradient of 5% to 30% ethyl acetate/hexane to give pure compound 840 as a yellow oil in 83% yield (0.93 g). ¹ H NMR(600MHz,CD ₃ CN)δ7.43-7.32(m,2H),7.19-7.10(m,2H),5.06-4.97(m,1H),4.36-4.29(m,1H),3.77-3.23(m,4H),2.67-2.39(m,2H),2.36-2.27(m,3H),1.70-1.55(m,6H),1.13-0.93(m,12H)。 ¹³ C NMR(151MHz,CD ₃ CN)δ138.08,137.85,134.25,133.76,131.16,130.77,129.34,128.85,119.28,86.81,86.73,86.00,85.94,64.40,62.96,62.94,61.09,58.37,58.28,58.24,58.13,43.69,43.60,43.59,43.51,27.99,27.56,27.54,24.59,24.52,24.47,24.43,24.39,24.31,24.27,24.22,23.83,23.80,20.73,20.69,20.56,20.51,20.41,20.36. ³¹ P NMR(243MHz,CD ₃ CN)δ 151.40,149.14。

Compound 842: compound 836 (0.50 g,1.95 mmol) was dissolved in anhydrous ethyl acetate (8 mL) under an inert atmosphere. N, N-diisopropylethylamine (0.51 mL,2.9 mmol) and 2-cyanoethyl N, N-diisopropylchlorophosphamide (0.65 mL,2.9 mmol) were added and the mixture was stirred at room temperature for 18 hours. The reaction was quenched, diluted with ethyl acetate (50 mL), washed with 5% NaCl (3X 50 mL) and saturated NaCl (1X 50 mL), and dried over Na ₂ SO ₄ Dried, filtered and concentrated. The crude residue was purified on a silica gel column with a gradient of 5% to 30% ethyl acetate/hexanes to give pure compound 842 as a yellow oil in 77% yield (0.68 g). ¹ H NMR(600MHz,CD ₃ CN)δ7.48-7.35(m,2H),6.92-6.83(m,2H),5.05-4.98(m,1H),4.34-4.24(m,1H),3.84-3.26(m,7H),2.66-2.42(m,2H),1.71-1.54(m,6H),1.15-0.95(m,12H)。 ¹³ C NMR(151MHz,CD ₃ CN)δ159.94,159.84,132.42,132.06,131.44,129.25,128.73,119.30,114.13,113.56,86.68,86.60,85.84,85.78,64.31,62.83,62.79,62.46,62.44,60.71,58.39,58.31,58.25,58.16,55.49,55.48,43.71,43.62,43.57,43.49,27.95,27.51,27.49,24.55,24.51,24.50,24.44,24.40,24.38,24.33,24.33,24.28,23.77,23.75,22.94,20.56,20.52,20.46,20.40. ³¹ P NMR(243MHz,CD ₃ CN)δ151.42,149.09。

Scheme 9

Compound 843: compound 816 (0.4 g,2.4 mmol) was dissolved in anhydrous ethyl acetate (12 mL) under an inert atmosphere. N, N-diisopropylethylamine (0.41 g,3.2 mmol) was added followed by 2-cyanoethyl N, N-diisopropylchlorophosphamide (0.75 g,3.2 mmol) and the mixture was stirred at room temperature for 3 hours. The reaction was quenched with saturated sodium bicarbonate solution and ethyl acetate. The organic phase was treated with anhydrous Na ₂ SO ₄ Drying, filtration, and concentration under reduced pressure gave a crude residue, which was purified by flash chromatography on silica gel to give pure compound 843 as a yellow oil, 89% yield (0.79 g). ¹ H NMR(400MHz,CD ₃ CN)δ3.91-3.42(m,6H),2.69-2.61(m,2H),1.52-1.43(m,9H),1.23-1.15(m,12H)。 ¹³ C NMR(101MHz,CD ₃ CN)δ119.62,92.89,92.76,92.58,92.46,60.15,60.10,59.76,59.74,59.11,59.07,58.91,58.87,52.19,52.15,51.96,44.17,44.15,44.05,44.02,26.90,26.68,26.63,25.10,25.02,24.96,24.94,24.88,23.39,22.91,21.04,20.96,19.92,19.81,19.75. ³¹ P NMR(162MHz,CD ₃ CN)δ150.00,149.81。

Scheme 10

Ketone 846: methyl propionate 845 (6.48 g,73.5 mmol), diphenyl ketone 844 (6.70 g,36.8 mmol) and zinc powder (9.62 g,147 mmol) were added to a 1L three-necked flask equipped with a reflux condenser under an argon atmosphere. To the mixture was added anhydrous THF (180 mL) with stirring. The suspension was cooled to 0-5 ℃ in an ice-water bath and titanium (IV) chloride (13.95 g,8.1ml,73.5 mmol) was slowly added to the mixture. The dark blue suspension was stirred at 25℃for 2 hours and then heated at 50℃for 6 hours. The mixture was cooled to room temperature and 1M HCl (800 mL) was added. The mixture was stirred at room temperature for 10 minutes, and extracted with ethyl acetate (250 ml×3). The organic layers were combined, washed with aqueous NaCl solution and MgSO ₄ And (5) drying. After filtering the solid and removing the solvent in vacuo, the residue was purified by flash column chromatography on silica gel (330 g,120mL/min, gradient of 30% DCM to 60% DCM/hexane) to give compound 846 (6.44 g, 78%). ¹ H NMR(600MHz,DMSO-d ₆ )δ0.93(t,3H,J＝6Hz),2.56(q，2h，J＝6Hz),5.39(s,1H),7.23-7.27(m,6H),7.31-7.34(m,4H)。 ¹³ C NMR(126MHz,DMSO-d ₆ )δ8.5,35.8,62.8,127.3,129.0,129.3,139.6,209.4。

