HK1189031B - Small molecule conjugates for intracellular delivery of nucleic acids - Google Patents

Description

Small molecule conjugates for intracellular delivery of nucleic acids

The present invention relates to the use of novel small molecule conjugates for the delivery of nucleic acids, such as siRNA. Delivery of nucleic acids into living cells is highly restricted by the complex membrane systems of the cells.

One method for delivering nucleic acids in vivo is to attach the nucleic acids to small targeting molecules or hydrophobic molecules such as lipids or sterols. However, delivery and activity of these conjugates is observed when administered to rodents, and the dosages of nucleic acid required are prohibitively large, often leading to undesirable toxic effects in vivo and costly and difficult to implement treatment regimens when switching to humans. Provided herein is the use of small molecule conjugates for the delivery of nucleic acids, such as siRNA, that successfully mediate the delivery of the nucleic acids into cells when the small molecule compound is conjugated to the nucleic acid. Surprisingly, it has been found that the nucleic acid dose sufficient for successful delivery is significantly reduced when using the novel compounds provided herein. Thus, the use of this compound provides a powerful tool for delivering nucleic acids that significantly limits in vivo toxicity.

In one embodiment, the invention relates to the use of a compound of formula (I) for delivering a nucleic acid

Wherein

Y is selected from- (CH)₂)₃-or-C (O) -N- (CH)₂-CH₂-O)_p-CH₂-CH₂-a linking group;

R¹is- (C1-6) alkyl;

-(CH₂) -a naphthyl group; or

-(CH₂)_m-phenyl, wherein phenyl is unsubstituted or substituted up to four times with substituents independently selected from the group consisting of:

-NO₂、

-CN、

halogen, halogen,

-O-(CH₂) -phenyl group,

-O- (C1-6) alkyl, or

-C(O)-NH₂；

R²Is hydrogen;

-(CH₂)_k-N-C(Ph)₃wherein the phenyl rings are unsubstituted or independently substituted with-O- (C1-4) alkyl;

-(CH₂)_k-C(O)-NH₂；

-(CH₂)_k-a phenyl group;

- (C1-6) alkyl, unsubstituted or substituted by-S-CH₃Once substituted;

R³is-NH-phenyl, wherein the phenyl group is further substituted with a substituent independently selected from the group consisting of:

-(CH₂) -OH; or

-(CH₂) -O-c (O) -O- (4-nitro-phenyl);

k is 1,2, 3,4, 5, 6;

m is 1,2, 3 or 4;

n is 0 or 1; and is

p is an integer of 1 to 20.

In another embodiment, there is provided the use of a compound of formula (I) having a particular configuration as shown in formula (Ia) for delivering a nucleic acid

Wherein all substituents R¹、R²、R³And Y and the variables k, m, n and p have the meanings indicated above.

In another embodiment, the invention relates to the use of a compound of formula (I) or (Ia) wherein Y is- (CH)₂)₃-; and all remaining substituent groups have the meanings described above.

In another embodiment, the invention relates to the use of a compound of formula (I) or (Ia) wherein Y is-C (O) -N- (CH)₂-CH₂-O)_p-CH₂-CH₂-; and all substituent groups have the meanings indicated above.

In another embodiment, there is provided the use of a compound of formula (I) or (Ia) for delivering a nucleic acid, wherein

Y is- (CH)₂)₃-；

R²Is- (CH)₂)_k-N-C(Ph)₃Wherein the phenyl rings are unsubstituted or independently substituted with-O- (C1-4) alkyl; and is

R³is-NH-phenyl, wherein the phenyl group is further substituted by- (CH)₂) -O-c (O) -O- (4-nitro-phenyl) substitution;

n is 0; and is

R¹And k has the meaning described above.

In another embodiment, there is provided the use of a compound of formula (I) or (Ia) for delivering a nucleic acid, wherein

Y is-C (O) -N- (CH)₂-CH₂-O)_p-CH₂-CH₂-；

R²Is- (CH)₂)_k-N-C(Ph)₃Wherein the phenyl rings are unsubstituted or independently substituted with-O- (C1-4) alkyl; and is

R³is-NH-phenyl, wherein the phenyl group is further substituted by- (CH)₂) -O-c (O) -O- (4-nitro-phenyl) substitution;

n is 0; and is

R¹K and p have the meanings indicated above.

