# Patent

## Title
Lipophilic conjugated iRNA agents

## Abstract

The invention relates to iRNA agents, which preferably include a monomer in which the ribose moiety has been replaced by a moiety other than ribose. The inclusion of such a monomer can allow for modulation of a property of the iRNA agent into which it is incorporated, e.g., by using the non-ribose moiety as a point to which a ligand or other entity, e.g., a lipophilic moiety is directly, or indirectly, tethered. The invention also relates to methods of making and using such modified iRNA agents.

## Background
TECHNICAL FIELD
The invention relates to iRNA agents, which preferably include a monomer in which the ribose moiety has been replaced by a moiety other than ribose. The inclusion of such a monomer can allow for modulation of a property of the iRNA agent into which it is incorporated, e.g., by using the non-ribose moiety as a point to which a ligand or other entity, e.g., a lipophilic moiety. e.g., cholesterol, a bile acid, or a fatty acid conjugate is directly, or indirectly, tethered. The invention also relates to methods of making and using such modified iRNA agents.
BACKGROUND
RNA interference or “RNAi” is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al. (1998) Nature 391, 806-811). Short dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi may involve mRNA degradation.
SUMMARY
The inventor has discovered, inter alia, that the ribose sugar of one or more ribonucleotide subunits of an iRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.
The carriers further include (i) at least one “backbone attachment point” (e.g., one or two backbone attachment points) and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a ligand, e.g., a targeting or delivery moiety, or a moiety which alters a physical property. One of the most preferred moieties is a moiety which promotes entry into a cell, e.g., a lipophilic moiety, e.g., cholesterol or a fatty acid conjugate. While not wishing to be bound by theory it is believed the attachment of a lipohilic agent increases the lipophilicity of an iRNA agent. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, it will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.
Incorporation of one or more RRMSs described herein into an RNA agent, e.g., an iRNA agent, particularly when tethered to an appropriate entity, can confer one or more new properties to the RNA agent and/or alter, enhance or modulate one or more existing properties in the RNA molecule. E.g., it can alter one or more of lipophilicity or nuclease resistance. Incorporation of one or more RRMSs described herein into an iRNA agent can, particularly when the RRMS is tethered to an appropriate entity, modulate, e.g., increase, binding affinity of an iRNA agent to a target mRNA, change the geometry of the duplex form of the iRNA agent, alter distribution or target the iRNA agent to a particular part of the body, or modify the interaction with nucleic acid binding proteins (e.g., during RISC formation and strand separation).
Accordingly, in one aspect, the invention features, an iRNA agent preferably comprising a first strand and a second strand, wherein at least one subunit having a formula (I) is incorporated into at least one of said strands:




wherein:
X is N(CO)R7, NR7 or CH2;
Y is NR8, O, S, CR9R10, or absent;
Z is CR11R12 or absent;
Each of R1, R2, R3, R4, R9, and R10 is, independently, H, OH, ORa, ORb, (CH2)nORa, (CH2)nORb, or (CH2)nOH provided that at least one of R1, R2, R3, R4, R9, and R10 is ORa, ORb, or OH and that at least one of R1, R2, R3, R4, R9, and R10 is (CH2)nORa, (CH2)nORb, or (CH2)nOH (when the RRMS is terminal, one of R1, R2, R3, R4, R9, and R10 will include OH or ORa and one will include ORb; when the RRMSS is internal, two of R1, R2, R3, R4, R9, and R10 will each include an ORb); further provided that preferably OH or ORa may only be present with (CH2)nORb and (CH2)nOH or (CH2)nORa may only be present with ORb;
Each of R5, R6, R11, and R12 is, independently, H, C1-C6 alkyl optionally substituted with 1-3 R13, or C(O)NHR7; or R5 and R11 together are C3-C8 cycloalkyl optionally substituted with R14;
R7 can be a ligand, e.g., R7 can be Rd, or R7 can be a ligand tethered indirectly to the carrier, e.g., through a tethering moiety, e.g., C1-C20 alkyl substituted with NRcRd; or C1-C20 alkyl substituted with NHC(O)Rd;
R8 is C1-C6 alkyl;
R13 is hydroxy, C1-C4 alkoxy, or halo;
R14 is NRcR7;
Ra is:




Each of A and C is, independently, O or S;
B is OH, O−, or




Rc is H or C1-C6 alkyl;
Rd is H or a ligand, e.g., a lipophilic ligand, e.g., cholesterol, a bile acid, a fatty acid or PEG; and
n is 1-4.
Embodiments can include one or more of the following features.
The iRNA agent can be 21 nucleotides in length and there can be a duplex region of about 19 pairs.
The iRNA agent can include a duplex region between 17 and 23 pairs in length.
R1 can be (CH2)nORa and R3 can be ORb; or R1 can be (CH2)nORa and R9 can be ORb; or R1 can be (CH2)nORa and R5 can be ORb; or R1 can be (CH2)nORa and R2 can be ORb.
R1 can be (CH2)nOH and R3 can be ORb; or R1 can be (CH2)nOH and R9 can be ORb; or R1 can be CH2OH and R5 can be ORb; or R1 can be (CH2)nOH and R2 can be ORb.
R1 can be (CH2)nORb and R3 can be ORb; or R1 can be (CH2)nORb and R9 can be ORb; or R1 can be (CH2)nORb and R5 can be ORb; or R1 can be (CH2)nORb and R2 can be ORb; or R1 can be (CH2)nORb and R3 can be ORa; or R1 can be (CH2)nORb and R9 can be ORa; or R1 can be (CH2)nORb and R5 can be ORa; or R1 can be (CH2)nOR and R2 can be ORa; or R1 can be (CH2)nORb and R3 can be OH; or R1 can be (CH2)nORb and R9 can be OH; or R1 can be (CH2)nORb and R5 can be OH; or R1 can be (CH2)nORb and R2 can be OH.
R1 can be ORa and R3 can be (CH2)nORb; or R1 can be ORa and R9 can be (CH2)nORb; or R1 can be ORa and R5 can be (CH2)nORb.
R1 can be OH and R3 can be (CH2)nORb; or R1 can be OH and R9 can be (CH2)nORb; or R1 can be OH and R5 can be (CH2)nORb.
R1 can be ORb and R3 can be (CH2)nORb; or R1 can be ORb and R9 can be (CH2)nORb; or R1 can be ORb and R5 can be (CH2)nORb; or R1 can be ORb and R3 can be (CH2)nORa; or R1 can be ORb and R9 can be (CH2)nORa; or R1 can be ORb and R5 can be (CH2)nORa; or R1 can be ORb and R3 can be (CH2)nOH; or R1 can be ORb and R9 can be (CH2)nOH; or R1 can be ORb and R5 can be (CH2)nOH.
R3 can be (CH2)ORa and R9 can be ORb; or R3 can be (CH2)nORa and R9 can be ORb.
R3 can be (CH2)nOH and R9 can be ORb; or R3 can be (CH2)nOH and R9 can be ORb.
R3 can be (CH2)nORb and R9 can be ORb; or can be (CH2)nORb and R9 can be ORb; or R3 can be (CH2)nORb and R9 can be ORa; or R3 can be (CH2)nORb and R4 can be ORa; or R3 can be (CH2)nORb and R9 can be OH; or R3 can be (CH2)nORb and R4 can be OH.
R3 can be ORb and R9 can be (CH2)nORb; or R3 can be ORb and R9 can be (CH2)nORa; or R3 can be ORb and R9 can be (CH2)nORb; or R3 can be ORb and R4 can be (CH2)nORb; or R3 can be ORb and R9 can be (CH2)nOH; or R3 can be ORb and R4 can be (CH2)nOH.
R3 can be ORa and R9 can be (CH2)nORb; or R3 can be ORa and R9 can be (CH2)nORb.
R3 can be OH and R9 can be (CH2)nORb; or R3 can be OH and R9 can be (CH2)nORb.
R9 can be (CH2)nORa and R10 can be ORb.
R9 can be (CH2)nOH and R10 can be ORb.
R9 can be (CH2)nORb and R10 can be ORb; or R9 can be (CH2)nORb and R10 can be ORa; or R9 can be (CH2)nORb and R10 can be OH.
In some embodiments, at least one of R3, R5 or R9 is OH.
In a preferred embodiment the ribose is replaced with a 4-hydroxyproline-derived scaffold, as shown in formula II.