Dibromo-ketone 847: 1, 1-diphenyl-butan-2-one (846) (7.2 g,32.1 mmol) was dissolved in anhydrous diethyl ether (45 mL) under argon. A solution of 12 drops of bromine (12.8 g,80.3 mmol) in anhydrous DCM (15 mL) was added to initiate the reaction. Once the color of the reaction mixture solution changed from orange to almost colorless, the remaining bromine solution was added dropwise over 35 minutes. The reaction mixture is reacted Stirred for an additional 2 hours, diluted with diethyl ether (120 mL) and slowly poured in portions into a stirred solution of 5% NaCl (150 mL). The organic layer was separated and washed with 5% NaCl (2X 150 mL), 5% sodium metabisulfite (1150 mL) and saturated NaCl (1X 150 mL). With Na ₂ SO ₄ The organic layer was dried, filtered, and concentrated in vacuo to give a pale yellow liquid which slowly solidified upon cooling to afford compound 847:95% purity, 11.2g (91%). ¹ H NMR(600MHz,DMSO-d ₆ )δ1.69(d,3H,J＝5Hz),4.99(q，2h，J＝5Hz),7.30-7.32(m,4H),7.41-7.48(m,6H)。 ¹³ C NMR(126MHz,DMSO-d ₆ )δ24.5,44.4,76.1,128.9,129.1,129.5,129.8,130.0,130.1,137.6,138.5,198.7。

Cyclic ketone 848: to 100mL RBF containing N-methylacetamide (15 mL) and heated to 33℃were added sodium sulfide nonahydrate (1.20 g,5.0 mmol) and sulfur (320 mg,10 mmol). The suspension was stirred at 35 ℃ for 24 hours to dissolve the solids. The reaction mixture was cooled to 30 ℃ and a solution of 847 (1.27 g,3.33 mmol) in N-methylacetamide (3 mL) was slowly added dropwise to the reaction mixture. The reaction was stirred at 30 ℃ for 3 hours and quenched by pouring into a stirred solution of 5% NaCl (60 mL). The mixture was extracted with ethyl acetate (50 mL), washed with 5% NaCl (3X 40 mL) and saturated NaCl (1X 40 mL). The organic layer was separated and taken up with Na ₂ SO ₄ Dried, filtered, and concentrated in vacuo to give a yellow oil, which was triturated with hexane (80 mL) to precipitate solid sulfur, which was removed by filtration. The resulting filtrate was concentrated in vacuo to give a yellow oil which was purified by flash column chromatography on silica gel (40 g,20ml/min, gradient elution with 5% to 35% ethyl acetate/hexanes). The product-containing fractions were combined and concentrated in vacuo to give compound 848 as a yellow oil: 265mg (27%). ¹ H NMR(600MHz,DMSO-d ₆ )δ1.08(d,3H,J＝5Hz),4.33(q,1H,J＝5Hz),7.30-7.39(m,8H),7.49-7.51(m,2H)。 ¹³ C NMR(126MHz,DMSO-d ₆ )δ11.3,51.6,68.24,125.9,126.1,126.3,126.4,126.8,126.9,137.7,140.9,205.5。

Scheme 11

Ketone 854: to a dried 100mL round bottom flask was added 1- (4-bromophenyl) -3-methyl-butan-2-one (853) (3.50 g,14.5 mmol), palladium (0) tetra-triphenylphosphine (1.34 g,1.2 mmol) and zinc cyanide (1.70 g,14.5 mmol). Anhydrous DMF (35 mL) was added, then the reaction mixture was degassed and heated overnight at 90 ℃ under argon. The mixture was cooled, diluted with 150mL EtOAc and washed with ammonium hydroxide (2M, 150 mL. Times.2), then saturated NaHCO ₃ (140 mL) and saturated NaCl (100 mL). The organic layer was separated, dried over sodium sulfate, filtered, and concentrated in vacuo to give 3.22g of crude residue. The crude residue was purified by flash column chromatography on silica gel (220 g,60ml/min, gradient of 20% to 35% ethyl acetate/hexanes) to give a colorless oil which slowly solidified to give compound 854 as a white solid: (2.13 g, 77%). ¹ H NMR(600MHz,CDCl ₃ )δ1.17(d,6H,J＝5Hz),2.74(m,1H),3.84(s,2H),7.31-7.33(m,2H),7.62-7.64(m,2H)。 ¹³ C NMR(126MHz,CDCl ₃ )δ18.2,41.0,47.1,110.9,118.8,130.4,132.3,139.8,210.2。