The term "(C1-6) alkyl" as used herein means a straight or branched chain saturated hydrocarbon containing from 1 to 6 carbon atoms. Preferred C1-6 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, 2-butyl and the like.

The term "halogen" as used herein means fluorine, chlorine, bromine or iodine, preferably fluorine and chlorine.

In general, the compounds of the invention for delivery of nucleic acids can be obtained using methods known to those of ordinary skill in the art of organic or pharmaceutical chemistry. Also, it is understood that the cholesterol moiety may be replaced by other natural or chemically synthesized compounds. The compounds of formula (a) are further reacted in steroids (such as cholanic acid, lithocholic acid, etc.) or other small molecules (such as vitamins) known to be effective in nucleic acid delivery, such as tocopherol (molecular therapy,2008,16,734)

For successful delivery of the nucleic acid, a compound of formula (I) or (Ia) is covalently linked to the nucleic acid. Preferably, the covalent bond is via a suitable functional group in the nucleic acid, e.g.a primary amine group, with R as defined above³In the part of-O-C (O) -O-)Activated carbonyl groups are produced by reaction. Thus, provided herein are conjugates comprising a compound of formula (I) or (Ia) and a nucleic acid.

The term "nucleic acid" as used herein refers to any form of DNA, including cDNA or RNA or fragments thereof, nucleotides, nucleosides, oligonucleotides (including antisense oligonucleotides, LNAs, and sirnas), which elicit a biological effect when administered in vivo in animals (including but not limited to birds and mammals, including humans). Preferably, the nucleic acid used herein is siRNA.

Conjugates comprising a compound of the invention covalently linked to a nucleic acid exhibit an increased ability to be taken up by cells compared to the nucleic acid alone. After delivery of the conjugate into cells and delivery to lysosomes, the corresponding nucleic acid is released by enzymatic cleavage. The cleavage preferably occurs when a dipeptide motif, preferably comprising the sequences alpha-or beta- (phenyl) alanine and lysine as present in the compounds of formula (I) or (Ia), is introduced into the conjugate (see scheme 1). More preferably, the conjugate comprises a dipeptide motif and a spacer, such as a p-aminobenzyl carbamate spacer (bioconjugategetechem. 2002,13,855), which spontaneously cleaves when the C-terminal amide bond of the dipeptide motif is cleaved, as exemplified by siRNA in scheme 2. Thus, the conjugate comprising a compound of formula (I) or (Ia) also relates to a dipeptide comprising a cholesterol conjugate. Enzymatic cleavage of nucleic acids from the dipeptide containing cholesterol conjugates of the invention is catalyzed by proteases inherent to the cell. An example of an intrinsic protease capable of cleaving the dipeptide motif present in the compound of formula (I) or (Ia) is cathepsin B. Cathepsin B is a cysteine protease known to be ubiquitous in mammalian cell lysosomes (bioconjugetechem.2002, 13,855; j.med.chem.2005,48,1344; nat.biotechnology2003,21,778). Thus, the dipeptide motif described above is also referred to as a cathepsin-cleavable dipeptide motif.

Thus, the invention also provides a method of delivering a nucleic acid into a cell, wherein the nucleic acid can subsequently be released from the conjugate to exhibit its therapeutic activity.

In other embodiments of the invention, there is provided a conjugate of a compound of formula (I) or (Ia) covalently linked to an siRNA for intracellular delivery.

Conjugates of formula (I) or (Ia) covalently linked to a nucleic acid are referred to herein as formula (II) or (IIa), respectively.

Scheme 1

Thus, in other embodiments, the present invention provides compounds of formula (II)

Wherein

R^aIs- (CH)₂)_k-NH₂；

R¹And k has the meaning given above for formula (I).