X is N(CO)R7 or NR7, Y is CR9R10, and Z is absent.
n can be 1.
A can be O or S.
R1 can be CH2ORb and R3 can be ORb; or R1 can be CH2ORa and R3 can be ORb; or R1 can be CH2ORb and R3 can be OH; or R1 can be CH2OH and R3 can be ORb; or R1 can be CH2ORb and R3 can be ORa.
R1 and R3 can be cis or R1 and R3 can be trans.
R1 can be CH2ORb and R9 can be ORa; or R1 can be CH2ORb and R9 can be OH; or R1 can be CH2ORb and R9 can be ORb; or R1 can be CH2ORa and R9 can be ORb; or R1 can be CH2OH and R9 can be ORb.
R1 and R9 can be cis or R1 and R9 can be trans.
R1 can be CH2ORb and R5 can be ORa; or R1 can be CH2ORb and R5 can be OH; or R1 can be CH2ORb and R5 can be ORb; or R1 can be CH2ORa and R5 can be ORb; or R1 can be CH2OH and R5 can be ORb.
R1 and R5 can be cis or R1 and R5 can be trans.
R7 can be (CH2)5NHRd or (CH2)5NHRd. Rd can be chosen from a folic acid radical; a cholesterol radical; a bile acid radical; a fatty acid radical (e.g., lithocholic-oleyl, lauroyl, docosnyl, stearoyl, palmitoyl, myristoyl, oleoyl, linoleoyl); a carbohydrate radical; a vitamin A radical; a vitamin E radical; a vitamin K radical; polyethylene glycol PEG). Preferably, Rd is a cholesterol radical. In some preferred embodiments, Rd is stearoyl, docosanyl, or lithocholic-oleyl. In some other preferred embodiments, Rd is PEG, e.g. PEG-5K, PEG-20K.
In a further aspect, this invention features an iRNA agent having a first strand and a second strand, wherein at least one subunit having a formula (I) or formula (II) is incorporated into at least one of said strands.
In one aspect, this invention features an iRNA agent having a first strand and a second strand, wherein at least two subunits having a formula (I) or formula (II) are incorporated into at least one of said strands.
In another aspect, this invention provides a method of making an iRNA agent described herein having a first strand and a second strand in which at least one subunit of formula (I) or formula (II) is incorporated in the strands. The method includes contacting the first strand with the second strand.
In a further aspect, this invention provides a method of modulating expression of a target gene, the method includes administering an iRNA agent described herein having a first strand and a second strand in which at least one subunit of formula (I) or formula (II) is incorporated in the strands to a subject.
RRMSs described herein may be incorporated into any single-stranded or double-stranded RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the RRMSs described herein. The tethered ligand on different RRMS can be the same or different. An RRMS can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An RRMS can be placed at or near (within 1, 2, or 3 positions of) the 3′ or 5′ end of the sense strand or at near (within 1, 2 or 3 positions of) the 3′ or 5′ end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5′ end of the antisense strand. An RRMS can be internal, and will preferably be positioned in regions not critical for antisense binding to the target.
In some embodiments, an iRNA agent has an RRMS at 3′ end of sense strand and 3′ end of antisense strand. In some embodiments, an iRNA agent has an RRMS at 5′ end of sense strand and 3′ end of antisense strand. In some embodiments, an iRNA agent has an RRMS at 3′ end of sense strand and 5′ end of antisense strand. In some embodiments, an iRNA agent has an RRMS at 3′ end of sense strand and at an internal position in the antisense strand. In some embodiments, an iRNA agent has an RRMS at 5′ end of sense strand and at an internal position in the antisense strand. In some embodiments, an iRNA agent has an RRMS at an internal position of sense strand and at 3′ end of antisense strand. In some embodiments, an iRNA agent has an RRMS at an internal position of sense strand and at 5′ end of antisense strand.
In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and at (or within 1, 2, or 3 positions of) the 3′ end of the sense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and an RRMS at the 5′ end of the sense strand, in which both ligands are located at the same end of the iRNA agent.
In certain embodiments, two ligands are tethered, preferably, one on each strand and are hydrophobic moieties. While not wishing to be bound by theory, it is believed that pairing of the hydrophobic ligands can stabilize the iRNA agent via intermolecular van der Waals interactions.
In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and an RRMS at the 5′ end of the sense strand, in which both RRMSs may share the same ligand (e.g., cholic acid) via connection of their individual tethers to separate positions on the ligand. A ligand shared between two proximal RRMSs is referred to herein as a “hairpin ligand.”
In other embodiments, an iRNA agent may have an RRMS at one or both of the 3′ end and/or 5′ end of the sense strand and an RRMS at an internal position of the sense strand. In other embodiments, an iRNA agent may have an RRMS at one or both of the 3′ end and/or 5′ end of the antisense strand and an RRMS at an internal position of the sense strand. An iRNA agent may have an RRMS at an internal position of the sense strand; or may have an RRMS at an internal position of the antisense strand; or may have an RRMS at an internal position of the sense strand and an RRMS at an internal position of the antisense strand.
In preferred embodiments the iRNA agent includes a first and second sequences, which are preferably two separate molecules as opposed to two sequences located on the same strand, have sufficient complementarity to each other to hybridize (and thereby form a duplex region), e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme.
It is preferred that the first and second sequences be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA agent contains first and second sequences, preferable paired to contain an overhang, e.g., one or two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides. Most embodiments will have a 3′ overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3′ overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5′ ends are preferably phosphorylated.
Other modifications to sugars, bases, or backbones described herein can be incorporated into the iRNA agents.
The iRNA agents can take an architecture or structure described herein. The iRNA agents can be palindromic, or double targeting, as described herein.
The iRNA agents can have a sequence such that a non-cannonical or other than cannonical Watson-Crick structure is formed between two monomers of the iRNA agent or between a strand of the iRNA agent and another sequence, e.g., a target or off-target sequence, as is described herein.
The iRNA agent can be selected to target any of a broad spectrum of genes, including any of the genes described herein.
In a preferred embodiment the iRNA agent has an architecture (architecture refers to one or more of overall length, length of a duplex region, the presence, number, location, or length of overhangs, single strand versus double strand form) described herein. E.g., the iRNA agent can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length and there is a duplex region of about 19 pairs. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.
In some embodiment the duplex region of the iRNA agent will have, mismatches. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
In addition to the RRMS-containing bases the iRNA agents described herein can include nuclease resistant monomers (NRMs).
In another aspect, the invention features an iRNA agent to which is conjugated a lipophilic moiety, e.g., cholesterol, a bile acid, or a fatty acid; (e.g., lithocholic-oleyl, lauroyl, docosnyl, stearoyl, palmitoyl, myristoyl, oleoyl, linoleoyl, dodecanoyl, lithocholyl, 5β-cholanyl, N,N-distearyl-lithocholamide, 1,2-di-O-stearoylglyceride); e.g., by conjugation to an RRMS of an iRNA agent. In a preferred embodiment, the lipophilic moiety enhances entry of the iRNA agent into a cell. In a preferred embodiment, the cell is part of an organism, tissue, or cell line, e.g., a primary cell line, immortalized cell line, or any type of cell line disclosed herein. Thus, the conjugated iRNA agent can be used to silence a target gene in an organism, e.g., a mammal, e.g., a human, or to silence a target gene in a cell line or in cells which are outside an organism.
The lipophilic moiety can be chosen, for example, from the group consisting of a lipid, cholesterol, oleyl, retinyl, cholesteryl residues, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid (i.e., palmitoyl), myristic acid (i.e., myristoyl), O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, lauroyl, docosnyl, stearoyl, oleoyl, linoleoyl, dimethoxytrityl, or phenoxazine. A preferred lipophilic moiety is cholesterol. In some embodiments, a preferred lipophilic moiety is stearoyl, docosanyl, or lithocholic-oleyl. The lipophilic moieties described herein are conjugated to the RRMS, for example, via a tether. When used herein, terms to describe the lipophilic moiety such as a free acid (such as a free fatty acid, e.g., palmitic acid) are used interchangeably with the radical term (e.g., palmitoyl) for the conjugated form of the lipophilic moiety.
The iRNA agent can have a first strand and a second strand, wherein at least one subunit having formula (I) or formula (II) is incorporated into at least one of the strands. The iRNA agent can have one or more of any of the features described herein. For example, when the subunit is of formula (I), Rd can be cholesterol; X can be N(CO)R7 or NR7, Y can be CR9R10, and Z can be absent, and R1 can be (CH2)nORb and R3 can be ORa or OH; X can be N(CO)R7 or NR7, Y can be CR9R10, and Z can be CR11R12, and R9 can be (CH2) % ORb and R10 can be ORa or OH; X can be N(CO)R7 or NR7, Y can be NR8, and Z can be CRR11R12, and R1 can be (CH2)nORb and R3 can be ORa or OH; X can be CH2; Y can be CR9R10; and Z can be CR11R12, in which R6 can be C(O)NHR7; or X can be CH2; Y can be CR9R10; and Z can be CR11R12 in which R11 or R12 can be C(O)NHR7 or R5 and R11 together can be C5 or C6 cycloalkyl substituted with N(CH3)R7.
In a preferred embodiment, the invention provides methods of silencing a target gene by providing an iRNA agent to which a lipophilic moiety is conjugated, e.g., a lipophilic conjugated iRNA agent described herein, to a cell. In a preferred embodiment the conjugated iRNA agent an be used to silence a target gene in an organism, e.g., a mammal, e.g., a human, or to silence a target gene in a cell line or in cells which are outside an organism.
In another aspect, the invention provides compositions of iRNA agents described herein, and in particular compositions of an iRNA agent to which a lipophilic moiety is conjugated, e.g., a lipophilic conjugated iRNA agent described herein. In a preferred embodiment the composition is a pharmaceutically acceptable composition.
The methods and compositions of the invention, e.g., the RRSM-containing iRNA agents described herein, can be used with any of the iRNA agents described herein. In addition, the methods and compositions of the invention can be used for the treatment of any disease or disorder described herein, and for the treatment of any subject, e.g., any animal, any mammal, such as any human.
The non-ribose scaffolds, as well as monomers and dimers of the RRMSs described herein are within the invention
An “RNA agent” as used herein, is an unmodified RNA, modified RNA, or nucleoside surrogate, all of which are defined herein, see the section herein entitled RNA Agents. While numerous modified RNAs and nucleoside surrogates are described herein, preferred examples include those which include one or more RRMS. Preferred examples are those which also a 2′ sugar modification, a modification in a single strand overhang, preferably a 3′ single strand overhang, or, particularly if single stranded, a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group.
An “iRNA agent” as used herein, is an RNA agent which can, or which can be cleaved into an RNA agent which can, down regulate the expression of a target gene, preferably an endogenous or pathogen target RNA. While not wishing to be bound by theory, an iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms. An iRNA agent can include a single strand or can include more than one strands, e.g., it can be a double stranded iRNA agent. If the iRNA agent is a single strand it is particularly preferred that it include a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features and advantages of the invention will be apparent from the description and drawings, and from the claims. This application incorporates all cited references, patents, and patent applications by references in their entirety for all purposes.



DESCRIPTION OF DRAWINGS
FIG. 1 a general synthetic scheme for incorporation of RRMS monomers into an oligonucleotide.
FIG. 2 is a representation of structures of lipophilic ligands and a hydroxyproline-amino linker conjugated to siRNA.
FIG. 3 is a trace of RP-HPLC titration of lipophilicity of conjugated siRNA. Traces are as follows: (1) Control sense strand (5′-GUCAUCACACUGAAUACCAAsU (SEQ ID NO:44)); (2) Sense strand with amino-linker; (3) Adamantaneformyl; (4) Lauroyl; (5) Lithocholyl; (6) Myristoyl; (7) Linoleoyl; (8) Palmitoyl; (9) Oleoyl; (10) 5β-cholanyl; (11)Stearoyl; (12) Cholesteryl; (13) Docosanoyl; (14) Vitamin E; (15) Thiocholesterol; (16) Lithocholic-Oleyl; (17) Cholesteryl-Cholesterol (bis-Cholesterol); (18) 1,2-Di-O-stearylglyceride and (19) N,N-Distearyl-lithocholamide.
FIGS. 4A and 4B are bar graphs depicting the percentage of relative apoB mRNA in liver and jejunum respectively upon treatment with lipophile-apoB-siRNA conjugates.
FIGS. 5A and 5B are bar graphs depicting the amount apoB mRNA in jejunum and liver respectively upon treatment with lipophile-apoB-siRNA conjugates relative to treatment with saline alone.
FIGS. 6A and 6B are bar graphs depicting the percentage of relative apoB mRNA in liver and jejunum respectively upon treatment with PEGylated siRNA. Mice were treated with 100 mg/kg iv single injection and sacrificed 72 h after injection.
FIG. 7 is a bar graph depicting the percentage of relative apoB mRNA in liver upon treatment with thiocholesterol conjugated siRNA.
FIG. 8 is a bar graph comparing the apo gene silencing in the liver upon treatment with cholesterol, PEG, vitamin E and cholanic acid conjugated siRNAs.
FIG. 9 is a bar graph comparing the apo gene silencing in the jejunum upon treatement with cholesterol, PEG, vitamin E and cholanic acid conjugated siRNAs.
FIG. 10 is a bar graph depicting the percentage of apoB mRNA relative to saline in liver and jejunum respectively upon treatment with cholesterol, dialkylglyceride and lithocholamide conjugated siRNAs.
FIG. 11 is a bar graph depicting the percentage of relative apoB mRNA in liver upon treatement with saline, siRNA 2, siRNA 5, and siRNA 6.
FIG. 12 is a bar graph depicting the percentage of relative apoB mRNA in jejunum upon treatement with saline, siRNA 2, siRNA 5, and siRNA 6.
FIG. 13 is a bar graph depicting the percentage of relative apoB mRNA in liver and Jejunum upon treatment with PBS and other exemplified PEG conjugated siRNA.



DETAILED DESCRIPTION
Double-stranded (dsRNA) directs the sequence-specific silencing of mRNA through a process known as RNA interference (RNAi). The process occurs in a wide variety of organisms, including mammals and other vertebrates.
It has been demonstrated that 21-23 nt fragments of dsRNA are sequence-specific mediators of RNA silencing, e.g., by causing RNA degradation. While not wishing to be bound by theory, it may be that a molecular signal, which may be merely the specific length of the fragments, present in these 21-23 nt fragments recruits cellular factors that mediate RNAi. Described herein are methods for preparing and administering these 21-23 nt fragments, and other iRNAs agents, and their use for specifically inactivating gene function. The use of iRNAs agents (or recombinantly produced or chemically synthesized oligonucleotides of the same or similar nature) enables the targeting of specific mRNAs for silencing in mammalian cells. In addition, longer dsRNA agent fragments can also be used.
Ligand-Conjugated Monomer Subunits and Monomers for Oligonucleotide Synthesis
Definitions
The term “halo” refers to any radical of fluorine, chlorine, bromine or iodine.
The term “alkyl” refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C1-C12 alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it. The term “haloalkyl” refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes alkyl moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may be optionally inserted with O, N, or S. The terms “aralkyl” refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of “aralkyl” include benzyl, 9-fluorenyl, benzhydryl, and trityl groups.
The terms “adeninyl, cytosinyl, guaninyl, thyminyl, and uracilyl” and the like refer to radicals of adenine, cytosine, guanine, thymine, and uracil.
A “protected” moiety refers to a reactive functional group, e.g., a hydroxyl group or an amino group, or a class of molecules, e.g., sugars, having one or more functional groups, in which the reactivity of the functional group is temporarily blocked by the presence of an attached protecting group. Protecting groups useful for the monomers and methods described herein can be found, e.g., in Greene, T. W., Protective Groups in Organic Synthesis (John Wiley and Sons: New York), 1981, which is hereby incorporated by reference.
General
An RNA agent, e.g., an iRNA agent containing a preferred, but nonlimiting ligand-conjugated monomer subunit, is presented as formula (II) below and in the scheme in FIG. 1. The carrier (also referred to in some embodiments as a “linker”) can be a cyclic or acyclic moiety and include one or two “backbone attachment points” (e.g., hydroxyl groups) and a ligand. The ligand can be directly attached (e.g., conjugated) to the carrier or indirectly attached (e.g., conjugated) to the carrier by an intervening tether (e.g., an acyclic chain of one or more atoms; or a nucleobase, e.g., a naturally occurring nucleobase optionally having one or more chemical modifications, e.g., an unusual base; or a universal base). The carrier therefore also includes a “ligand or tethering




attachment point” for the ligand and tether/tethered ligand, respectively.

The ligand-conjugated monomer subunit may be the 5′ or 3′ terminal subunit of the RNA molecule, i.e., one of the two —P(O)(O−)W groups may be a hydroxyl group, and the “W” of the other —P(O)(O−)W group may be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the ligand-conjugated monomer subunit may occupy an internal position, and both “W” groups may be one or more unmodified or modified ribonucleotides. More than one ligand-conjugated monomer subunit may be present in a RNA molecule, e.g., an iRNA agent. Preferred positions for inclusion of a tethered ligand-conjugated monomer subunits, e.g., one in which a lipophilic moiety, e.g., cholesterol, a bile acid radical, or a fatty acid conjugate radical (e.g., lithocholic-oleyl, lauroyl, docosnyl, stearoyl, palmitoyl, myristoyl oleoyl, linoleoyl) is tethered to the carrier are at the 3′ terminus, the 5′ terminus, or an internal position of the sense strand.
The modified RNA molecule of formulas (II) can be obtained using oligonucleotide synthetic methods known in the art. In a preferred embodiment, the modified RNA molecule of formula (II) can be prepared by incorporating one or more of the corresponding monomer compounds (see, e.g., A, B, and C below and in the scheme in FIG. 1) into a growing sense or antisense strand, utilizing, e.g., phosphoramidite or H-phosphonate coupling strategies.
The monomers, e.g., a ligand-conjugated monomer, generally include two differently functionalized hydroxyl groups (OFG1 and OFG2), which are linked to the carrier molecule (see A below and in FIG. 1), and a ligand/tethering attachment point. As used herein, the term “functionalized hydroxyl group” means that the hydroxyl proton has optionally been replaced by another substituent. In some embodiments, the functionalized hydroxyl group (OFG) is simply an OH, for example a deprotected OH group. As shown in representative structures B and C below and in FIG. 1, one hydroxyl group (OFG1) on the carrier is functionalized with a protecting group (PG). The other hydroxyl group (OFG2) can be functionalized with either (1) a liquid or solid phase synthesis support reagent (solid circle) directly or indirectly through a linker, L, as in B, or (2) a phosphorus-containing moiety, e.g., a phosphoramidite as in C. The tethering attachment point may be connected to a hydrogen atom, a suitable protecting group, a tether, or a tethered ligand at the time that the monomer is incorporated into the growing sense or antisense strand (see variable “R” in A below). Thus, the tethered ligand can be, but need not be attached to the monomer at the time that the monomer is incorporated into the growing strand. In certain embodiments, the tether, the ligand or the tethered ligand may be linked to a “precursor” ligand-conjugated monomer subunit after a “precursor” ligand-conjugated monomer subunit has been incorporated into the strand. The wavy line used below (and elsewhere herein) refers to a connection, and can represent a direct bond between the moiety and the attachment point or a tethering molecule which is interposed between the moiety and the attachment point. Directly tethered means the moiety is bound directly to the attachment point. Indirectly tethered means that there is a tether molecule interposed between the attachment point and the moiety.
The OFG protecting group may be selected as desired, e.g., from T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991). The protecting group is preferably stable under amidite synthesis conditions, storage conditions, and oligonucleotide synthesis conditions.