Dibromo-ketone 855: 1- (4-cyanophenyl) -3-methyl-butan-2-one 854 (2.10 g,11.2 mmol) is dissolved in anhydrous diethyl ether (12 mL) under argon. A solution of 12 drops of bromine (3.85 g,24.1 mmol) in anhydrous DCM (5 mL) was added to initiate the reaction. Once the color of the reaction mixture solution changed from orange to almost colorless, the remaining bromine solution was added dropwise over 25 minutes. The reaction was then stirred for an additional 2 hours, diluted with diethyl ether (40 mL) and slowly poured in portions into a stirred solution of 5% NaCl (55 mL). The organic layer was separated, washed with 5% NaCl (55 mL. Times.2), 5% sodium metabisulfite (1X 55 mL) and saturated NaCl (1X 55 mL), and dried over Na ₂ SO ₄ Drying, filtration and concentration in vacuo afforded compound 855 (-95% purity) as a yellow liquid that slowly solidified upon cooling: 3.35g (86%). ¹ H NMR(600MHz,CDCl ₃ )δ11.79(s,3H),2.05(s,3H),6.04(s,1H),7.59(d，2h，J＝8Hz),7.73(d，2h，J＝8Hz)。 ¹³ C NMR(126MHz,CDCl ₃ )δ27.2,28.5,41.5,62.2,111.1,116.3,128.2,130.5,138.9,194.1。

Cyclic ketone 856: to 100mL RBF containing N-methylacetamide (15 mL) and heated to 33℃were added sodium sulfide nonahydrate (1.20 g,5.0 mmol) and sulfur (320 mg,10 mmol). The suspension was stirred at 35 ℃ for 24 hours to dissolve the solids. The reaction mixture was cooled to 30℃and a solution of 855 (1.27 g,3.33 mmol) in N-methylacetamide (3 mL) was slowly added dropwise to the reaction mixture. The reaction was stirred at 30 ℃ for 3 hours and quenched by pouring into a stirred solution of 5% NaCl (60 mL). The mixture was extracted with ethyl acetate (50 mL), washed with 5% NaCl (3X 40 mL) and saturated NaCl (1X 40 mL). The organic layer was separated with Na ₂ SO ₄ Dried, filtered, and concentrated in vacuo to give a yellow oil, which was triturated with hexane (80 mL) to precipitate solid sulfur, which was removed by filtration. The resulting filtrate was concentrated in vacuo to give a yellow oil which was purified by flash column chromatography on silica gel (40 g,20ml/min, gradient elution with 5% to 35% ethyl acetate/hexanes). The product containing fractions were combined and concentrated in vacuo to give 856 as a yellow oil: 265mg (27%). ¹ H NMR(600MHz,DMSO-d ₆ )δ1.56(s,3H),1.60(s,3H),5.50(s,1H),7.52-7.54(m,2H),7.86-7.88(m,2H)。 ¹³ C NMR(126MHz,DMSO-d ₆ )δ24.5,24.8,57.5,58.3,111.6,119.0,130.5,133.2,142.0,209.7。

Scheme 12

Dibromo-ketone 861: 1- (4-bromophenyl) -3-methyl-butan-2-one 853 (2.00 g,8.3 mmol) was dissolved in anhydrous diethyl ether (12 mL) under argon. A solution of 12 drops of bromine (3.98 g,24.9 mmol) in anhydrous DCM (6 mL) was added to initiate the reaction. Once the color of the reaction mixture changed from orange to almost colorless, the remaining bromine solution was added dropwise over 25 minutes. The mixture was stirred for an additional 2 hours, diluted with diethyl ether (40 mL) and slowly poured in portions into a stirred solution of 5% NaCl (60 mL). The organic layer was separated, washed with 5% NaCl (60 mL. Times.2), 5% sodium metabisulfite (60 mL. Times.1) and saturated NaCl (60 mL. Times.1), and dried over Na ₂ SO ₄ Drying, filtration, and concentration under vacuum afforded compound 861 (95% purity) as a yellow liquid that slowly solidified upon cooling: 3.15g (95%). ¹ H NMR(600MHz,CDCl ₃ )δ1.74(s,3H),1.98(s,3H),6.00(s,1H),7.17-7.45(m,4H)。 ¹³ C NMR(126MHz,CDCl ₃ )δ29.3,30.5,44.5,63.8,123.6,130.9,132.0,134.9,196.6。

Cyclic ketone 862: to a 100mL round bottom flask containing N-methylacetamide (14 mL) and heated to 33℃was added sodium sulfide nonahydrate (1.00 g,4.2 mmol) and sulfur (0.268 g,8.4 mmol). The suspension was stirred at 35 ℃ for 24 hours to dissolve the solids. The mixture was cooled to 30℃and a solution of compound 861 (1.11 g,2.8 mmol) in N-methylacetamide (3 mL) was slowly added dropwise. The reaction mixture was stirred at 30 ℃ for 3 hours and quenched by pouring into a stirred solution of 5% nacl (50 mL). The mixture was extracted with ethyl acetate (50 mL), the organic layer was separated, and washed with 5% NaCl (3X 40 mL) and saturated NaCl (1X 40 mL). With Na ₂ SO ₄ The organic layer was dried, filtered, and concentrated in vacuo to give a yellow oil, which was triturated with hexane (80 mL) to precipitate sulfur, which was removed by filtration. The filtrate was concentrated in vacuo to give a crude residue as a yellow oil which was purified by silica gel flash column chromatography (40 g,20ml/min, elution with a gradient of 5% to 35% ethyl acetate/hexanes). The product containing fractions were combined and concentrated in vacuo to give compound 862 (80% purity) as a yellow oil: (156 mg, 18%). ¹ H NMR(600MHz,CDCl ₃ )δ1.60(s,3H),1.69(s,3H),4.75(s,1H),7.19-7.20(m,2H),7.51-7.55(m,2H)。