In a more particular embodiment, the present invention provides a compound of formula (IIa)

Wherein

R^aIs- (CH)₂)_k-NH₂；

R¹And k has the meaning given above for formula (I).

In a preferred embodiment, the nucleic acid in formula (II) or (IIa) is an siRNA.

In another preferred embodiment, the biologically active substance in formula (II) or (IIa) is a protein or peptide.

The compounds of formula (II) or (IIa) may have therapeutically advantageous properties. Thus, in other embodiments, there is provided a compound of formula (II) or (IIa) for use as a medicament.

Another embodiment of the invention is a pharmaceutical composition comprising a conjugate of a compound of formula (I) or (Ia) covalently attached to a nucleic acid.

In another embodiment of the present invention, there is provided a pharmaceutical composition comprising a compound of formula (IIa) together with a pharmaceutically acceptable excipient.

The following embodiments illustrate conjugates of compounds of formula (I) or (Ia) covalently linked to siRNA. It will be appreciated that these embodiments may also be applied to other types of nucleic acids as defined above.

The covalent attachment of the siRNA to the compound of formula (I) or (Ia) is via a suitable nucleophilic group (i.e., a primary amine group) in the siRNA to R of said compound of formula (I) or (Ia)³Is achieved by reaction of activated-c (o) -groups. Activation of the-C (O) -group is obtained by p-nitrophenoxycarbonate as shown in scheme 2 below.

(procedure 2)

The p-nitrophenyl activated carbonate may be reacted, for example, with an siRNA with a primary amine bearing a suitable nucleophilic group, such as a hexylamino linker, to form a carbamate linkage, thereby yielding an siRNA conjugate. After the siRNA is absorbed into the cell and transferred to the lysosome, the compound of formula (II) or (IIa) in which the biologically active substance is siRNA is cleaved by protease activity via a1, 6-elimination reaction, releasing the siRNA, as shown in scheme 3. The cholesterol moiety of the conjugate of the compound of formula (II) or (IIa) alters the PK properties of the siRNA, whereby systemic administration silences the gene in vivo.

In one embodiment, the compound of formula (II) or (IIa) wherein the nucleic acid is an siRNA is administered in combination with a delivery polymer. The delivery polymer provides a means to rupture the cell membrane and modulate endosomal release. In another embodiment, the delivery polymer and the siRNA conjugate of the invention are non-covalently linked and synthesized separately, and may be supplied in separate containers or in a single container. Delivery polymers for oligonucleotides such as siRNA are well known in the art.

For example, Rozema et al, in U.S. patent publication 20040162260, exemplify a method of reversibly modulating membrane disruptive activity of membrane-activated polyamines. Reversible regulation provides a means to limit activity on the endosomes of target cells, thus limiting toxicity. Their method relies on the reaction between an amine on a polyamine and 2-propionic acid-3-methyl maleic anhydride. This modification converts the polycation into a polyanion by converting a primary amine into a group comprising a carboxyl group and reversibly inhibits the membrane activity of the polyamine.

Scheme 3

To enable delivery of the nucleic acid in combination with a delivery vehicle, the nucleic acid is covalently linked to a delivery polymer. A new generation of delivery polymers is described in U.S. provisional patent application 61/307490. Among other things, a membrane active polyamine is provided that includes an amphiphilic terpolymer formed from the random polymerization of an amine-containing monomer, a less hydrophobic monomer, and a more hydrophobic monomer. This new generation of delivery polymers does not require the association of polynucleotides and polymers by covalent linkage or by charge-charge interaction.

Non-limiting examples of delivery polymers for administration in combination with the siRNA conjugates of the present invention are membrane active polyamines and poly (vinyl ethers) (PBAVE), dynamic polyconjugates (DPC; Rozema et al 2007) and modified DPC as disclosed in U.S. provisional patent application 61/307490.

In other embodiments, novel chemical siRNA modification patterns for in vivo functional delivery are provided. This novel chemical siRNA modification pattern is particularly useful with delivery vehicles that exhibit relatively strong endosomal/lysosomal retention.