Tethered Ligands
A wide variety of entities, e.g., ligands, can be tethered to an iRNA agent, e.g., to the carrier of a ligand-conjugated monomer subunit. Examples are described below in the context of a ligand-conjugated monomer subunit but that is only preferred, entities can be coupled at other points to an iRNA agent.
Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether, to the carrier. In preferred embodiments, the ligand is attached to the carrier via an intervening tether. As discussed above, the ligand or tethered ligand may be present on the ligand-conjugated monomer when the ligand-conjugated monomer is incorporated into the growing strand. In some embodiments, the ligand may be incorporated into a “precursor” ligand-conjugated monomer subunit after a “precursor” ligand-conjugated monomer subunit has been incorporated into the growing strand. For example, a monomer having, e.g., an amino-terminated tether, e.g., carrier-(CH2)nNH2 may be incorporated into a growing sense or antisense strand. In a subsequent operation, i.e., after incorporation of the precursor monomer subunit into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor ligand-conjugated monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor ligand-conjugated monomer subunit tether.
In preferred embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g, molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.
Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; nuclease-resistance conferring moieties; and natural or unusual nucleobases. General examples include lipophiles, lipids (e.g., cholesterol, a bile acid, or a fatty acid (e.g., lithocholic-oleyl, lauroyl, docosnyl, stearoyl, palmitoyl, myristoyl oleoyl, linoleoyl), steroids (e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics.
The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., polyethylene glycol (PEG), PEG-40K, PEG-20K and PEG-5K.
Other examples of ligands include lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, glycerol (e.g., esters and ethers thereof, e.g., C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl; e.g., lauroyl, docosnyl, stearoyl, oleoyl, linoleoyl 1,3-bis-O(hexadecyl)glycerol, 1,3-bis-O(octaadecyl)glycerol), geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dodecanoyl, lithocholyl, 5β-cholanyl, N,N-distearyl-lithocholamide, 1,2-di-O-stearoylglyceride, dimethoxytrityl, or phenoxazine) and PEG (e.g., PEG-5K, PEG-20K, PEG-40K).
In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).
The iRNA agents of the invention are particularly useful when targeted to the liver. An iRNA agent can be targeted to the liver by incorporation of a monomore derivitzed with a ligand which targets to the liver. For example, a liver-targeting agent can be a lipophilic moiety. Preferred lipophilic moieties include lipid, cholesterols, oleyl, retinyl, or cholesteryl residues. Other lipophilic moieties that can function as liver-targeting agents include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, oleoylic acid, linoleoylic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, lauroylic acid, docosynylic acid, stearoylic acid, or phenoxazine. In some embodiments, a preferred lipophilic moiety is stearoyl, docosanyl, or lithocholic-oleyl.
An iRNA agent can also be targeted to the liver by association with a lipoprotein such as HDL or LDL, e.g., lactosylated LDL. Polymeric carriers complexed with sugar residues can also function to target iRNA agents to the liver.
Exemplary Ligand Conjugated Monomers




Exemplary structures of lipophilic ligand and Hydroxyproline-amino linker conjugated to siRNA are provided in FIG. 2.
The lipohilic moiety can be attached at the 3′ terminus, the 5′terminus, or internally, preferably on the sense strand. The lipohilic moiety can be attached to an RRMS, e.g., a pyrroline-based RRMS which is at the 3′ terminus, the 5′terminus, or internal, in the sense strand. The attachment can be direct or through a tethering molecule. Tethers, spacers or linkers discussed herein can be used to attach the moiety to the RRMS.
iRNA Agent Structure
The monomers described herein can be used to make oligonucleotides which are useful as iRNA agents, e.g., RNA molecules, (double-stranded; single-stranded) that mediate RNAi, e.g., with respect to an endogenous gene of a subject or to a gene of a pathogen. In most cases the iRNA agent will incorporate monomers described herein together with naturally occurring nucleosides or nucleotides or with other modified nucleosides or nucleotides. The modified monomers can be present at any position in the iRNA agent, e.g., at the termini or in the middle region of an iRNA agent or in a duplex region or in an unpaired region. In a preferred embodiment iRNA agent can have any architecture, e.g., architecture described herein. E.g., it can be incorporated into an iRNA agent having an overhang structure, a hairpin or other single strand structure or a two-strand structure, as described herein.
The RRMS-containing 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 a fragment thereof, can mediate down regulation of the target gene. (For ease of exposition the term nucleotide or ribonucleotide is sometimes used herein in reference to one or more monomeric subunits of an RNA agent. It will be understood herein that the usage of the term “ribonucleotide” or “nucleotide”, herein can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.) Thus, the iRNA agent is or includes a region which is at least partially, and in some embodiments fully, complementary to the target RNA. It is not necessary that there be perfect complementarity between the iRNA agent and the target, but the correspondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., mRNA.
In addition to homology to target RNA and the ability to down regulate a target gene, an iRNA agent will preferably have one or more of the following properties:

    
    
        (1) if single stranded it will have a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group;
        (2) it will, despite modifications, even to a very large number, or all of the nucleosides, have an antisense strand that can present bases (or modified bases) in the proper three dimensional framework so as to be able to form correct base pairing and form a duplex structure with a homologous target RNA which is sufficient to allow down regulation of the target, e.g., by cleavage of the target RNA;
        (3) it will, despite modifications, even to a very large number, or all of the nucleosides, still have “RNA-like” properties, i.e., it will possess the overall structural, chemical and physical properties of an RNA molecule, even though not exclusively, or even partly, of ribonucleotide-based content. For example, an iRNA agent can contain, e.g., a sense and/or an antisense strand in which all of the nucleotide sugars contain e.g., 2′ fluoro in place of 2′ hydroxyl. This deoxyribonucleotide-containing agent can still be expected to exhibit RNA-like properties. While not wishing to be bound by theory, the electronegative fluorine prefers an axial orientation when attached to the C2′ position of ribose. This spatial preference of fluorine can, in turn, force the sugars to adopt a C3′-endo pucker. This is the same puckering mode as observed in RNA molecules and gives rise to the RNA-characteristic A-family-type helix. Further, since fluorine is a good hydrogen bond acceptor, it can participate in the same hydrogen bonding interactions with water molecules that are known to stabilize RNA structures. (Generally, it is preferred that a modified moiety at the 2′ sugar position will be able to enter into H-bonding which is more characteristic of the OH moiety of a ribonucleotide than the H moiety of a deoxyribonucleotide. A preferred iRNA agent will: exhibit a C3′-endo pucker in all, or at least 50, 75, 80, 85, 90, or 95% of its sugars; exhibit a C3′-endo pucker in a sufficient amount of its sugars that it can give rise to a the RNA-characteristic A-family-type helix; will have no more than 20, 10, 5, 4, 3, 2, or 1 sugar which is not a C3′-endo pucker structure. These limitations are particularly preferably in the antisense strand;
        (4) regardless of the nature of the modification, and even though the RNA agent can contain deoxynucleotides or modified deoxynucleotides, particularly in overhang or other single strand regions, it is preferred that DNA molecules, or any molecule in which more than 50, 60, or 70% of the nucleotides in the molecule, or more than 50, 60, or 70% of the nucleotides in a duplexed region are deoxyribonucleotides, or modified deoxyribonucleotides which are deoxy at the 2′ position, are excluded from the definition of RNA agent.
    
    


A “double stranded (ds) iRNA agent” as used herein, is an iRNA agent which includes more than one, and preferably two, strands in which interchain hybridization can form a region of duplex structure.
The antisense strand of a double stranded iRNA agent should be equal to or at least, 14, 15, 16, 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It should be equal to or less than 200, 100, or 50, nucleotides in length. Preferred ranges are 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.
The sense strand of a double stranded iRNA agent should be equal to or at least 14, 15, 16, 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It should be equal to or less than 200, 100, or 50, nucleotides in length. Preferred ranges are 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.
The double strand portion of a double stranded iRNA agent should be equal to or at least, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotide pairs in length. It should be equal to or less than 200, 100, or 50, nucleotides pairs in length. Preferred ranges are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.
In many embodiments, the ds iRNA agent is sufficiently large that it can be cleaved by an endogenous molecule, e.g., by Dicer, to produce smaller ds iRNA agents, e.g., sRNAs agents
It may be desirable to modify one or both of the antisense and sense strands of a double strand iRNA agent. In some cases they will have the same modification or the same class of modification but in other cases the sense and antisense strand will have different modifications, e.g., in some cases it is desirable to modify only the sense strand. It may be desirable to modify only the sense strand, e.g., to inactivate it, e.g., the sense strand can be modified in order to inactivate the sense strand and prevent formation of an active sRNA/protein or RISC.
It is preferred that the sense and antisense strands be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA agent contains sense and antisense strands, preferable paired to contain an overhang, e.g., one or two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides. Most embodiments will have a 3′ overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3′ overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5′ ends are preferably phosphorylated. Preferred lengths for the duplexed region is between 15 and 30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in length.
The isolated iRNA agents described herein, including ds iRNA agents and sRNA agents can mediate silencing of a target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes a protein. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. In general, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also be targeted.
As used herein, the term “oligonucleotide” refers to a nucleic acid molecule (RNA or DNA) preferably of length less than 100, 200, 300, or 400 nucleotides.
RNA agents discussed herein include otherwise unmodified RNA as well as RNA which have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al., (1994) Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183-2196. Such rare or unusual RNAs, often termed modified RNAs (apparently because the are typically the result of a post transcriptionally modification) are within the term unmodified RNA, as used herein. Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, preferably different from that which occurs in the human body. While they are referred to as modified “RNAs,” they will of course, because of the modification, include molecules which are not RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to the presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone.
Much of the discussion below refers to single strand molecules. In many embodiments of the invention a double stranded iRNA agent, e.g., a partially double stranded iRNA agent, is required or preferred. Thus, it is understood that that double stranded structures (e.g. where two separate molecules are contacted to form the double stranded region or where the double stranded region is formed by intramolecular pairing (e.g., a hairpin structure)) made of the single stranded structures described below are within the invention. Preferred lengths are described elsewhere herein.
As nucleic acids are polymers of subunits or monomers, many of the modifications described below occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or the a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many, and infact in most cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal regions, e.g. at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an RNA. E.g., a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal regions, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.
Exemplary modification include the following:
(a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S—P═S); these modifications can be used to promote nuclease resistance;
(b) 2′-O alkyl, e.g., 2′-OMe, 3′-O alkyl, e.g., 3′-OMe (at terminal and/or internal positions); these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand, the 3′ modifications can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;
(c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe and with P═O or P═S) these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;
(d) L sugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;
(e) modified sugars (e.g., locked nucleic acids (LNA's), hexose nucleic acids (HNA's) and cyclohexene nucleic acids (CeNA's)); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;
(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines), these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand;
(g) cationic groups and Zwitterionic groups (preferably at a terminus), these modifications can be used to promote nuclease resistance;
(h) conjugate groups (preferably at terminal positions), e.g., naproxen, biotin, a lipophilic moiety such as cholesterol, a bile acid, or a fatty acid conjugate, ibuprofen, folic acid, peptides, and carbohydrates; these modifications can be used to promote nuclease resistance or to target the molecule, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC. The invention is further illustrated by the following examples, which should not be construed as further limiting.
EXAMPLES
Example 1
Synthesis of Lipophilic Conjugate Building Blocks
The amino derivative 100 was synthesized by Pd/C mediated hydrogenation of Cbz group and used as such with out any further purification (Scheme 1). All the lipid derivatives (102a-k) were prepared by the reaction of acid and amine with HBTU in the presence of DIEA in a mixture of DCM/DMF. All the hydroxyl derivatives (102a-k) were converted to the corresponding succinate by the reaction of succinic anhydride and DMAP in DCM and it was loaded on to Long chain alkyl amine-CPG using the HBTU/DIEA peptide coupling method or PPh3/DMAP/DTNP method (Scheme 2).
Synthesis of 6-amino-1-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-hexan-1-one 100
The Cbz protected amine 100 (33.56 g, 50.33 mmol) was dissolved in a mixture of MeOH/Ethyl acetate (1:8, 250 mL) and degassed with argon. Pd/C (3.3 g, 10 wt % Degussa wet type) was added and degassed and purged with hydrogen. The mixture was stirred under hydrogen (Balloon pressure) for 7 hrs. Reaction was monitored by TLC (10% MeOH/DCM, PMA stain). The TLC of the reaction mixture showed the complete disappearance of the starting Cbz protected amine. The reaction mixture was filtered through a pad of celite, washed with EtOAc (100 mL) and the combined filtrates were concentrated. The residue was dried under high vacuum overnight to provide the product as a foam (26.8 g). It was directly used for the next reaction with out further purification. 1H NMR (DMSO-d6, 400 MHz) δ=7.36-7.24 (m, 4H), 7.23-7.15 (m, 5H), 6.92-6.82 (m, 4H), 4.40 (m, 0.7H), 4.29 (m, 0.3H), 4.20-4.08 (m, 1H), 3.71 (s, 6H), 3.62-3.42 (m, 1H), 3.36-3.14 (m, 2H), 3.04-2.96 (m, 2H), 2.56-2.44 (m, 2H), 2.24-2.10 (m, 2H), 2.16-1.80 (m, 3H) 1.54-1.05 (m, 7H). 13C NMR DMSO-d6) 171.06, 170.94, 158.11, 157.99, 145.11, 144.79, 135.87, 135.76, 135.50, 135.42, 129.61, 129.58, 128.88, 128.19, 127.88, 127.77, 127.59, 126.74, 126.58, 125.30, 113.19, 113.09, 85.78, 85.12, 79.20, 68.58, 67.45, 65.23, 63.36, 59.76, 55.60, 55.05, 55.01, 54.98, 54.91, 53.46, 41.52, 38.04, 36.31, 34.25, 33.17, 32.60, 26.22, 26.17, 24.77, 24.41, 22.61, 21.03, 20.74, 14.07. MS. Molecular weight calculated for C32H40N2O5, Cal. 532.67, Found