Scheme 13

Ketone 868: to a 1L three-necked flask equipped with a reflux condenser were added methyl 2-methylpropionate compound 867 (10.2 g,100 mmol), diphenyl ketone compound 844 (9.10 g,49.9 mmol) and zinc powder (13.06 g,199.8 mmol) under an argon atmosphere. Anhydrous THF (200 mL) was added with stirring and the suspension cooled to0-5℃and titanium (IV) chloride (18.9 g,10.9mL,100 mmol) was slowly added. The dark blue suspension was stirred at 25 ℃ for 2 hours and then heated at 50 ℃ overnight. The reaction mixture was cooled to room temperature and 1M HCl (800 mL) was added. The mixture was stirred at room temperature for 10 minutes, and extracted with ethyl acetate (300 ml×3). The organic layers were combined, washed with aqueous NaCl solution, and dried over MgSO ₄ And (5) drying. After filtering the solid and removing the solvent in vacuo, the crude residue was purified by silica gel flash column chromatography (330 g,120ml/min,30% to 60% DCM/hexanes gradient) to give compound 868: (8.87 g, 74%). ¹ H NMR(600MHz,CDCl ₃ )δ1.15(d,6H,J＝6Hz),2.83(m,1H),5.33(s,1H),7.25-7.29(m,6H),7.32-7.35(m,4H)。 ¹³ C NMR(126MHz,CDCl ₃ )δ18.6,41.0,62.2,127.1,128.7,129.0,138.6,212.1。

EXAMPLE 2 Synthesis of oligonucleotides containing modified phosphate prodrugs at the 5' -end of the oligonucleotides

All oligonucleotides were synthesized as described herein or as further described in table 7. Oligonucleotides were synthesized on a 1 or 10. Mu. Mol scale using standard solid phase oligonucleotide protocols using a solid phase oligonucleotide with a template of 500-Controlled Pore Glass (CPG) solid supports and commercially available phosphoramidites from ChemGENs. The phosphoramidite solution is a 0.15M solution in anhydrous acetonitrile with 15% thf as a co-solvent for 2 '-O-methyluridine, 2' -O-methylcytidine, and modified phosphate ester prodrugs. The modified phosphate ester prodrug monomers are coupled on a synthesizer or manually. For manual coupling, an activator (0.25M anhydrous ACN solution of 5-ethylsulfanyl-1H-tetrazole (ETT)) was added followed by an equal volume of prodrug solution. The solution was mixed for 20 minutes. After coupling, the column is placed on ABI for oxidation or sulfidation. An oxidizing solution (0.02M iodine in THF/pyridine) or a sulfiding solution (0.1M 3- (dimethylaminomethylene) amino-3H-1, 2, 4-dithiazole-3-thione (DDTT) in pyridine) was fed to the column for 1 minute or 30 seconds, respectively, and then kept in solution for 10 minutes. The process is repeated for vulcanization. After completion of Solid Phase Synthesis (SPS), the CPG solid support was washed with anhydrous acetonitrile Washed and dried with argon. The oligonucleotides were deprotected by incubation with 5% Diethanolamine (DEA) in ammonia for 2 hours at room temperature.

The crude oligonucleotide was purified by TSKgel SuperQ-5PW (20) resin with phosphate buffer containing sodium bromide (ph=8.5) using strong anion exchange at 65 ℃. The appropriate fractions were combined and desalted by SEC.

TABLE 7 Single oligonucleotide chain with modified phosphate prodrugs at the 5' end

Capital letters are followed by F-2' -deoxy-2 ' -fluoro (2 ' -F) sugar modifications; lower case letter-2 '-O-methyl (2' -OMe) sugar modification; s-Phosphorothioate (PS) linkages; VP-vinyl phosphonate;

prodrug-

Wherein X is O or S.

siRNA duplex with cyclic disulfide phosphate modification at the 5' -end of the antisense strand was synthesized and listed in Table 8.

Table 8: siRNA with modified phosphate prodrug at the 5' -end of antisense strand

Capital letters are followed by F-2' -F sugar modifications; lower case letter-2' -OMe sugar modification; s-Phosphorothioate (PS) linkages; VP-vinyl phosphonate; the prodrugs were the same as in table 7 above; ligand-

Example 3 in vitro evaluation of siRNA duplex containing modified phosphate prodrug at the 5' -terminus

Transfection procedure: siRNA duplex (Table 9) containing modified phosphate prodrug at the 5' end was transfected into primary mouse hepatocytes using RNAiMAX concentrations of 0.1, 1, 10 and 100nm and analyzed 24 hours after transfection. The percentage of residual F12 messenger was determined by qPCR. The results are plotted against the control, as shown in figure 1.

Free uptake procedure: siRNA duplex containing modified phosphate prodrug at the 5' end (Table 9) were incubated with primary mouse hepatocytes at 0.1, 1, 10 and 100nm concentrations in cell culture medium and analyzed after 48 hours. The percentage of residual F12 messenger was determined by qPCR. The results are plotted against the control, as shown in figure 2.

TABLE 9 siRNA duplex for in vitro evaluation

Capital letters are followed by F-2' -F sugar modifications; lower case letter-2' -OMe sugar modification; s-Phosphorothioate (PS) linkages; VP-vinyl phosphonate; the prodrugs and ligands are the same as in table 8 above.

Transfection procedure: siRNA duplex containing modified phosphate prodrug at the 5' end was transfected into primary mouse hepatocytes using RNAiMAX concentrations of 0.1, 1, 10 and 100nm and analyzed 24 hours after transfection. The percentage of residual F12 messenger was determined by qPCR. The results are plotted against the control, as shown in figure 3.