It has been found that stabilization of sirnas against degradation by endonucleolytic/lysosome-localized nucleases such as DNAseII strongly promotes target knockdown (knockdown). This stabilization can directly affect the amount of siRNA released into the cytoplasm of the cell where the RNAi machinery is located. Only the cytoplasmic siRNA portion triggers RNAi action.

In addition to poor pharmacokinetic properties, sirnas are sensitive to nucleases in biological environments when siRNA without a protective delivery vehicle is administered directly into the circulatory system. Thus, many sirnas degrade rapidly in the extracellular tissues and bloodstream or after cellular uptake (endosomes).

It is well known that the nuclease located in the endosome/lysosome compartment is DNaseII. The enzyme is active at pH below 6-6.5 and has the strongest activity at pH in the range of 4.5-5, reflecting the acidic environmental conditions present in the endosome/lysosome compartment. The subsequent RNA degradation pathway initiated by dnasei has been identified in vitro and is disclosed in the present invention:

A. the RNA strand, which contains at least one 2 '-OH nucleotide, is rapidly degraded by the cyclic pentavalent phosphorus intermediate, resulting in a 2' -3 'cyclic phosphate ester at the 5' -cleavage product. The formation of the pentavalent intermediate may be inhibited by nucleotides lacking a2 ' -OH group, such as 2 ' -deoxy, 2 ' -O-methyl (2 ' -OMe) or2 ' -deoxy-2 ' -fluoro (2 ' -F) nucleotides.

B. In addition, RNA is not dependent on the 5 ' -terminal nucleotide 2 ' -modified 5 ' -nucleic acid exo path degradation. This degradation pathway can be inhibited by non-nucleotide moieties at the 5' -terminus, such as cholesterol, aminoalkyl linkers, or phosphorothioates at the bonds between the first nucleotides.

C.5' -phosphates also protect and slow down the kinetics of exonucleolytic cleavage, but are unable to completely block this pathway. This is most likely due to cleavage of the 5' -phosphate by phosphatase activity inherent to the phosphatase or DNaseII enzyme preparation used in the stability assay.

D. Optimal protection is achieved with oligonucleotides lacking any 2 ' -OH nucleotide in the chain, starting with a2 ' -OMe nucleotide at the 5 ' -end linked to a second nucleotide via a thiosulfate (PTO) linkage. Other nucleotides lacking a2 ' -OH group at the end may also prevent 5 ' -exo-degradation, but to a lesser extent compared to 2 ' -OMe modification.

Thus, the inventors of the present invention found that siRNA can be significantly stabilized when using the following design, in which an antisense strand with a modification pattern is used to provide an oligonucleotide: 5' - (w) - (Z1) - (Z2) - (Z3) n_a-3 'and having modification mode 5' - (Z3) n_s-a sense strand of 3', wherein

w is independently 5 '-phosphate or 5' -phosphorothioate or H,

z1 is independently a 2' -modified nucleoside.

Z2 is independently a2 '-deoxynucleoside or a 2' -fluoro-modified nucleoside,

z3 is independently a 2' -modified nucleoside,

n_ais 8-23 and n_sIs 8 to 25.

In a preferred embodiment, the oligonucleotide is modified with a modification pattern of 5' - (w) - (Z1) - (Z2) - (Z3) n_a3 'antisense strand with modification pattern 5' - (Z3) n_s-3 'sense strand provided wherein Z1 is a 2' -fluoro-modified nucleoside or 2-deoxynucleoside, and all other substituents and variable n_aAnd n_sHave the meaning as described above.

In a preferred embodiment, the oligonucleotide is modified with a modification pattern of 5' - (w) - (Z1) - (Z2) - (Z3) n_a3 'antisense strand with modification pattern 5' - (Z3) n_s-3 ' sense strand wherein Z3 is a2 ' -O-methyl modified nucleoside, 2 ' -fluoroModified nucleosides or 2-deoxynucleosides, and all other substituents and the variable n_aAnd n_sHave the meaning as described above.