General Procedure for the Synthesis of Amides 102
The carboxylic acid 101 (10 mmol) and HBTU (4.2 g, 1.1 eq.) were dissolved in DMF (30 mL) under argon. DIEA (3.81 g, 30 mmol) was added and stirred for 5 minutes. To this solution amine 100 (6.10 g, 10.7 mmol) was added and the reaction mixture was stirred at room temperature overnight. The progress of the reaction was monitored by TLC (10% MeOH/DCM). Ice-cold water was added and extracted with ethyl acetate, washed with NaHCO3 solution, water and brine. Solvents were removed and residue was purified by chromatography (during the packing of column please add few drops of TEA, First elute with ethyl acetate, followed by 5% MeOH/DCM) to get the product.
Preparation of Adamentane carboxylic acid (6-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-amide 102a: Using the above procedure adamentane carboxylic acid (1.80 g, 10 mmol) was coupled with the amine 100 (6.00 g, 11.27 mmol) using HBTU (4.55 g, 1.2 eq.) and DIEA (5.21 mL, 3.00 eq). After work-up and column purification the pure amide 102a was isolated as a white solid (Yield, 6.70 g, 96%). 1H NMR (DMSO-d6, 400 MHz) δ=7.33-7.25 (m, 5H), 7.23-7.13 (m, 5H), 6.87-6.80 (m, 4H), 4.97 (d, J=3.90 Hz, 1H), 4.88 (d, J=3.90 Hz, 1H), 4.39-4.05 (m, 2H), 3.71 (s, 6H), 3.58-3.41 (m, 1H), 3.32-3.12 (m, 2H), 3.01-2.93 (m, 3H), 2.20-1.05 (m, 25H). 13C NMR (DMSO-d6, 400 MHz) δ=171.81, 170.91, 170.80, 16=58.09, 157.96, 145.06, 144.71, 135.86, 135.71, 135.45, 135.41, 129.67, 129.57, 129.52, 129.52, 127.80, 127.69, 127.63, 127.56, 126.52, 113.14, 113.05, 85.79, 85.12, 68.59, 67.46, 63.34, 55.06, 55.01, 54.96, 54.96, 54.93, 38.27, 38.24, 36.31, 35.43, 34.17, 30.89, 29.08, 29.03, 28.73, 28.68, 28.59, 26.63, 26.60, 26.20, 25.33, 25.19, 24.14, 22.02, 21.97, 13.87
Preparation of Dodecanoic acid (6-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-amide 102b: Using the above procedure Dodecanoic acid (2.00 g, 10 mmol) was coupled with the amine 100 (6.10 g, 11.45 mmol) using HBTU (4.54 g, 1.2 eq.) and DIEA (5 mL, 3.00 eq). After work-up and column purification the pure amide 102b was isolated as a pale yellow solid (Yield, 5.2 g, 71%). 1H NMR (DMSO-d6, 400 MHz) δ=7.73-7.68 (m, 1H), 7.42-7.31 (m, 4H), 7.27-7.09 (m, 5H), 6.91-6.80 (m, 4H), 4.97 (d, J=3.90 Hz, 1H), 4.94 (d, J=3.90 Hz, 1H), 4.41-4.05 (m, 2H), 3.71 (s, 6H), 3.59-3.41 (m, 1H), 3.35-3.11 (m, 2H), 3.05-2.91 (m, 3H), 2.22-1.78 (m, 6H), 1.51-1.0 (m, 24H), 0.82 (t, J=6.34 Hz, 3H). 13C NMR (DMSO-d6, 400 MHz) δ=171.85, 170.93, 170.83, 158.10, 157.97, 145.09, 144.75, 135.87, 135.73, 135.47, 135.42, 129.60, 129.55, 127.85, 127.78, 127.57, 126.63, 126.53, 113.18, 113.09, 85.78, 85.11, 68.58, 67.45, 63.33, 55.60, 54.99, 54.96, 53.43, 38.21, 38.02, 36.30, 35.43, 34.17, 32.54, 31.31, 29.09, 29.03, 29.01, 28.97, 28.79, 28.74, 28.67, 26.19, 25.33, 24.55, 24.15, 22.10, 21.21, 13.94.
Preparation of myristic acid (6-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-amide 102c: Using the above procedure Myristic acid (2.30 g, 10 mmol) was coupled with the amine 100 (6.10 g, 11.45 mmol) using HBTU (4.54 g, 1.2 eq.) and DIEA (5 mL, 3.00 eq). After work-up and column purification the pure amide 102c was isolated as a pale yellow solid (Yield, 6.10 g, 80%). 1H NMR (DMSO-d6, 400 MHz) δ=7.72-7.65 (m, 1H), 7.35-7.26 (m, 4H), 7.27-7.12 (m, 5H), 6.90-6.80 (m, 4H), 4.99 (d, J=3.91 Hz, 1H), 4.89 (d, J=3.91 Hz, 1H), 4.41-4.10 (m, 2H), 3.71 (s, 6H), 3.57-3.42 (m, 1H), 3.37-3.11 (m, 2H), 3.05-2.91 (m, 3H), 2.25-1.80 (m, 6H), 1.52-1.08 (m, 28H), 0.83 (t, J=6.34 Hz, 3H). 13C NMR (DMSO-d6, 400 MHz) δ=171.84, 170.93, 170.82, 158.11, 157.98, 145.09, 144.75, 135.87, 135.72, 135.47, 135.42, 129.60, 129.55, 127.84, 127.75, 127.72, 126.56, 113.17, 113.08, 85.78, 85.12, 68.58, 67.45, 65.19, 63.33, 55.61, 55.06, 54.99, 53.44, 39.08, 38.87, 38.22, 38.02, 36.31, 35.44, 34.18, 32.54, 31.32, 29.09, 29.04, 28.99, 28.80, 28.74, 28.64, 26.20, 25.34, 24.55, 24.15, 22.11, 13.93.
Preparation of Palmitic acid (6-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-amide 102d: Using the above procedure Palmitic acid (2.57 g, 10 mmol) was coupled with the amine 100 (6.10 g, 11.45 mmol) using HBTU (4.54 g, 1.2 eq.) and DIEA (5 mL, 3.00 eq). After work-up and column purification the pure amide 102d was isolated as a white solid (Yield, 6.15 g, 79%). 1H NMR (DMSO-d6, 400 MHz) δ=7.74-7.66 (m, 1H), 7.37-7.22 (m, 4H), 7.21-7.12 (m, 5H), 6.87-6.79 (m, 4H), 4.98 (d, J=3.90 Hz, 1H), 4.90 (d, J=3.90 Hz, 1H), 4.40-4.07 (m, 2H), 3.71 (s, 6H), 3.59-3.42 (m, 1H), 3.34-3.13 (m, 2H), 3.02-2.87 (m, 3H), 2.20-1.80 (m, 6H), 1.46-1.08 (m, 32H), 0.82 (t, J=6.35 Hz, 3H). 13C NMR (DMSO-d6, 400 MHz) δ=171.84, 170.92, 170.81, 158.11, 157.98, 145.09, 144.74, 135.87, 135.72, 135.46, 135.41, 129.59, 129.55, 127.84, 127.74, 127.57, 126.55, 113.16, 113.07, 85.78, 85.12, 68.59, 67.45, 65.19, 63.33, 59.74, 55.61, 55.06, 54.95, 53.44, 44.58, 39.08, 38.87, 38.22, 38.02, 36.30, 35.44, 34.18, 32.54, 31.32, 29.09, 29.05, 29.00, 28.81, 28.74, 28.69, 26.20, 25.34, 24.55, 24.15, 22.12, 21.20, 14.06, 13.92.
Synthesis of octadecanoic acid (6-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-amide 102e. Using the above described procedure, stearic acid 101e (2.84 g, 10 mmol) was coupled with the amine 100 and after work-up and column purification the pure amide was isolated as a foam (7.17 g, 90%). 1H NMR (DMSO-d6, 400 MHz) δ=7.76-7.65 (m, 1H), 7.34-7.10 (m, 9H), 6.90-6.79 (m, 4H), 4.96 (d, J=3.90 Hz, 0.7H), 4.88 (d, J=3.90 Hz, 0.3H), 4.40-4.05 (m, 2H), 3.71 (s, 6H), 3.59-3.42 (m, 1H), 3.34-3.13 (m, 2H), 3.10-2.87 (m, 3H), 2.20-1.80 (m, 6H), 1.50-1.08 (m, 36H), 0.83 (t, J=6.1 Hz, 3H). 13C NMR (DMSO-d6, 400 MHz) δ=171.83, 170.92, 170.82, 162.29, 158.10, 157.97, 145.08, 144.74, 135.86, 135.42, 135.47, 129.59, 129.54, 127.84, 127.76, 127.57, 126.56, 113.18, 113.09, 85.78, 85.10, 68.57, 67.44, 65.19, 63.32, 54.97, 40.12, 38.21, 36.29, 35.77, 35.42, 34.16, 31.30, 30.75, 29.04, 28.78, 28.72, 28.65, 26.18, 25.33, 24.14, 22.10, 13.95.
Synthesis of octadec-9-enoic acid (6-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-amide 102f. Using the above described procedure, oleic acid 101f (2.83 g, 10 mmol) was coupled with the amine 100 and after work-up and column purification the pure amide was isolated as aa oil (7.7 g, 98%). 1H NMR (DMSO-d6, 400 MHz) δ 7.71-7.67 (m, 1H), 7.34-7.10 (m, 9H), 6.90-6.79 (m, 4H), 5.33-5.26 (m, 2H), 4.98 (d, J=3.90 Hz, 0.7H), 4.88 (d, J=3.90 Hz, 0.3H), 4.40-4.05 (m, 2H), 3.71 (s, 6H), 3.59-3.42 (m, 1H), 3.34-3.20 (m, 2H), 3.18-3.10 (m, 1H), 3.02-2.92 (m, 3H), 2.20-1.80 (m, 6H), 1.50-1.04 (m, 39H), 0.83 (t, J=6.1 Hz, 3H). 13C NMR (DMSO-d6, 400 MHz) δ=171.81, 170.92, 170.83, 158.09, 157.96, 145.08, 144.74, 135.87, 135.72, 135.42, 129.61, 129.54, 127.86, 127.74, 127.57, 126.56, 113.18, 113.09, 85.78, 85.10, 68.57, 67.44, 65.19, 63.32, 54.97, 54.92, 48.59, 45.71, 40.12, 38.21, 38.02, 36.29, 35.41, 34.16, 32.53, 31.28, 29.09, 28.84, 28.69, 28.66, 28.59, 28.55, 26.58, 26.56, 26.19, 25.32, 24.54, 24.14, 22.10, 13.94.
Preparation of Linoleic acid (6-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-amide 102g: Using the above procedure Linoleic acid (2.80 g, 10.5 mmol) was coupled with the amine 100 (6.10 g, 11.45 mmol) using HBTU (4.55 g, 1.2 eq.) and DIEA (5.21 mL, 3.00 eq). After work-up and column purification the pure amide 102g was isolated as pale yellow gummy liquid (Yield, 6.10 g, 78%). 1H NMR (DMSO-d6, 400 MHz) δ=7.71-7.67 (m, 1H), 7.33-7.25 (m, 4H), 7.23-7.13 (m, 5H), 6.87-6.80 (m, 4H), 5.35-5.24 (m, 4H), 4.97 (d, J=3.90 Hz, 1H), 4.88 (d, J=3.90 Hz, 1H), 4.39-4.05 (m, 2H), 3.72 (s, 6H), 3.58-3.41 (m, 1H), 3.32-3.12 (m, 2H), 3.01-2.93 (m, 3H), 2.72-2.67 (m, 2H), 2.20-1.80 (m, 9H), 1.46-1.08 (m, 23H), 0.83 (t, J=6.34 Hz, 3H). 13C NMR (DMSO-d6, 400 MHz) δ=176.63, 170.97, 170.86, 162.22, 158.09, 157.97, 145.07, 144.73, 135.87, 135.73, 135.48, 135.43, 129.60, 129.57, 129.55, 127.81, 127.71, 127.57, 127.55, 126.69, 126.53, 113.15, 113.07, 113.06, 85.79, 85.12, 68.61, 67.47, 63.36, 55.07, 55.01, 54.96, 54.93, 38.60, 38.56, 38.29, 38.18, 38.02, 36.30, 36.16, 36.01, 35.71, 34.17, 32.55, 30.70, 29.06, 29.02, 27.73, 27.70, 27.53, 26.08, 26.04, 24.49, 24.10.
Synthesis of docosanoic acid (6-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-amide 102h. Using the above described procedure, docosanoic acid 101h (3.41 g, 10 mmol) was coupled with the amine 100 and after work-up and column purification the pure amide was isolated as a foam (3.5 g, 41%). 1H NMR (DMSO-d6, 400 MHz) δ=7.69 (m, 1H), 7.32-6.80 (m, 13H), 4.98 (d, J=3.90 Hz, 0.7H), 4.88 (d, J=3.90 Hz, 0.3H), 4.40-4.05 (m, 2H), 3.71 (s, 6H), 3.59-3.42 (m, 1H), 3.34-3.13 (m, 2H), 3.10-2.87 (m, 3H), 2.20-1.80 (m, 6H), 1.50-1.08 (m, 44H), 0.81 (t, J=6.1 Hz, 3H). 13C NMR (DMSO-d6, 400 MHz) δ=171.78, 170.89, 170.79, 162.30, 162.25, 158.09, 157.97, 145.08, 144.73, 135.86, 135.70, 135.45, 135.40, 129.59, 129.53, 127.82, 127.72, 127.56, 126.69, 126.52, 113.14, 113.05, 85.78, 85.10, 68.58, 67.44, 65.19, 63.32, 55.60, 55.05, 54.96, 54.93, 38.22, 38.01, 36.30, 35.76, 35.43, 34.16, 32.53, 31.35, 30.74, 29.09, 28.87, 28.78, 28.73, 26.20, 25.35, 24.13, 22.13, 13.88.
Synthesis of lithocholic acid methyl ester 3
To a solution of the lithocholic acid 1 (25.1 g, 66.4 mmol) in anhydrous methanol (100 mL), 4M HCl in 1,4-dioxane (100 mL) was added and the reaction mixture stirred at ambient temperature overnight. The reaction mixture became heterogeneous. The reaction mixture was diluted with dichloromethane (300 mL) and the organic layer was washed with saturated NaHCO3 (2×200 mL). The organic layer was concentrated and dried under high vacuum overnight to provide the pure ester as a white solid (25.7 g, 97%). The analytical and spectral data were consistent with that of reported values (Rensen, Patrick C. N.; van Leeuwen, Steven H.; Sliedregt, Leo A. J. M.; Van Berkel, Theo J. C.; Biessen, Erik A. L. Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asialoglycoprotein Receptor. Journal of Medicinal Chemistry (2004), 47(23), 5798-5808.).
Synthesis of 3-octadec-9-enylcarbamoyloxy-lithocholic acid methyl ester 4