TABLE 10 siRNA duplex for in vitro evaluation in FIG. 3

Capital letters are followed by F-2' -F sugar modifications; lower case letter-2' -OMe sugar modification; s-Phosphorothioate (PS) linkages; the structures of prodrug Pmds and ligand L96 are the same as in table 8 above.

Example 4.Dtt reduction assay to examine the cleavable nature of cyclic phosphate prodrug analogues.

Dithiothreitol (DTT) was studied to reduce 5' -cyclic modified phosphate prodrugs to 5' phosphates or 5' phosphorothioates by incubating 100 μm modified oligonucleotides (23-nt length) with 100mM DTT in 1 x PBS. The amount of full length (non-reduced) oligonucleotides was observed by LCMS analysis (Agilent Single-head MS or Novatia HTSC) until 72 hours after incubation.

TABLE 11 MS data from Novat LCMS

TABLE 12 MS data from Agilent LCMS

Capital letters are followed by F-2' -F sugar modifications; lower case letter-2' -OMe sugar modification; s-Phosphorothioate (PS) linkages; the structure of the prodrug is the same as that of table 8 above.

Representative LCMS spectra of oligonucleotides tested in the DTT reduction assay are shown in fig. 4A-J.

Example 5 glutathione assays to check the cleavable nature of cyclic prodrug analogs.

The modified oligonucleotides (11-nt or 23-nt long) were added at 100. Mu.M to a solution of 250. Mu.g (6.25U/mL) glutathione-S-transferase (GST) (Sigma catalog G6511) and 0.1mg/mL NADPH (Sigma catalog 481973) in 0.1M Tris pH 7.2 from horse liver. Glutathione (GSH) (MP Biomedicals, inc. Catalog # 101814) was added to the mixture at a final concentration of 10mM. Immediately after GSH addition, the samples were injected onto a Dionex DNAPac PA200 column (4X 250 mM) at 30℃and on a 35-65% anion exchange gradient (20 mM sodium phosphate, 10-15% CH) ₃ CN,1M sodium bromide pH 11) was run at 1mL/min for 6.5 min.

Glutathione mediated lysis kinetics were monitored every hour for 24 hours. The area under the main peak per hour was normalized to the area at the 0h time point (first injection). The first order decay kinetics was used to calculate half-life. In a daily assay run, a control sequence containing a modified oligonucleotide (23-nt long) with a 5' thiol modifier C6 (Glen Research catalog number 10-1936-02) was run between N6 and N7. Each set of sequences was also run once with a second control sequence containing a modified oligonucleotide (23-nt long) with the same 5' thiol modifier C6 at N1. Half-life is reported relative to the half-life of the control sequence. Glutathione and GST were prepared as 100mM and 10mg/mL stock solutions, respectively, in water and aliquoted into 1mL tubes and stored at-80 ℃. The assay was performed daily using new aliquots.

TABLE 13 half-life of oligonucleotide 5' -modified phosphate prodrugs after incubation with glutathione

Oligonucleotide ID	Sequence(s)	Half-life (h)
			A-515432	(Pmd)dTdTdTdTdTdTdTdTdTdT	>24
A-515433	(Pmds)dTdTdTdTdTdTdTdTdTdT	>24
			A-801703 (control 1)	Q51uUfaUfaGfaGfcAfagaAfcAfcUfgUfuuu	<1
A-801704 (control 2)	uUfaUfaGfQ51GfcAfagaAfcAfcUfgUfuuu	4.2
			A-1875173	(Cymd)usCfsacuUfuAfUfugagUfuUfcugugscsc	>24
A-1875172	(Cymds)usCfsacuUfuAfUfugagUfuUfcugugscsc	>24
			A-1875175	(Pd)usCfsacuUfuAfUfugagUfuUfcugugscsc	>24
A-1875174	(Pds)usCfsacuUfuAfUfugagUfuUfcugugscsc	>24
			A-1875179	(Pmmd)usCfsacuUfuAfUfugagUfuUfcugugscsc	>24
A-1875178	(Pmmds)usCfsacuUfuAfUfugagUfuUfcugugscsc	>24
			A-1875180	(Ptmd)usCfsacuUfuAfUfugagUfuUfcugugscsc	>24
A-1875181	(Ptmds)usCfsacuUfuAfUfugagUfuUfcugugscsc	>24

Capital letters are followed by F-2' -deoxy-2 ' -fluoro (2 ' -F) sugar modifications; lower case letter-2 '-O-methyl (2' -OME) sugar modification; s-Phosphorothioate (PS) linkages; the structure of the prodrug is the same as that of table 9 above; q51-

Example 6 in vivo evaluation of siRNA duplex containing modified phosphate prodrug at the 5' end

In vivo study procedure: 0.3mg/mL of siRNA duplex containing modified phosphate prodrug at the 5' end in table 14 was administered to C57bl6 female mice (n=3/group). Serum was collected at days 11, 22 and 35 post-dose and analyzed by ELISA to determine relative F12 protein levels. The results are shown in FIG. 5.

TABLE 14 siRNA duplex used in the in vivo evaluation shown in FIG. 5

In vivo study procedure: c57bl6 female mice (n=3/group) were administered 0.1mg/mL or 0.3mg/mL of siRNA duplex containing modified phosphate prodrug at the 5' end in table 15. Serum was collected at days 11, 22 and 35 post-dose and analyzed by ELISA to determine relative F12 protein levels. The results are shown in FIG. 6.