In a preferred embodiment, the oligonucleotide is modified with a modification pattern of 5' - (w) - (Z1) - (Z2) - (Z3) n_a3 'antisense strand with modification pattern 5' - (Z3) n_s-3 'sense strand provided wherein Z1 is a 2' -fluoro-modified nucleoside or2 deoxynucleoside and Z3 is a2 '-O-methyl modified nucleoside, 2' -fluoro-modified nucleoside or2 deoxynucleoside with all other substituents and the variable n_aAnd n_sHave the meaning as described above.

Nucleosides in the nucleic acid sequence of oligonucleotides having novel modification patterns can be linked by 5 '-3' phosphodiester or 5 '-3' phosphorothioate linkages.

As used herein, an "antisense" strand is a strand of siRNA that is complementary to a target mRNA and binds to that mRNA when the siRNA is expanded.

The sense strand of the siRNA comprising the novel modification pattern is complementary to the antisense strand.

The siRNA comprising the novel modification patterns proved particularly advantageous when covalently linked to delivery polymers as exemplified by Rozema et al (DPC; Rozema et al 2007.) the efficacy and duration of action can be significantly improved using the siRNA modification strategy described in the present invention.

In another embodiment, the sirnas comprising the novel modification patterns are particularly useful when conjugated to small molecules that alter the pharmacokinetic properties of the siRNA, such as cholesterol or the compounds of formulae (I) and (Ia) provided herein. In one embodiment, conjugates of small molecules and oligonucleotides are provided, wherein the oligonucleotides have the following modification patterns: having modification patterns of 5' - (w) - (Z1) - (Z2) - (Z3) n_a3 'antisense strand with modification pattern 5' - (Z3) n_sThe sense strand of (A), wherein the substituents and the variable n_aAnd n_sHave the meaning as described above. In one embodiment, the small molecule is cholesterol.In another embodiment, the small molecule is a compound of formula (I) or (Ia) to give a compound of formula (II) or (IIa).

Preferably, the siRNA conjugate is administered in combination with a delivery polymer. Suitable delivery polymers are described above.

In one embodiment, the sirnas comprising the novel modification patterns are particularly useful when conjugated to ligands known to bind to specific receptors capable of internalizing the conjugate into cells. In particular, the sialoglycoprotein receptor (ASGPR) expressed on hepatocytes is a well-known receptor capable of clearing (endocytosis or lysosomal degradation) desialylated proteins from the circulatory system. It has been shown that N-acetyl-D-galactosamine has a high affinity for this receptor, especially when it is multivalent and when the galactose residues have a suitable spacing (jbiol bhem,2001,276,37577). In order to utilize the high energy receptor for receptor-mediated phagocytosis of nucleic acids, synthetic ligands shown below were prepared to be covalently linked to siRNA containing a novel modification pattern. Since this type of cellular phagocytosis leads to lysosomal degradation of internalized materials, the siRNA must be prepared in a manner that is stable in the lysosome, and this problem has now been addressed by the new modification model outlined above.

Likewise, it will be appreciated that the targeting ligand of formula III conjugated to a nucleic acid such as an siRNA, as shown in formula IV, may be replaced by other natural or chemically synthesized compounds (antagonists or agonists) that exhibit high affinity for cell surface expression receptors. Examples include folate (ann.n.y.acad.sci.,2009,1175,32) or PSMA binding molecules (NatureBiotech,2006,24,1005; MolPharm,2009,6,780) that are ligands of folate receptors expressed on a variety of cancer cells.

The ligand of ASGPR is linked to the nucleic acid via an amide bond. The formation of the amide bond can be performed with the aid of N-hydroxy-succinimide (NHS) chemistry. The ligands used in the conjugation reaction are shown below (formula III). To interact with ASGPR, the O-acetate group on the saccharide residue needs to be removed as shown in siRNA (formula IV).

In one embodiment of the invention, there is provided a conjugate of a compound of formula IV and an oligonucleotide, wherein the oligonucleotide has the following modification pattern: having modification patterns of 5' - (w) - (Z1) - (Z2) - (Z3) n_a3 'antisense strand with modification pattern 5' - (Z3) n_sThe sense strand of (A), wherein the substituents and the variable n_aAnd n_sHave the meaning as described above. The conjugates are also referred to as GalNAc palmitoyl conjugates. Preferably, the GalNAc palmitoyl conjugate is administered in combination with a delivery polymer. Suitable delivery polymers are described above.