To a solution of the lithocholic acid methyl ester 3 (13.9 g, 35.6 mmol) in anhydrous dichloromethane (200 mL), triethylamine (10 mL) and disuccinimoyldicarbonate (13.7 g, 53.4 mmol) were added and the reaction mixture was stirred at ambient temperature overnight. The reaction mixture was then concentrated to dryness and the residue was re-dissolved in dichloromethane (300 mL). The organic layer was washed with saturated NaHCO3 (3×100 mL). The organic layer was dried (Na2SO4), filtered and evaporated to dryness to obtain the succinimoylcarbonate of lithocholic acid methyl ester as a white foam. The oleyl amine (9.5 g, 35.6 mmol) was dissolved in anhydrous dichloromethane (50 mL) and cooled to 0° C. To this mixture anhydrous pyridine (10 mL) and the above carbonate in anhydrous CH2Cl2 (20 mL) was added. The reaction mixture was allowed to warm to ambient temperature and stirred for 4 h. The TLC showed the completion of the reaction and the reaction mixture was diluted with CH2Cl2 (200 mL) and washed with saturated NaHCO3, brine and dried over anhyd. Na2SO4. The organic layer was concentrated and dried under high vacuum overnight to provide the crude product which was further purified by column chromatography (silica gel, 1:1 EtOAc:hexanes) to obtain the pure product as a white foam (13.7 g, 55%). 1H NMR (DMSO-d6, 400 MHz) δ 6.92 (t, J=5.2 Hz, 1H), 5.34-5.29 (m, 2H), 4.45-4.41 (m, 1H), 3.55 (s, 6H), 2.93-2.88 (m, 2H), 2.34-2.16 (m, 2H), 1.97-1.40 (m, 14H), 1.40-1.10 (m, 40H), 1.10-0.90 (m, 6H), 0.90-0.80 (m, 9H), 0.59 (s, 3H). 13C NMR (DMSO-d6, 400 MHz) δ=173.38, 168.34, 155.47, 149.82, 129.28, 128.85, 128.79, 81.65, 77.43, 72.98, 55.49, 55.29, 54.87, 54.82, 50.39, 41.68, 40.87, 40.75, 34.72, 34.67, 34.26, 34.13, 33.66, 33.56, 33.47, 31.78, 31.53, 30.81, 30.75, 30.69, 29.95, 29.91, 28.96, 28.67, 28.60, 28.56, 28.51, 28.42, 28.40, 28.24, 28.19, 28.14, 28.04, 27.86, 27.14, 26.12, 26.06, 25.89, 25.76, 25.37, 25.25, 25.21, 24.62, 23.16, 22.44, 22.19, 21.61, 19.78, 17.32, 13.22, 11.08.
Synthesis of 3-octadec-9-enylcarbamoyloxy-lithocholic acid 101j
To a solution of the ester 4 (13.7 g, 20 mmol) in THF (100 mL) at 0° C., an aqueous solution of lithium hydroxide [2.52 g (60 mmol) in 10 mL of water] was added and the reaction mixture was stirred at ambient temperature for 20 h after which the TLC showed the complete disappearance of the starting ester. The reaction mixture was acidified to pH 6 by drop wise addition of acetic acid and the reaction mixture was diluted with water (100 mL) and the mixture was extracted with EtOAc (3×100 mL) and the organic layers were combined, washed with water (200 mL), brine (200 mL) and dried over Na2SO4. Concentration of the organic layer provided the pure product as a foam (10.6 g, 80%). 1H NMR (CDCl3, 400 MHz) δ 11.15 (bs, 1H), 5.44-5.25 (m, 2H), 4.58 (bs, 2H), 3.22-3.04 (m, 2H), 2.43-2.21 (m, 2H), 2.06-1.20 (m, 14H), 1.40-1.10 (m, 40H), 1.10-0.90 (m, 6H), 0.96-0.84 (m, 9H), 0.64 (s, 3H). 13C NMR (DMSO-d6, 400 MHz) δ=179.98, 156.50, 130.18, 130.01, 76.91, 74.78, 56.72, 56.19, 42.96, 42.09, 41.13, 40.64, 40.37, 36.00, 35.52, 35.28, 34.77, 32.88, 32.82, 32.12, 32.00, 31.17, 30.99, 30.21, 29.98, 29.95, 29.91, 29.87, 29.74, 29.66, 29.53, 29.47, 28.44, 28.39, 27.43, 27.40, 27.24, 26.96, 26.56, 24.39, 23.54, 22.90, 21.03, 18.46, 14.35, 12.26.
Synthesis of 3-(tert-butyl-diphenyl-silyloxy)-lithocholic acid methyl ester 2
To a solution of the lithocholic acid methyl ester 3 (11.72 g, 30 mmol) in anhydrous acetonitrile (100 mL), imidazole (4.1 g, 60 mmol) was added and the solution was cooled in an ice bath. To this cold solution, TBDPS chloride was added drop wise with stirring. The reaction mixture was allowed to warm ton ambient temperature and stirred overnight. The TLC of the reaction mixture (5% EtOAc in hexanes) showed the complete disappearance of the starting alcohol. The reaction mixture was concentrated, the residue was quenched with water (200 mL) and extracted with CH2Cl2 (3×100 mL). The combined organic layers were washed with brine and concentrated to get the crude product. The thus obtained crude product was purified by column (silica gel) chromatography using 0-5% EtOAc in hexanes. The pure product fractions were concentrated to obtain the product as a clear liquid (19.6 g, 100%). 1H NMR (DMSO-d6, 400 MHz) δ 7.34-7.25 (m, 4H), 7.25-7.15 (m, 4H), 6.88-6.82 (m, 2H), 5.30 (t, J=4.6 Hz, 1H), 3.31 (s, 3H), 2.34-2.16 (m, 2H), 1.90-0.97 (m, 27H), 0.92 (s, 3H), 0.90 (m, 3H), 0.64 (s, 3H).
Synthesis of 3-(tert-butyl-diphenyl-silyloxy)-lithocholic acid 101i
To a solution of the ester 2 (19.6 g, 31 mmol) in THF (100 mL) at 0° C., an aqueous solution of lithium hydroxide [6.5 g (155 mmol) in 20 mL of water] was added and the reaction mixture was stirred at ambient temperature for 20 h after which the TLC showed the complete disappearance of the starting ester. The reaction mixture was acidified to pH 6 by drop wise addition of acetic acid and the reaction mixture was diluted with water (100 mL) and the mixture was extracted with EtOAc (3×100 mL) and the organic layers were combined, washed with water (200 mL), brine (200 mL) and dried over Na2SO4. Concentration of the organic layer provided the pure product as a foam (18.8 g, 98%). 1H NMR (DMSO-d6, 400 MHz) δ 11.95 (bs, 1H), 7.80-7.15 (m, 10H), 3.53 (bs, 1H), 2.34-2.16 (m, 2H), 1.90-0.90 (m, 27H), 0.94 (s, 3H), 0.74 (m, 3H), 0.55 (s, 3H).
Synthesis of 3-didodecylcarbamoyloxy-lithocholic acid methyl ester 5
In a procedure similar to that of the carbamate 4 lithocholic acid methyl ester 2 (3.76 g, 10 mmol) was converted to the corresponding carbamate 5. After work-up and column purification, the pure product was isolated as a colorless oil (6.4 g, 83%). 1H NMR (CDCl3, 400 MHz) δ 4.62 (m, 1H), 3.55 (s, 6H), 3.20-3.06 (m, 2H), 2.34-2.16 (m, 2H), 1.97-1.40 (m, 14H), 1.40-1.10 (m, 40H), 1.10-0.90 (m, 6H), 0.90-0.80 (m, 9H), 0.59 (s, 3H). 13C NMR (CDCl3, 400 MHz) δ=175.04, 156.31, 72.98, 56.69, 56.23, 51.71, 42.95, 42.17, 40.68, 36.02, 35.59, 34.82, 32.15, 31.29, 29.88, 29.82, 29.59, 28.41, 28.40, 28.24, 28.19, 28.14, 28.04, 23.61, 22.92, 18.49, 14.35, 12.25
Synthesis of 3-didodecylcarbamoyloxy-lithocholic acid 101k
To a solution of the ester 5 (6.4 g, 8.3 mmol) in THF (50 mL) at 0° C., an aqueous solution of lithium hydroxide [0.42 g (10 mmol) in 5 mL of water] was added and the reaction mixture was stirred at ambient temperature for 20 h after which the TLC showed the complete disappearance of the starting ester. The reaction mixture was acidified to pH 6 by drop wise addition of acetic acid and the reaction mixture was diluted with water (100 mL) and the mixture was extracted with EtOAc (3×100 mL) and the organic layers were combined, washed with water (200 mL), brine (200 mL) and dried over Na2SO4. Concentration of the organic layer provided the pure product as a foam (6 g, 95%). 1H NMR (CDCl3, 400 MHz) δ 11.35 (bs, 1H), 4.62 (m, 1H), 3.55 (s, 6H), 3.20-3.06 (m, 2H), 2.34-2.16 (m, 2H), 1.97-1.40 (m, 14H), 1.40-1.10 (m, 40H), 1.10-0.90 (m, 6H), 0.90-0.80 (m, 9H), 0.59 (s, 3H). 13C NMR (CDCl3, 400 MHz) δ=175.04, 156.31, 72.98, 56.69, 56.23, 51.71, 42.95, 42.17, 40.68, 36.02, 35.59, 34.82, 32.15, 31.29, 29.88, 29.82, 29.59, 28.41, 28.40, 28.24, 28.19, 28.14, 28.04, 23.61, 22.92, 18.49, 14.35, 12.25.
Synthesis of 3-(tert-butyl-diphenyl-silyloxy)-lithocholic acid (6-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-amide 102i. Using the above described procedure, 3-(tert-butyl-diphenyl-silyloxy)-lithocholic acid 101i (2.46 g, 4 mmol) was coupled with the amine 100 and after work-up and column purification the pure amide was isolated as an oil (3.2 g, 70%). 1H NMR (DMSO-d6, 400 MHz) δ 7.71 (bs, 1H), 7.30-7.15 (m, 19H), 6.98-6.82 (m, 4H), 5.33 (bs, 1H), 5.30 (t, J=4.6 Hz, 1H), 4.98 (d, J=3.9 Hz, 0.7H), 4.90 (d, J=3.9 Hz, 0.3H), 4.46-4.14 (m, 1H), 3.71 (s, 3H), 3.60-3.15 (m, 2H), 3.10-2.88 (m, 2.34-2.16 (m, 2H), 1.90-0.90 (m, 27H), 0.94 (s, 3H), 0.74 (m, 3H), 0.55 (s, 3H). 13C NMR (DMSO-d6) δ 172.25, 170.95, 170.85, 162.32, 158.10, 157.98, 155.80, 145.09, 144.75, 135.89, 135.73, 135.47, 129.62, 127.78, 127.58, 126.59, 113.10, 85.12, 72.99, 68.58, 63.33, 56.00, 55.58, 54.99, 45.71, 42.24, 41.24, 38.25, 36.30, 35.79, 35.30, 34.92, 34.65, 34.16, 32.43, 31.64, 31.32, 30.77, 29.38, 29.11, 28.87, 28.82, 28.74, 28.61, 28.52, 26.58, 26.18, 24.17, 23.83, 23.07, 22.13, 20.38, 18.27, 13.96, 11.85. MS. Molecular weight calculated for C72H96N2O7Si, Cal. 1128.7°, Found
Synthesis of 3-octadec-9-enylcarbamoyloxy-lithocholic acid (6-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-amide 102j. Using the above described procedure, 3-octadec-9-enylcarbamoyloxy-lithocholic acid 101j (2.68 g, 4 mmol) was coupled with the amine 100 and after work-up and column purification the pure amide 102j was isolated as an oil (4.45 g, 95%). 1H NMR (DMSO-d6, 400 MHz) δ 7.94 (bs, 1H), 7.58 (dd, J=1.3 and 7.65 Hz, 4H), 7.45-7.12 (m, 15H), 6.90-6.82 (m, 4H), 4.98 (d, J=3.9 Hz, 0.7H), 4.89 (d, J=3.9 Hz, 0.3H), 4.46-4.14 (m, 2H), 3.71 (s, 3H), 3.60-3.15 (m, 2H), 3.10-2.88 (m, 2.34-2.16 (m, 2H), 1.90-0.90 (m, 42H), 0.96 (s, 3H), 0.86-0.80 (m, 3H), 0.74 (m, 3H), 0.55 (s, 3H). 13C NMR (DMSO-d6) δ 172.23, 170.91, 170.82, 158.09, 157.96, 145.07, 144.72, 135.87, 135.71, 135.45, 135.40, 135.19, 134.10, 134.01, 129.69, 129.58, 129.52, 127.84, 127.75, 127.68, 127.56, 126.73, 126.56, 113.16, 113.08, 85.77, 85.10, 73.11, 68.57, 67.44, 65.17, 63.32, 55.81, 55.61, 55.05, 54.98, 54.95, 42.21, 41.37, 38.21, 38.01, 36.30, 36.14, 35.25, 34.90, 34.82, 32.50, 31.69, 30.38, 29.06, 27.72, 26.78, 26.16, 25.98, 24.54, 24.13, 23.77, 22.96, 20.40, 18.66, 18.28, 11.81.
Synthesis of 3-didodecylcarbamoyl-lithocholic acid (6-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-amide 102k. Using the above described procedure, 3-didodecylcarbamoyl-lithocholic acid 101k (2.68 g, 4 mmol) was coupled with the amine 100 and after work-up and column purification the pure amide was isolated as an oil (4.45 g, 95%). 1H NMR (CDCl3, 400 MHz) δ 7.45-7.12 (m, 9H), 6.90-6.82 (m, 4H), 5.90 (d, J=3.9 Hz, 0.7H), 5.81 (d, J=3.9 Hz, 0.3H), 4.46-4.14 (m, 2H), 3.71 (s, 3H), 3.60-3.15 (m, 2H), 3.10-2.88 (m, 6H), 2.34-2.16 (m, 9H), 1.90-0.90 (m, 56H), 0.96 (s, 3H), 0.86-0.80 (m, 9H), 0.74 (m, 3H), 0.55 (s, 3H).
General Procedure for the Synthesis of Succinates 103
To a solution of the alcohol 102 (3 mmol) in anhydrous dichloromethane (100 mL), DMAP (1.1 g, 9 mmol) was added followed by the addition of succinic anhydride (0.6 g, 6 mmol). The reaction mixture was stirred at room temperature overnight. The TLC of the reaction mixture showed the complete disappearance of the starting alcohol. The reaction mixture was cooled in ice and to it few drops of NEt3 was added. The reaction mixture was washed with a ice-cold solution of 10% aqueous citric acid (2×50 mL) followed by water, brine and dried over anhydrous sodium sulfate. Concentration of the organic layer provided the product 103 which was used as such in the next step with out any further purification.