TABLE 15 siRNA duplex for in vivo evaluation in FIG. 6

EXAMPLE 7.5 in vivo Metabolic stability and determination of the phosphate

Metabolic stability study procedure: c57bl6 female mice (n=2/group) were dosed with 10mg/kg of siRNA duplex containing modified phosphate prodrugs at the 5' end in table 16. Livers were collected 5 days after dosing and analyzed by LC-MS.

TABLE 16 siRNA duplex for metabolic evaluation

The results of the metabolic stability study are shown in tables 17 and 18.

TABLE 17 siRNA metabolite found after in vivo studies

TABLE 18 percentage of 5' -phosphate, phosphorothioate or hydroxyl groups found on the antisense strand after 5 days in mice

Duplex ID	5’PO(％)	5’PS(％)	5’OH(％)
				AD-326760	0	0	5
AD-1023155	0	0	8
				AD-1023156	0	0	18
AD-1023152	1.6	1.3	1
				AD-1023157	0	0	0

The possible in vivo cytosolic unmasking mechanism of the 5 '-phosphate is presented in fig. 7 for the 5' cyclic disulfide modified phosphate prodrug.

Example 8: in vivo evaluation of siRNA duplex containing modified phosphate prodrug at 5' end in CNS

In vivo study procedure of CNS (IT-intrathecal administration): female rats (n=3/group) were administered 0.1 mg/rat, 0.3 mg/rat or 0.9 mg/rat SOD1 siRNA duplex containing modified phosphate prodrug at the 5' end in table 19. Brains were collected 14 or 84 days after IT administration and dissected in different areas for qPCR analysis to determine relative SOD1 mRNA levels. The results are shown in FIGS. 8-13.

In vivo study procedure of CNS (ICV-intracranial ventricular administration): SOD1siRNA duplex containing modified phosphate prodrug at the 5' end in table 19 was administered 100 μg to C57bl6 female mice (n=4/group). Brains were collected 7 days after ICV administration and the right hemisphere was used for qPCR analysis to determine relative SOD1 mRNA levels. The results are shown in fig. 14.

TABLE 19 siRNA for CNS research

Capital letters are followed by F-2' -deoxy-2 ' -fluoro (2 ' -F) sugar modifications; lower case letter-2 '-O-methyl (2' -OME) sugar modification; s-Phosphorothioate (PS) linkages; uhd:2 '-O-hexadecyluridine (2' -C16); VP-vinyl phosphonate; prodrugs:

Pmmds(((4 sr,5 sr) -3, 5-trimethyl-1, 2-dithiolan-4-ol) phosphodiester;

cPmmds(((4 sr,5 rs) -3, 5-trimethyl-1, 2-dithiolan-4-ol) phosphodiester (cis Pmmds));

PdAr1s(((4 sr,5 rs) -5-phenyl-3, 3-dimethyl-1, 2-dithiolan-4-ol) phosphoric acid diester;

PdAr3s(((4 sr,5 rs) -5- (4-methoxyphenyl) -3, 3-dimethyl-1, 2-dithiolan-4-ol) phosphoric acid diester); />

PdAr5s(((4 sr,5 rs) -5- (4-methoxyphenyl) -3, 3-dimethyl-1, 2-dithiolan-4-ol) phosphoric acid diester);

PdAr2sPdAr4sPdAr6s

Pmmd/PmmdsPmds

Cymd/Cymds(x is O/S); ptmd/Ptmds (++>X is O/S), pd/PdsX is O/S).

As shown in fig. 8-11, siRNA duplex containing Pmmds and cpmds prodrugs at the 5 '-end showed similar activity and duration in CNS tissues as siRNA duplex containing 5' -VP control.

As shown in fig. 12-13, siRNA duplex containing PdAr1, pdAr3 and PdAr5 prodrugs at the 5 '-end showed better or at least comparable activity in CNS tissue compared to siRNA duplex containing 5' -VP control.

In conclusion, metabolically stable 5' -phosphate mimics such as 5' -VP can improve siRNA activity in extrahepatic tissues, while the endogenous 5' -phosphorylation efficiency of modified siRNAs is lower. The novel 5' modified phosphate prodrugs described herein exhibit stability in plasma and endosomal environments. These 5 'modified phosphate prodrugs are unmasked in the cytosol to present the natural 5' -phosphate (or phosphorothioate) required for efficient RISC loading. Sirnas containing novel 5 'modified phosphate prodrugs at the 5' end exhibit activity comparable to or even better than sirnas containing stable 5 '-phosphate mimetic designs, e.g., 5' -VP.

For example, the activity of siRNA containing 5' modified phosphate prodrugs of the following list,

is generally comparable to the activity of siRNA containing 5' -VP. siRNAs containing 5' modified phosphate prodrugs of the following list,

generally have improved stability over 5'-VP containing siRNAs and have better or comparable activity than 5' -VP containing siRNAs.

EXAMPLE 9 introduction of modified phosphate prodrugs for masking internucleotide phosphate linkages to mask charges

As shown in schemes 14-19, different cyclic phosphate prodrug derivatives can be introduced to the phosphate groups as temporary protecting groups for any internucleotide phosphate groups on the sense or antisense strand or both.

Scheme 14

Scheme 15

Scheme 16

Scheme 17

Scheme 18

Scheme 19

Example 10 use of modified phosphate prodrugs to generate cleavable siRNA conjugates with different targeting ligands

As shown in scheme 20, different targeting ligands can be introduced into the siRNA duplex by cyclic phosphate derivatives. These derivatives will cleave after the siRNA enters the cytosol.