It has been found that for these modification patterns, cleavable linkers prove advantageous compared to stably linked small molecule ligands. Possible cleavable linkers are dipeptide linkers or cleavable RNA-linkers comprising nucleotides containing a 2' -OH as exemplified in scheme 1. This cleavable RNA linker is particularly useful when combined with siRNA with the new modification pattern (all 2' -modified siRNA) as described above.

In general, the nuclease cleavage site may be introduced by a 3 ' -or 5 ' -overhang of nucleotides comprising at least one 2 ' -OH on the sense or antisense strand. The final active siRNA species was generated by intracellular nuclease treatment. Also, it is possible to use defined cleavage sites realized by 2' -OH nucleotides within the base pair region. This can be done using at least one 2 ' -OH nucleotide complementary to the corresponding strand or by introducing at least one mismatched 2 ' -OH nucleotide or hairpin/bulge loop of nucleotides comprising at least one 2 ' -OH.

The use of a defined cleavage site by introducing a 2' -OH nucleotide, as opposed to other cleavable linker chemistries, leads to a more versatile conjugation approach. By introducing selective cleavage sites at the 3 'and/or 5' -end or within the double stranded structure on one or both strands of the siRNA, it is possible to generate multiple conjugates.

Thus, in one embodiment, there is provided a conjugate of a small molecule and an oligonucleotide, wherein

a) The small molecule comprises a nucleotide linker comprising 1-10, preferably 1-5, most preferably 1-3 2' OH nucleotides;

b) the oligonucleotides have the following modification patterns: having modification patterns of 5' - (w) - (Z1) - (Z2) - (Z3) n_a3 'antisense strand with modification pattern 5' - (Z3) n_sThe sense strand of (A), wherein the substituents and the variable n_aAnd n_sHave the meaning as described above; and is

c) The oligonucleotide is covalently linked to a small molecule through a nucleotide linker.

The nucleotidic linker is cleaved by an intracellular nuclease such as DNaseII, for example in the endosome, after the conjugate is internalized into the endosome, thus releasing the siRNA.

Preferably, the conjugate is administered in combination with a delivery polymer. Suitable delivery polymers are described above.

In another embodiment of the present invention, there is provided a compound of formula (V). The compounds comprise a cholesterol moiety and a nucleotide linker comprising 1-10, preferably 1-5, most preferably 1-3 2' OH-nucleotides. The nucleotide linker is for covalent attachment of an oligonucleotide, e.g., an siRNA, to a compound of formula (V). Preferably, the oligonucleotides have a novel modification pattern as outlined above. Thus in another embodiment, there is provided a conjugate of a compound of formula (V) and an oligonucleotide, wherein the oligonucleotide is covalently linked to the nucleotide linker of the compound of formula (V).

The nucleotidic linker is cleaved by intracellular nucleases, such as DNaseII, after the conjugate of the compound of formula (V) and oligonucleotide is internalized into the endosome, thus releasing the siRNA.

Preferably, the conjugate of the compound of formula (V) and the oligonucleotide is administered in combination with a delivery polymer. Suitable delivery polymers are described above.

In another embodiment, the conjugate of the delivery polymer and the compound of formula (V) and oligonucleotide of the invention is not covalently linked, but is synthesized separately and may be provided in separate containers or in a single container.

Definition of

The term "small molecule" as used herein refers to an organic or inorganic molecule, synthetic or found in nature, generally having a molecular weight of less than 10,000 g/mole, optionally less than 5,000 g/mole, and optionally less than 2,000 g/mole.

The term "peptide" as used herein refers to any polymeric compound resulting from the formation of an amide bond between the α -carboxyl group of one D-or L-amino acid and the α -amino group of another D-or L-amino acid. The term "protein" as used herein refers to polypeptides of a particular sequence of more than about 50 residues.