Synthesis of succinic acid mono-{5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-(6-octadecanoylamino-hexanoyl)-pyrrolidin-3-yl}ester 103e. Using the above described procedure, the amide 102e (2.4 g, 3 mmol) was treated with succinic anhydride and after work-up the amide was isolated as a foam (2.69 g, 99%) and this was used in the next step without further purification.
Synthesis of succinic acid mono-{5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-(6-octadec-9-enoylamino-hexanoyl)-pyrrolidin-3-yl}ester 103f. Using the above described procedure, the amide 102f (2.4 g, 3 mmol) was treated with succinic anhydride and after work-up the amide was isolated as a foam (2.8 g, 99%) and this was used in the next step without further purification.
Synthesis of succinic acid mono-{5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-(6-docosanoylamino-hexanoyl)-pyrrolidin-3-yl}ester 103h. Using the above described procedure, the amide 102h (2.56 g, 3 mmol) was treated with succinic anhydride and after work-up the amide was isolated as a foam (2.8 g, 99%) and this was used in the next step without further purification.
Synthesis of succinic acid 103i. Using the above described procedure, the amide 102i (2.82 g, 2.5 mmol) was treated with succinic anhydride and after work-up the amide was isolated as a foam (3 g, 99%) and this was used in the next step without further purification.
Synthesis of succinic acid 103j. Using the above described procedure, the amide 102j (2.36 g, 2 mmol) was treated with succinic anhydride and after work-up the amide was isolated as a foam (2.56 g, 99%) and this was used in the next step without further purification.
Synthesis of succinic acid 103k. Using the above described procedure, the amide 102k (2.8 g, 2.2 mmol) was treated with succinic anhydride and after work-up the amide was isolated as a foam (3 g, 99%) and this was used in the next step without further purification.
Preparation of Long alkyl chain CPG 104a: Hydroxy derivative 102a (Adamentane carboxylic acid) (2.58 g, 3.71 mmol) was dissolved in DCM (20 mL) to that Succinic anhydride (0.742 g, 2 eq) and DMAP (1.36 g, 3 eq.) were added and stirred overnight. TLC showed completion of reaction. The reaction mixture was diluted with DCM (20 mL), washed successively with cold dilute citric acid and water (2 times), dried over sodium sulfate. Solvents were removed and dried under high vacuum to get the succinate 103a. PPh3 (1.073 g, 1.1 eq.), DMAP (0.498 g, 1.1 eq.) and the succinate from the previous step were dissolved in a mixture of acetonitrile and DCM (20 mL). A solution of DTNP (1.208 g, 1.05 eq.) in DCM (8 mL) was added to the above solution. The mixture was slowly shaken for 3-4 minutes. Long chain alkyl amine-CPG (lcaa CPG, 20.0 g, 133 μmol/g) was added to the mixture and gently shaken for 2 h. The CPG was filtered, successively washed with DCM, mixture of MeOH/DCM (1:9) and DCM until filtrate remained colorless and dried. The dried CPG was transferred into another flask treated with Ac2O in pyridine (25%) in the presence of TEA (1 mL) for 15 min. under gentle shaking. Finally the CPG was filtered, washed with DCM, DCM:MeOH (9:1), followed by DCM and ether. The CPG 104a was dried under vacuum overnight and the loading was measured as reported (20.51 g, loading 69 μmol/g).
Preparation of Long alkyl chain CPG 104b: Compound 102b (2.02 g, 2.76 mmol) was dissolved in DCM (20 mL) to that Succinic anhydride (0.556 g, 2 eq) and DMAP (1.02 g, 3 eq.) were added and stirred overnight. The reaction mixture was diluted with DCM (20 mL), washed with cold dilute citric acid and water (2 times), dried over sodium sulfate. Solvents were removed and residue was directly used for next reaction with out further purification. Compound 103b from the previous step and HBTU (1.25 g, 1.2 eq.) were dissolved in a mixture of DCM/DMF (1:1) to that DIEA (1.5 mL) was added and kept for 5 minutes. Long chain alkyl amine-CPG (lcaa CPG, 13.30 g, 133 μmol/g) was added to above solution and shaken for 2 hrs. CPG was filtered off, washed with DMF, a mixture of DCM/MeOH, DCM and dried. CPG was transferred to the same flask and treated with 25% Ac2O in Py (with small amount of TEA) and shaken well for 1⁄2 h. The solid was filtered off, washed with DCM, a mixture of DCM/MeOH, DCM and diethyl ether. CPG was transferred to a bottle and dried overnight under high vacuum and the loading was measured as reported (Yield=13.63 g, Loading 53 μmol/g).
Preparation of Long alkyl chain CPG 104c: Compound 102 (2.16 g, 2.84 mmol) was dissolved in DCM (20 mL) to that Succinic anhydride (0.571 g, 2 eq) and DMAP (1.03 g, 3 eq.) were added and stirred overnight. The reaction mixture was diluted with DCM, washed with cold dilute citric acid and water (2 times), dried over sodium sulfate. Solvents were removed and residue was directly used for next reaction with out further purification. Compound 103c from the previous step and HBTU (1.28 g, 1.2 eq.) were dissolved in a mixture of DCM/DMF (1:1) to that DIEA (1.5 mL, 3 eq) was added and kept for 5 minutes. Long chain alkyl amine-CPG (lcaa CPG, 13.40 g, 133 μmol/g) was added to above solution and shaken for 2 hrs. CPG was filtered off, washed with DMF, a mixture of DCM/MeOH, DCM and dried. CPG was transferred to the same flask and treated with 25% Ac2O in Py (with small amount of TEA) and shaken well for 1⁄2 h. The solid was filtered off, washed with DCM, a mixture of DCM/MeOH, DCM and diethyl ether. CPG was transferred to a bottle and dried overnight under high vacuum and the loading was measured as reported (Yield=13.69 g, Loading 47.27 μmol/g).
Preparation of Long alkyl chain CPG 104d: Compound 102d (2.08 g, 2.64 mmol) was dissolved in DCM (20 mL) to that Succinic anhydride (0.532 g, 2 eq) and DMAP (0.966 g, 3 eq.) were added and stirred overnight. The reaction mixture was diluted with DCM (20 mL), washed with cold dilute citric acid and water (2 times), dried over sodium sulfate. Solvents were removed and residue was directly used for next reaction with out further purification. Compound 103d from the previous step and HBTU (1.20 g, 1.2 eq.) were dissolved in a mixture of DCM/DMF (1:1) to that DIEA (1.5 mL) was added and kept for 5 minutes. Long chain alkyl amine-CPG (lcaa CPG, 14.2 g, 133 μmol/g) was added to above solution and shaken for 2 hrs. CPG was filtered off, washed with DMF, a mixture of DCM/MeOH, DCM and dried. The dried CPG was transferred into another flask treated with Ac2O in pyridine (25%) in the presence of TEA (1 mL) for 15 min. under gentle shaking. The solid was filtered off, washed with DCM, a mixture of DCM/MeOH, DCM and diethyl ether. CPG was transferred to a bottle and dried overnight under high vacuum and the loading was measured as reported (Yield=14.05 g, Loading 39.2 μmol/g).
General Procedure for the Synthesis of CPG's 104e, 104f, 104h, 104i, 104j and 104k
The succinic acids 103 (3 mmol) was dissolved in anhydrous DMF (30 mL) and to it diisopropylethylamine (10 eq.) was added followed by HATU (1.3 eq.) and the mixture was stirred for 5 minutes. To this solution CPG-NH2 (lcaa CPG, 133 μmol/g) was added and the resulting mixture was shaken in a shaker for 2 h. The residue was filtered and washed with 20 mL of DMF, 200 mL of dichloromethane, 400 mL of 5% MeOH in dichloromethane and 300 mL of dichloromethane. The thus obtained solid was dried and the powder was transferred to a RB flask and to it capping agent (30 mL, of 25% acetic anhydride in pyridine) was added and the resulting heterogeneous mixture was again shaken in the shaker for 1 h. The mixture was then filtered and washed with 200 mL of CH2Cl2, 100 mL of 5% MeOH in CH2Cl2, 200 mL of CH2Cl2 and 200 mL of anhydrous ether. The residue was dried under vacuo overnight to get a free flowing powder. The loading was calculated by measuring the absorption in a DU 800 spectrophotometer as reported.a 
The CPG 104e was synthesized using the above procedure and treatment of 2.69 g of 103e with 15 g of CPG provided 15.67 g of product and 61 μM/g loading was observed.
The CPG 104f was synthesized using the above procedure and treatment of 2.83 g of 103f with 1°g of CPG provided 19.3 g of product and 70 μM/g loading was observed.
The CPG 104h was synthesized using the above procedure and treatment of 2.75 g of 103h with 15 g of CPG provided 15.85 g of product and 68 μM/g loading was observed.
The CPG 104i was synthesized using the above procedure and treatment of 3 g of 103i with 1°g of CPG provided 10.4 g of product and 70 μM/g loading was observed.
The CPG 104j was synthesized using the above procedure and treatment of 2.56 g of 103j with 1°g of CPG provided 11.8 g of product and 25 μM/g loading was observed.
Example 2
Synthesis of siRNA-Lipophile Conjugates
Sense and antisense strands of control and corresponding 3′-lipophile conjugated sense strands were individually synthesized and purified according to the standard oligonucleotide synthesis and deprotection protocols. Commercially available 5′-O-(4,4′-dimethoxytrityl)-2′-O-t-butyldimethylsilyl-3′-O-(2-cyanoethyl-N,N-diisopropyl) RNA and the corresponding 2′-O-methyl phosphoramidite monomers of 6-N-benzoyladenosine (ABz), 4-N-acetylcytidine (CAc), 2-N-isobutyrylguanosine (GiBu), and uridine (U) were used for unmodified and 2′-O-methylsugar modified RNA synthesis. The 2′-deoxy-2′-fluoro sugar modified pyrimidines were introduced to the RNA by using 5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-2′-fluoro-3′-O-(2-cyanoethyl-N,N-diisopropyl) phosphoramidite monomers of 4-N-acetylcytidine (CAc) and uridine (U).
All lipophile bearing solid supports to conjugate the lipophiles to 3′-end of sense strand were synthesized as described in Example 1. Each individual lipophile conjugated sense strand was synthesized from the corresponding hydroxyprolinol-lipophile solid support. Beaucage reagent was used as an oxidant to obtain the phosphorothioate backbone modification. The lipophile conjugated sense strands were purified by reverse phase-high performance liquid chromatography (RP-HPLC) on an in-house packed Source 15RPC resin reverse-phase column. The buffers were 20 mM NaOAc in 10% CH3CN (buffer A) and 20 mM NaOAc in 70% CH3CN (buffer B). The unconjugated RNA oligonucleotides were purified by anion-exchange HPLC. Fractions containing full-length oligonucleotides were pooled, desalted and lyophilized. Analytical HPLC, CGE and ES LC-MS established the integrity of the compounds. For duplex generation, equimolar amounts of sense and antisense strand were heated in 1×PBS at 95° C. for 5 min and slowly cooled to room temperature. Annealing of equimolar mixture of sense and antisense strands in 1×PBS buffer afforded the desired siRNA.