Scheme 20

Claims

1. A compound comprising the structure of formula (I):

wherein saidThe structure is as follows:

wherein:

R ₁ is O or S, and is bonded to theP atoms of (c);

R ₃ And R is ₅ Each independently is H, halogen, OR ¹³ OR alkylene-OR ¹³ N (R ') (R ') or alkylene-N (R ') (R "), alkyl, C (R) ¹⁴ )(R ¹⁵ )(R ¹⁶ ) Or alkylene-C (R) ¹⁴ )(R ¹⁵ )(R ¹⁶ ) Alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, each of which may optionally be substituted with one or more R ^sub Group substitution; or R is ₃ And R is ₅ With adjacent carbon atoms and two sulfur atomsThe children together form a second ring;

g is O, N (R'), S or C (R) ¹⁴ )(R ¹⁵ )；

n is an integer from 0 to 6;

2. The compound according to claim 1, wherein in said compound In (a):

R ₁ is O;

g is CH ₂ ；

n is 0 or 1;

R 'and R' are each independently H or C ₁ -C ₆ An alkyl group.

3. The compound of claim 1, wherein theThe structure is as follows:

4. the compound of claim 1, wherein theThe structure is as follows:

5. the compound of claim 3 or 4, wherein R ₂ Is an optionally substituted aryl group.

6. The compound of claim 3 or 4, wherein R ₂ Is optionally substituted C _1-6 An alkyl group.

7. The compound of claim 1, wherein theHaving a structure selected from one of the following groups of formula Ia), ib) and II):

Ia)：

Ib)：

and

II)：

8. a compound according to any one of claim 1 to 3,wherein said The structure is as follows:

wherein the method comprises the steps of

X ₁ And Z ₁ Each independently is H, OH, OM, OR ¹³ 、SH、SM、SR ¹³ C (O) H, S (O) H or alkyl, each of which may optionally be substituted with one or more R ^sub Group substitution, N (R') (R), B (R) ¹³ ) ₃ 、BH ₃ ^- Se; or D-Q, wherein D is independently at each occurrence absent, O, S, N (R'), alkylene, each of which may optionally be substituted with one or more R ^sub Group substitution, and Q is independently at each occurrence a nucleoside or oligonucleotide;

Y ₁ s, O or N (R');

m is an organic or inorganic cation; and is also provided with

9. A compound according to any one of claims 1-3, wherein the The structure is as follows:

Wherein:

X ₁ and Z ₁ Each independently is OH, OM, SH, SM, C (O) H, S (O) H, C optionally substituted with one or more hydroxy or halogen groups ₁ -C ₆ Alkyl or D-Q;

d is independently at each occurrence C which is absent, O, S, NH, optionally substituted with one or more halogen groups ₁ -C ₆ An alkylene group; and is also provided with

Y ₁ Is S or O.

10. The compound according to claim 9, wherein X ₁ Is OH or SH; and Z is ₁ Is D-Q.

11. A compound according to any one of claims 1-3, wherein the The structure is as follows:

wherein:

X ₂ is N (R') (R ");

Z ₂ is X ₂ 、OR ¹⁸ Or D-Q;

R ¹⁸ is H or cyano-substituted C ₁ -C ₆ An alkyl group; and is also provided with

R 'and R' are each independentlyIs C ₁ -C ₆ An alkyl group.

12. The compound of claim 11, wherein theHas a structure selected from the group consisting of:

13. the compound of claim 1, wherein the compound has a structure selected from the group consisting of:

14. the compound of claim 8, wherein theHas a structure of (P-I), and the-having a structure selected from:

wherein X is O or S.

15. The compound of claim 1, wherein one or more targeting ligands are attached to the compound optionally through one or more linkers R of (2) ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ 、R ₇ 、R ₈ And R is ₉ Any one of them.

16. The compound of claim 15, wherein the ligand is selected from the group consisting of antibodies, ligand binding portions of receptors, ligands of receptors, aptamers, carbohydrate-based ligands, fatty acids, lipoproteins, folic acid, thyroid stimulating hormone, melanotropin, surface active protein a, mucins, glycosylated polyamino acids, transferrin, bisphosphonic acids, polyglutamic acid, polyaspartic acid, lipophilic portions, cholesterol, steroids, bile acids, vitamin B12, biotin, fluorophores, and peptides.

17. An oligonucleotide comprising one or more structures of formula (II):

wherein the saidThe structure is as follows:

or a salt thereof,

wherein:

* Represents a bond to the oligonucleotide,

y is absent, =O or=S, X is-OH, -SH OR X ', wherein X' is-OR ¹³ or-SR ¹³ ；

R ₁ Is O or S, and is bonded to a P atom of the group-P (Y) (X) -;

g is O, N (R'), S or C (R) ¹⁴ )(R ¹⁵ )；

n is an integer from 0 to 6;

R ¹⁴ 、R ¹⁵ and R is ¹⁶ Each independently is H, halogen, haloalkyl, alkyl, alkylaryl, aryl, heteroaryl, arylalkyl, hydroxy, alkoxy, aryloxy, N (R')(R”)；

Wherein, when saidHaving the structure of formula (C-III), at least oneAttached to the 5' end of the nucleoside or oligonucleotide.

18. The oligonucleotide of claim 17, wherein the Having a structure selected from one of the following groups of formula Ia), ib) and II):

Ia)：

Ib)：

and

II)：

19. the oligonucleotide of claim 17, wherein the Having a structure selected from one of the groups of formula (III):

20. the oligonucleotide of claim 17, wherein the The group has a structure selected from the group consisting of:

or a salt thereof, wherein X is O or S.