The term "dipeptide motif" as used herein refers to any motif comprising an amide bond formed by an alpha or beta amino group of a first D-or L-amino acid and an alpha-carboxyl group of a second D-or L-amino acid.

The term "amino acid" as used herein refers to any molecule comprising an amine and a carboxyl functional group. Thus, the term "amino acid" refers to natural, unnatural and synthetic amino acids. Any natural amino acid used in the present invention is represented herein in its conventional abbreviations.

The term "ligand" as used herein refers to a moiety capable of covalently or otherwise chemically binding to a nucleic acid. The term "ligand" in the context of the present invention is preferably a compound of formula (I) or (Ia) covalently linked to a nucleic acid.

The term "nucleic acid" as used herein means an oligomer or polymer composed of nucleotides, such as deoxyribonucleotides or ribonucleotides, or synthetically prepared compounds (e.g., PNAs as described in U.S. patent No. 5,948,902 and references cited therein) that can hybridize to naturally occurring nucleic acids in a similar sequence-specific manner as two naturally occurring nucleic acids, e.g., can participate in watson-crick base pairing interactions. Non-naturally occurring nucleic acids are oligomers or polymers comprising a sequence of nucleobases that does not occur in nature, or a substance comprising a functionalized equivalent of a naturally occurring nucleobase, saccharide or intersugar linkage, such as a Peptide Nucleic Acid (PNA), a Threose Nucleic Acid (TNA), a Locked Nucleic Acid (LNA) or a Glycerol Nucleic Acid (GNA). The term includes oligomers containing the naturally occurring nucleobases adenine (a), guanine (G), thymine (T), cytosine (C) and uracil (U), as well as oligomers containing base analogs or modified nucleobases. Nucleic acids can be derived from many natural sources, such as viruses, bacteria, and eukaryotic DNA and RNA. Other nucleic acids may be derived from synthetic routes and include any of a variety of oligonucleotides prepared for use as research reagents, diagnostic reagents, or effective and defined therapeutic agents. The term includes oligomers containing single-stranded nucleic acids or double-stranded nucleic acids.

The term "2 '-modification" as used herein refers to a β -D-ribonucleoside or a β -D-ribonucleoside comprising a naturally occurring nucleobase with a 2' -OH group replaced by H, F, O-CH3 or other substituents known in the art.

The term "2 '-OH-nucleotide" as used herein refers to a β -D-ribonucleoside comprising a naturally occurring nucleobase having a 2' -OH group.

The term "5' -phosphate ester" as used herein refers to the formula-O-P (= O) (OH) OH. In another aspect, the phosphate is modified in which one of the O or OH groups is replaced with S, referred to herein as "5' -phosphorothioate".

The term "phosphorothioate" as used herein refers to an internucleotide linkage in which one non-bridging oxygen is replaced by thio.

The term "delivery polymer" as used herein refers to a polymer that is used for functional delivery of nucleic acids. In the context herein, the delivery polymer is administered covalently linked to or in combination with a biologically active substance conjugated to a compound described herein and mediates endosomal escape following internalization into the cell and uptake into the endosome. The term "polymer" as used herein means any compound made up of two or more monomeric units covalently bonded to each other, wherein the monomeric units may be the same or different, and the polymer may be a homopolymer or a heteropolymer. Representative polymers include peptides, polysaccharides, nucleic acids, and the like, wherein the polymer may be naturally occurring or synthetic. Non-limiting examples of such delivery polymers are for example those reviewed in INTERNATIONALIOURNALIFEPHARMACEUTICAL RESERCHANDDEVELOPMENT, 10/Vol.2/Vol.8/article No. 2, 2010. Non-limiting examples of delivery polymers for delivering nucleic acids are disclosed in EP applications 10165502.5 and 10191030.5, PCT publication WO2008/0022309, and US provisional application 61/307490 and references cited therein; the contents of which are incorporated herein by reference in their entirety.

As used herein, a "pharmaceutical composition" comprises a conjugate of the invention, a pharmaceutical carrier or diluent, and any other media or agents necessary for formulation.