TABLE 1







Lipophile conjugated siRNA duplexes.














SEQ ID



Target
Duplex
Sequence
NO:
Ligand














ApoB
1
5′-GUCAUCACACUGAAUACCAAUsHyp-NH2-3′
1
Amino-linker




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2



ApoB
2
5′-GUCAUCACACUGAAUACCAAUsHyp-L1-3′
3
C27; Cholesterol




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2



ApoB
3
5′-GUCAUCACACUGAAUACCAAUsHyp-L2-3′
4
C11;




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2
1-Adamentaneformyl


ApoB
4
5′-GUCAUCACACUGAAUACCAAUsHyp-L3-3′
5
C12; Lauroyl




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2



ApoB
5
5′-GUCAUCACACUGAAUACCAAUsHyp-L4-3′
6
C14; Myristoyl




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2



ApoB
6
5′-GUCAUCACACUGAAUACCAAUsHyp-L5-3′
7
C16; Palmitoyl




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2



ApoB
7
5′-GUCAUCACACUGAAUACCAAUsHyp-L6-3′
8
C18; Stearoyl




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2



ApoB
8
5′-GUCAUCACACUGAAUACCAAUsHyp-L7-3′
9
C18; Oleoyl




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2



ApoB
9
5′-GUCAUCACACUGAAUACCAAUsHyp-L8-3′
10
C18; Linoleoyl




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2



ApoB
10
5′-GUCAUCACACUGAAUACCAAUsHyp-L9-3′
11
C22; Docosanoyl




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2



ApoB
11
5′-GUCAUCACACUGAAUACCAAUsHyp-L10-3′
12
C43; Lithocholic-




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2
oleyl


ApoB
12
5′-GUCAUCACACUGAAUACCAAUsHyp-L11-3′
13
C24; Lithocholyl




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2



ApoB
13
5′-GUCAUCACACUGAAUACCAAUsHyp-L12-3′
14
C24 5β-Cholanyl




5′-AUTJGGUAUUCAGUGUGAUGAcsasC-3′
2



ApoB
14
5′-GUCAUCACACUGAAUACCAAUsHyp-L13-3′
15
C29; Vitamin E




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2



ApoB
15
5′-GUCAUCACACUGAAUACCAAUsHyp-L14-3′
16
C60; N,N-Disteary1-




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2
lithocholamide









Example 3
HPLC Titration of Hydrophobicity of Lipophile-siRNA Conjugates
Each sense strand bearing the ligand was individually injected to an analytical HPLC loaded with a reverse-phase C4 Delta Pack column (from Waters) and eluted under reverse-phase condition at 35° C. with a flow rate of 1.2 mL/min.: Grad: A(100%) 0-3 min and A(100%)->B(100%) from 3 to 43 min; A: 50 mM TEAA in H2O and B: 100% MeCN. The analysis of each duplex was performed under identical condition and the retention times of the duplexes were compared for relative hydrophobicity, shown in FIG. 3. Unconjugated and amino-linker bearing sense strands were used as reference.
Example 4
In Vivo siRNA Activity
Various siRNA conjugates were analyzed in an in vivo assay. The general study design for each of the siRNA conjugates is as provided below:
Female mice BL6, 8-10 weeks
5-6 Animals/group
Dosage 50 mg/kg once daily for 3 days, iv injection and sacrificed 24 h after last injection 100 mg/kg iv single injection and sacrificed 72 h after injection
Collect blood and harvest liver and jejunum
Liver and Jejunum apoB mRNA levels were evaluated in the following siRNA conjugates 3′ Chol (siRNA 2); 3′ Dodecyl (siRNA 4); 3′ Stearoyl (siRNA 7); 3′Docosanoyl (siRNA 10) and 3′Lithocholic-oleyl (siRNA 11). The results of the in vivo assay are provided in FIGS. 4A and 4B, for Liver and Jejunum respectively.
In vivo siRNA activity of lipophile-apoB-siRNA conjugates siRNA 2, siRNA 14, and siRNA 13 (Table 1 above) were evaluated. FIG. 5A depicts ApoB mRNA levels in the Jejunum, and FIG. 5B ApoB mRNA levels in the Liver. Both figures show mRNA levels after systemic administration of lipophile conjugated siRNA in vivo. Female BL6 mice 8-12 weeks old; 6 mice/group; single iv injection 100 mg/kg; sacrificed 72 h after injection: siRNA 2 (Chol-siRNA); siRNA 14 (VitaminE-siRNA) and siRNA 13 (5β-cholanyl-siRNA).
In vivo apoB gene silencing of PEGylated siRNA was evaluated in liver and jejunum, the results of which are depicted in FIGS. 6A (Liver) and 6B (Jejunum). The apoB mRNA levels were evaluated after systemic administration of PEG conjugated siRNA in vivo. Female BL6 mice 8-12 weeks old; 6 mice/group; single iv injection 100 mg/kg; sacrificed 72 h after injection: 5167 (Chol-siRNA, positive control); 3027 (PEG5000-siRNA) and 3028 (PEG20,000-siRNA).
In vivo liver apoB gene silencing of thiocholesterol conjugated siRNA was evaluated, the results of which are depicted in FIG. 7. Female BL6 mice 6-8 weeks old; 6 mice/group; iv injection 20 and 100 mg/kg; sacrificed 72 h after injection. siRNA 18, siRNA 16, siRNA 19, and siRNA 2, as depicted in the Table depicting exemplary siRNA-lipophile conjugates, Table 1, above, were used in this study.
In vivo liver apoB gene silencing of cholesterol, PEG, vitamin E and cholanic acid conjugated siRNAs were evaluated, the results of which are depicted in FIG. 8. Female C57/BL6 mice 6-8 weeks old; 6 mice/group; iv injection 100 mg/kg; sacrificed 72 h after injection. siRNA 2, siRNA 20, siRNA 21, siRNA14, and siRNA 13, as depicted in the Table 1 above, were used in this study.
In vivo jejunum apoB gene silencing of cholesterol, PEG, vitamin E and cholanic acid conjugated siRNAs were evaluated, the results of which are depicted in FIG. 9. Female C57/BL6 mice 6-8 weeks old; 6 mice/group; iv injection 100 mg/kg; sacrificed 72 h after injection. siRNA 2, siRNA 20, siRNA 21, siRNA 14, and siRNA 13, as depicted in the Table 1 above, were used in this study.
In vivo apoB gene silencing of cholesterol, dialkylglyceride and lithocholamide conjugated siRNAs were evaluated in liver and jejunum, the results of which are depicted in FIGS. 10A and 10B respectively. Female C57/BL6 mice 6-8 weeks old; 6 mice/group; iv injection. siRNA 2, siRNA 17, and siRNA 15, as depicted in the Table 1 above, were used in this study.
Relative apoB mRNA expression in liver was evaluated after treatment once daily for three days at 50 mg/kg of saline, siRNA 2, siRNA 5, and siRNA 6, as depicted in the Table 1 above. The results are depicted in FIG. 11. Relative apoB mRNA expression in jejunum was evaluated after treatment once daily for three days at 50 mg/kg of saline, siRNA 2, siRNA 5, and siRNA 6, as depicted in the Table above. The results are depicted in FIG. 12.
Example 5
siRNA-PEG Conjugate or Pegylated siRNA







TABLE 2







Oligonucleotide-PEG conjugate: Analytical data

















HPLC


Seq


MW
MW*
Retention


No
Sequence 5′-3′
Modifications
Expected
Obs.
time















3194
GCACAUAGGAGAGAUGACGUUs-HP-NH2 
Hydroxyprolinol amine
7107.46
7107.2
37.497



(SEQ ID NO: 17)






3195
GCACAUAGGAGAGAUGACGUUs-HP-NH2-
Hydroxyprolinol amine,
27213.19
27718-29656
31.283



20KPEG (SEQ ID NO: 18)
20K PEG





3164
GsUCAUCACACUGAAUACCAAU-HP-NH2 
Hydroxyprolinol amine
6932.33
6932.15
19.733



(SEQ ID NO: 19)






3170
GsUCAUCACACUGAAUACCAAU-HP-NH-5KPEG 
Hydroxyprolinol amine, 
11746.19
11906-13316
16.822



(SEQ ID NO: 20)
5K PEG





3171
GsUCAUCACACUGAAUACCAAU-HP-NH-20KPEG 
Hydroxyprolinol amine, 
26746.19
28116-2935
16.164



(SEQ ID NO: 21)
20K PEG





2936
NH2-HP-CUUACGCUGAGUACUUCGAdTsdT 
Hydroxyprolinol amine
6915.3
6915.01
20.506



(SEQ ID NO: 22)






3187
5KPEG-NH-HP-CUUACGCUGAGUACUUCGAdTsdT 
Hydroxyprolinol amine, 
12021.46
11847-13256
17.829



(SEQ ID NO: 23)
5K PEG





3188
20KPEG-NH-HP-CUUACGCUGAGUACUUCGAdTsdT 
Hydroxyprolinol amine, 
27021.46
27440-29289
16.921



(SEQ ID NO: 24)
20K PEG





2937
CsUUACGCUGAGUACUUCGAdTdT-HP-NH2 
Hydroxyprolinol amine
6915.3
6915.06
20.537



(SEQ ID NO: 25)