21. The oligonucleotide of any one of claims 17-20, comprising a structure having the formula: or a salt thereof. />

22. The oligonucleotide of claim 17, wherein the Has a structure selected from the group consisting of:

wherein indicates a bond to the phosphorus atom of the-P (X) (Y) -, group.

23. The oligonucleotide of claim 22, wherein the Has a structure selected from the group consisting of:Wherein X is O or S.

24. The oligonucleotide of claim 17, wherein the oligonucleotide contains at least one at the 5' -end of the oligonucleotide

25. The oligonucleotide of claim 24, wherein the first nucleotide at the 5' -end of the oligonucleotide has the structure:

Or a salt thereof, wherein:

the base is an optionally modified nucleobase;

R ^s is saidAnd is also provided with

R is H, OH, O-methoxyalkyl, O-methyl, O-allyl, CH ₂ -allyl, fluoro, O-N-methylacetamido (O-NMA), O-dimethylaminoethoxyethyl (O-DMAEOE), O-aminopropyl (O-AP) or ara-F.

26. The oligonucleotide of claim 24, wherein the first nucleotide at the 5' -end of the oligonucleotide has the structure:

or a salt thereof.

27. The oligonucleotide of claim 24, wherein the first nucleotide at the 5' -end of the oligonucleotide has the structure:

or a salt thereof.

28. The oligonucleotide of any one of claims 25-27, wherein the base is uridine.

29. The oligonucleotide of any one of claims 25-28, wherein R is methoxy or hydrogen.

30. The oligonucleotide of claim 17, wherein the oligonucleotide contains at least one at the 3' -end of the oligonucleotide

31. The oligonucleotide of claim 17, wherein the oligonucleotide contains at least one at an internal position of the oligonucleotide

32. The oligonucleotide of claim 17, wherein the oligonucleotide is a single stranded oligonucleotide.

33. The oligonucleotide of claim 17, wherein the oligonucleotide is a double-stranded oligonucleotide comprising a sense strand and an antisense strand.

34. The oligonucleotide of claim 33, wherein the oligonucleotide contains at least one at the antisense strand, sense strand, or both strands of the oligonucleotide

35. The oligonucleotide of claim 34, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, wherein the strand forms a double-stranded region of 21 consecutive base pairs with a single-stranded overhang of 2 nucleotides in length at the 3' -end.

36. The oligonucleotide of claim 33, wherein the oligonucleotide contains at least one at the 5' -end of the antisense strandAnd at least one targeting ligand at the 3' -end of the sense strand.

37. The oligonucleotide of claim 17, wherein the oligonucleotide contains one or more 2'-O modifications selected from 2' -deoxy, 2 '-O-methoxyalkyl, 2' -O-methyl, 2 '-O-allyl, 2' -C-allyl, 2 '-fluoro, 2' -O-N-methylacetamido (2 '-O-NMA), 2' -O-dimethylaminoethoxyethyl (2 '-O-DMAEOE), 2' -O-aminopropyl (2 '-O-AP), and 2' -ara-F.

38. The oligonucleotide of claim 33, wherein the sense strand and the antisense strand comprise no more than ten 2' -fluoro modified nucleotides.

39. The oligonucleotide of claim 33, wherein the sense and antisense strands comprise at least 50%, at least 60%, or at least 70% 2' -OMe modified nucleotides.

40. The oligonucleotide of claim 33, wherein the sense strand or antisense strand comprises at least two phosphorothioate linkages at the 5 '-terminus or at the 3' -terminus.

41. A pharmaceutical composition comprising the oligonucleotide of claim 17 and a pharmaceutically acceptable excipient.

42. A method for reducing or inhibiting expression of a target gene in a subject, comprising:

administering the oligonucleotide of claim 17 to the subject in an amount sufficient to inhibit expression of the target gene.

43. A method for modifying an oligonucleotide, comprising:

contacting the oligonucleotide with the compound under conditions suitable for reacting the compound of claim 1 with the oligonucleotide, wherein the oligonucleotide comprises a free hydroxyl group.

44. The method of claim 43, wherein the free hydroxyl group is part of a 5 '-terminal nucleotide or part of a 3' -terminal nucleotide.

45. The method of claim 43, wherein the oligonucleotide comprises a 5'-OH group or a 3' -OH group.

46. A method for preparing a modified oligonucleotide, comprising:

under conditions suitable for forming a modified oligonucleotide comprising a group of formula (B):

wherein Y is O or S; and X is-OH, -SH or X',

oxidizing a first oligonucleotide comprising a group of formula (a):

or a salt thereof, wherein:

R ^S is that

47. The method of claim 46, wherein the first nucleotide at the 5' -end of the first oligonucleotide is according to formula (C):

or a salt thereof, wherein:

the base is an optionally modified nucleobase; and is also provided with

R is H, OH, O-methoxyalkyl, O-methyl, O-allyl, CH ₂ -allyl, fluoro, O-N-methylacetamido (O-NMA), O-dimethylaminoethoxyethyl (O-DMAEOE), O-aminopropyl (O-AP) or ara-F, and

The first nucleotide at the 5' -end of the modified oligonucleotide has the structure of formula (D):

48. the method of claim 46, wherein the first nucleotide at the 5' -end of the modified oligonucleotide has the structure of formula (E) or (F):

or a salt thereof, wherein x represents a bond to a subsequent nucleotide.