3172
CsUUACGCUGAGUACUUCGAdTdT-HP-NH-5KPEG 
Hydroxyprolinol amine, 
12021.46
11932-13342
17.578



(SEQ ID NO: 26)
5K PEG





3173
CsUUACGCUGAGUACUUCGAdTdT-HP-NH-20KPEG 
Hydroxyprolinol amine, 
27021.46
27967-29865
17.087



(SEQ ID NO: 27)
20K PEG









PEGylation of siRNA
General. The reagent solutions for RNA synthesis were made from products of the Aldrich Chemical Co. The phosphoramidites were products of Pierce Nucleic Acid Technologies. The hydroxyprolinol-phthalamideamine supports were synthesized in-house. The PEG was a product of NOF Corp. Ion exchange preparative chromatography was performed on TSKgel-SuperQ-5PW (Tosoh). Ion exchange analytical chromatography was performed on a DNAPac Pa100 (Dionex). Electron spray ionization mass spectra were recorded with an Agilent 1100 MSD-SL.
HPLC Techniques. The RNA was analyzed by ion-exchange chromatography (column, DNAPac Pa100, 4×250 mm, analytical), heated to 30° C., flow rate 1.5 mL min−1, buffer A=0.02°M Na2HPO4 in 10% CH3CN, pH 11; buffer B=buffer A+1 M NaBr in 10% CH3CN, pH 11, linear gradient from 0 to 75% in 53 min. The LC/ESI-MS conditions were as follows: column XTerra C8 (2.1×30 mm, 2.5 μm), linear gradient from 5 to 35% in 2 min and from 35 to 70% in 30.5 min, flow rate 0.200 mL min−1, buffer A=400 mM HFIP/16.3 mM TEA in H2O, buffer B=100% methanol. The RNA was purified by ion-exchange chromatography (5 cm in-house packed column, TSKgel-SuperQ-5PW, 20 μm), heated to 75° C., flow rate 50 mL min−1, buffer A=0.020M Na2HPO4 in 10% CH3CN, pH 8.5; buffer B=buffer A+1 M NaBr in 10% CH3CN, pH 8.5, linear gradient from 20 to 55% in 120 min.
RNA synthesis. The protected RNA was assembled on an AKTA Oligo Pilot 100 on a 100-150 μmol scale using custom in-house support and phosphoramidite chemistry. Phosphoramidites were used as 0.2 mol L−1 solutions in dry CH3CN, with a 900 s coupling time and the manufacturer's recommended synthesis protocols were used. After synthesis, the support-bound RNA was treated with aqueous CH3NH2 (40%) for 90 minutes at 45° C., cooled, filtered and washed with DMSO (3×40 mL). The filtrate was then treated with TEA.3HF (60 mL) for 60 minutes at 40° C., and quenched with aq. NaOAc (0.05M, pH 5.5, 200 mL). The synthesis was followed by analytical ion-exchange HPLC, preparative HPLC, then desalting on Sephadex G-25.
PEG Conjugation.
A) Initial reaction conditions. The purified and desalted RNA was lyophilized. RNA (1 mg) was dissolved in aq NaHCO3 (0.1M, 200L, pH 8.1) and DMF (200L each). 5 K (13 equivalents, 10 mg) or 20 KPEG (3.4 equivalents, 10 mg, Sunbright ME-20°HS, NOF Corp.) was added directly to reaction vial and vortexed thoroughly. The reaction was continued overnight at 4° C., and was followed by analytical ion-exchange HPLC. When the reaction reached >85% completion, it was quenched with aq. NaOAc (0.05M, pH 5.5) until the pH was  ̃7.
B) Borate buffer conjugation. The purified and desalted RNA was lyophilized. A sample of RNA (1 mg) was dissolved in sodium borate buffer (200 μL, 0.05M, pH10). 5KPEG (3 mg, 4.5 equivalents Sunbright ME-50HS, NOF Corp.) was dissolved in CH3CN (200 μL). The RNA solution was added to the PEG solution and vortexed thoroughly. The reaction continued for one hour at room temperature, and was followed by analytical ion-exchange HPLC. When reaction reached >85% completion, it was quenched with aq. NaOAc (0.05M, pH 5.5) until the pH was  ̃7.
C) PEG linker (AS and HS) comparison (Figure Exp-1). A sample of RNA (1 mg) was dissolved in aq, NaHCO3 (0.1M, 200L, pH 8.1) and DMF (200L). 5 KPEG (13.5 eq, 10 mg, Sunbright ME-5°HS or Sunbright ME-50AS, NOF Corp.) was added directly to the reaction vial and vortexed thoroughly. The reaction continued overnight at 4° C., and was followed by analytical ion-exchange HPLC. When the reaction reached >85% completion, it was quenched with aq. NaOAc (0.05M, pH 5.5) until the pH was  ̃7.
D) Final optimized PEG conjugation. The purified and desalted RNA was lyophilized. A sample of RNA (50 mg) was dissolved in aq. NaHCO3 (0.1M, 2 mL pH 8.1) and DMF (1 mL). 20KPEG (2.7 eq, 400-520 mg Sunbright ME-200HS, NOF Corp.) was dissolved in CH3CN (2 mL). The RNA solution was added to the PEG solution and vortexed thoroughly. H2O (250 mL) was added to the reaction to decrease turbidity. The reaction continued for one hour at room temperature, and was followed by analytical ion-exchange HPLC. When the reaction reached >85% completion, it was quenched with aq. NaOAc (0.05M, pH 5.5) until the pH was  ̃7.
PEG Starting Material Structure.




Structure of mPEG-NHS ester used for postsynthetic conjugation to siRNA
Exemplary PEGylated siRNAs are provided in Table 3 below:







TABLE 3







PEGylated siRNAs














SEQ ID



Target
Duplex
Sequence
No:
Ligand














ApoB
5167
5′-GUCAUCACACUGAAUACCAAUsHyp-Chol-3′
28
Cholesterol




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2



ApoB
3027
5′-GsUCAUCACACUGAAUACCAAU-Hyp-PEG5K
29
PEG-5K




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2



ApoB
3028
5′-GsUCAUCACACUGAAUACCAAU-Hyp-PEG20K
30
PEG-20K




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2



ApoB
3245
5′-GsUCAucAcAcuGAAuAccAAU-Hyp-PEG40K-3′
31
PEG-40K




5′-AUUGGUAUUCAGUGUGAUGAcsasC-3′
2



Luc
3246
5′-CsUUACGCUGAGUACUUCGAdTdT-Hyp-PEG5K
32
PEG-5K




5′-UCGAAGUACUCAGCGUAAGdTdT-3′
33



Luc
3247
5′-CsUUACGCUGAGUACUUCGAdTdT-Hyp-PEG20K
34
PEG-20K




5′-UCGAAGUACUCAGCGUAAGdTdT-3′
33



Luc
3248
5′-CsUUACGCUGAGUACUUCGAdTdT-Hyp-PEG40K-3′
35
PEG-40K




5′-UCGAAGUACUCAGCGUAAGdTdT-3′
33



Luc
1695
5′-CUUACGCUGAGUACUUCGAdTdT-Hyp-Chol-3′
36
Cholesterol




5′-UCGAAGUACUCAGCGUAAGdTdT-3′
33









Relative ApoB mRNA was evaluated in Liver and Jejunum with dosing of PEG siRNA conjugates as provided in Table 3 above, using procedures similar to those described in Example 4. The results are depicted in FIG. 13.
Example 6
In Vivo Down-Modulation of Endogenous CNPase mRNA Levels by CNS Administration of Lipophilic Conjugates of siRNAs Targeting CNPase in Rats
To assess the in vivo biological activity of lipophilic conjugates of siRNAs in the CNS, including, behenoyl, lithocholic acid, dialkylglyceride, and Vitamin E conjugated siRNAs targeting CNPase (2′,3′-cyclic nucleotide 3′-phosphodiesterase), a gene expressed specifically in oligodendrocytes within the CNS, dsRNAs AD-3236, AD-3237, AD-3238, AD-3181, AD-3282 and AD-3178 (Table 4) were synthesized in which liphophilic molecules were placed on the 3′-end of the sense strand (outside of the dsRNA's nucleotide region that targets the CNPase mRNA).







TABLE 4







Sequences of dsRNAs AD-3236, AD-3237, AD-3238, AD-3181,


AD-3182 and AD-3178











Duplex
Sense strand
SEQ
Antisense strand sequence
SEQ


name
sequence(5′′-3′′)
ID NO:
(5′′-3′′)
ID NO:





AD-3236
GGccuuGAccucuuAGAGATT-
37
UCUCuAAGAGGUcAAGGCCTsT
38



Behenoyl





AD-3237
GGccuuGAccucuuAGAGATT-
39
UCUCuAAGAGGUcAAGGCCTsT
38



Lithocholic acid





AD-3238
GGccuuGAccucuuAGAGATT-
40
UCUCuAAGAGGUcAAGGCCTsT
38



Dialkylglycerides





AD-3181
GGccuuGAccucuuAGAGATT-
41
UCUCuAAGAGGUcAAGGCCTsT
38



Vitamin E





AD-3182
GGccuuGAccucuuAGAGATT-
42
UCUCuAAGAGGUcAAGGCCTsT
38



Stearoyl





AD-3178
GGccuuGAccucuuAGAGATT-
43
UCUCuAAGAGGUcAAGGCCTsT
38



cholesterol









In rats, different amounts of siRNA or phosphate-buffered saline (PBS, vehicle control) was administered by continuous intraparenchymal infusion into the corpus callosum over 3 days. Male Sprague-Dawley rats, approximately 250-300 g body weight, received stereotaxic implantation of 30-gauge infusion cannulae (Plastics One, Roanoke, Va.) such that unilateral infusions were targeted to the corpus callosum (anteroposterior +0.7 mm, mediolateral +2.2 mm, relative to bregma; dorsoventral −3 mm, relative to skull surface). Mini-osmotic pumps (manufactured by Alzet, Inc. model 2ML1) were primed overnight according to the manufacturer's specifications, implanted subcutaneously, and connected via catheters, to deliver (6 rats per treatment group) PBS or different concentrations of siRNAs at 10 uL/hr over 3 days. At the end of the 3 day infusion period, animals were sacrificed, brains were removed, and ipsilateral corpus callosum encompassing the infusion site was dissected. 1 mm thick coronal brain slabs were obtained over a distance of 12 mm anterior to posterior and then ipsilateral corpus callosum tissue samples were collected from each brain slab. Tissue was flash frozen and then stored at −80° C. For branched DNA analysis, frozen corpus callosum tissue pieces were lysed in 1 ml of Tissue and Cell Lysis solution (Epicentre, Madison, Wis.) containing Proteinase K (Ambion, Austin, Tex.). CNPase and MBP (myelin basic protein) mRNA were quantified using the Quantigene Explore Kit according to the manufacturer's instructions (Panomics, Fremont, Calif.). For each tissue sample, the ratio of CNPase/MBP (normalized CNPase mRNA level) was calculated as an average of three determinations. These average ratios from each individual animal were then averaged to obtain a group (treatment) average.
The siRNA duplexes, AD-3236, AD-3237 and AD-3238 were found to be effective in vivo in down-modulating CNPase mRNA levels after intraparenchymal CED infusion with 2.16 mg over 3 days (10 uL/hr at 3 mg/ml) in rats. AD-3236, AD-3237, and AD3238 reduced the normalized CNPase mRNA levels (normalized to MBP) in corpus callosum tissue by 39%, 28% and 42%, respectively, relative to the PBS control group. Similarly, intraparenchymal CNS infusion of the Vitamin E conjugated siRNA AD-3181 and Stearoyl conjugated siRNA AD-3182 over 3 days (also 10 ul/hr at 3 mg/ml for a total of 2.16 mg administered dose) reduced CNP mRNA levels (normalized to MBP) by 41% and 51% respectively compared to PBS control animals. These results demonstrate that the different lipophilic conjugated siRNAs AD-3222, AD-3236, AD-3237, AD-3238, AD-3181 and AD-3182 following intraparenchymal CNS infusion down-modulate CNPase mRNA levels within the corpus callosum.

## Claims

1. An iRNA agent comprising a first strand and a second strand, wherein at least one subunit having a formula (II) is incorporated into at least one of said strands, and at least one of said strands leads to the cleavage of a complementary target sequence via a RISC dependent mechanism:




wherein:
X is NC(O)R7, or NR7;
R3 and R9 are each independently H, OH, ORa, or ORb, provided that at least one of R3 or R9 is OH, ORa, or ORb;
R5 is H or C1-C6 alkyl;
R1 is CH2OH or CH2ORb, provided that at least one of R1, R3, or R9 is ORb or CH2ORb;
R7 is C1-C20 alkyl substituted with NRcRd or NHC(O)Rd;




each of A and C is, independently, O or S; and




B is OH, O−, or
Rc is H or C1-C6 alkyl; and
Rd is a bile acid radical, a fatty acid conjugate radical or PEG.

2. The compound of claim 1, wherein R1 is CH2ORb and R9 is OH.

3. The compound of claim 2, wherein R1 and R9 are trans.

4. The compound of claim 3, wherein A is O.

5. The compound of claim 3, wherein A is S.

6. The compound of claim 1, wherein R7 is (CH2)5NHRd or (CH2)5NHC(O)Rd.

7. The compound of claim 1, wherein Rd is lithocholic-oleyl, lauroyl, docosanoyl, stearoyl, palmitoyl, myristoyl, oleoyl, linoleoyl or dodecanoyl.

8. The compound of claim 1, wherein Rd is lauroyl.

9. The compound of claim 1, wherein Rd is dodecanoyl.

10. The compound of claim 1, wherein Rd is stearoyl.

11. The compound of claim 1, wherein Rd is palmitoyl.

12. The compound of claim 1, wherein Rd is myristoyl.

13. The compound of claim 1, wherein Rd is oleoyl.

14. The compound of claim 1, wherein Rd is linoleoyl.

15. The compound of claim 1, wherein Rd is lithocholic-oleyl.

16. The compound of claim 1, wherein Rd is docosanoyl.

17. The compound of claim 1, wherein Rd is PEG.

18. The compound of claim 1, wherein Rd is PEG-5K.

19. The compound of claim 1, wherein Rd is PEG-20K.

20. The compound of claim 1, wherein Rd is PEG-40K.

21. An iRNA agent comprising a first strand and a second strand, wherein at least one subunit having a formula (II) is incorporated into at least one of said strands, and at least one of said strands leads to the cleavage of a complementary target sequence via a RISC dependent mechanism:




wherein:
X is NC(O)R7, or NR7;
each of R3, R5 and R9 is, independently, H, OH, ORa, or ORb, provided that at least one of R3, R5, or R9 is OH, ORa, or ORb;
R1 is CH2OH or CH2ORb, provided that at least one of R1, R3, R5, or R9 is ORb or CH2ORb;
R7 is C1-C20 alkyl substituted with NRcRd or NHC(O)Rd;




each of A and C is, independently, O or S; and




B is OH, O−, or
Rc is H or C1-C6 alkyl; and
Rd is a bile acid radical, a fatty acid conjugate radical or PEG.

22. The compound of claim 21, wherein R1 is CH2ORb and R9 is OH.

23. The compound of claim 21, wherein R7 is (CH2)5NHRd or (CH2)5NHC(O)Rd.

24. The compound of claim 21, wherein Rd is lithocholic-oleyl, lauroyl, docosanoyl, stearoyl, palmitoyl, myristoyl, oleoyl, linoleoyl or dodecanoyl.

25. The compound of claim 21, wherein Rd is PEG, PEG-5K, PEG-20K, or PEG-40K.