As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and other agents that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

The conjugates of the invention can be administered by a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and/or mode of administration will vary depending on the desired result. When the conjugate of the present invention is administered by some route of administration, it must be coated with a substance that prevents its inactivation or administered in combination therewith. For example, the conjugate may be administered to the individual in a suitable carrier or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art.

The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, typically by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

These carriers also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Prevention of the presence of microorganisms may be ensured by methods of sterilization (as described above) as well as inclusion of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, and the like). It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like in the composition. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In addition to the chosen route of administration, the conjugates of the invention and/or the pharmaceutical compositions of the invention, which can be used in a suitable hydrated form, are formulated into pharmaceutically acceptable dosage forms according to conventional methods known to those skilled in the art.

The actual dosage level of the active ingredient in the pharmaceutical compositions of this invention will vary with the amount of active ingredient to be obtained which is effective to produce the desired therapeutic response for a particular patient, composition and mode of administration and which is non-toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular composition employed in accordance with the present invention, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and/or substances used in combination with the particular compound employed, the age, sex, body weight, condition, general physical condition and previous medical history of the patient, and like factors well known in the medical arts.

Pharmaceutical compositions must be sterile and fluid to the extent that the composition can be delivered by injection. In addition to water, the carrier is preferably an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the dispersion and by the use of surfactants. In many cases, it is preferred to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.

Brief Description of Drawings

Figure 1 shows the combined in vivo administration of an siRNA conjugate comprising a compound of formula (I) or (Ia) and a delivery polymer.

Figure 2 shows the combined in vivo administration of an siRNA conjugate comprising a compound of formula (I) or (Ia) and a delivery polymer.

Figure 3 shows the combined in vivo administration of an siRNA conjugate comprising a compound of formula (I) or (Ia) and a delivery polymer.

Figure 4 shows the combined in vivo administration of an siRNA conjugate comprising a compound of formula (I) or (Ia) and a delivery polymer.

FIG. 5a shows antisense strand mediated silencing of siRNA with all 2' -modifications. COS7 cells were co-transfected with EGFP-directed siRNA AT 3nM and psiCHECK 2-AT. The knockdown activity of the siRNA was evaluated by detecting the activity of the flower worm (renilla) relative to the firefly luciferase from the reporter construct. siRNAs were classified by knock-down deactivation of unmodified (2-19-2) reference siRNAs.

Figure 5b shows gene silencing of sense strand mediated siRNA with all 2' -modifications. COS7 cells were co-transfected with EGFP-directed siRNA AT 3nM and psiCHECK 2-AT. The knockdown activity of the siRNA was evaluated by detecting luciferase expression from the reporter construct. siRNAs were classified by knock-down deactivation of unmodified (2-19-2) reference siRNAs.

FIG. 6a shows the reduction of serum FVII activity following intravenous injection of various 2' -modified siRNAs covalently linked to a delivery polymer in a non-human primate.

Figure 6b shows the development of prothrombin time after treatment with 2' -modified siRNA covalently conjugated to a delivery polymer in a non-human primate.

Examples

The following examples are merely reference examples for the purpose of illustrating the synthesis of compounds for delivering nucleic acids. They are not meant to form part of the present invention.

Example 1

Step 13- [ (3S,8S,9S,10R,13R,14S,17R) -17- ((R) -1, 5-dimethyl-hexyl) -10, 13-dimethyl-2, 3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] a]Phenanthren-3-yloxy]-propylamine

The title amine was prepared from its nitrile precursor according to literature procedures [ Lollo et al, WO2001/070415 ].

Step 2N- {3- [ (3S,8S,9S,10R,13R,14S,17R) -17- ((R) -1, 5-dimethyl-hexyl) -10, 13-dimethyl-2, 3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] a]Phenanthren-3-yloxy]-propyl } -amberAmic acid (succinaminocacid)

In a 2L round bottom flask, 3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propan-1-amine (21.15g,47.7mmol, eq: 1.00) and Huenig base (12.3g,16.6ml,95.3mmol, eq: 2.00) were combined with OEt (845ml) to give a colorless solution. A solution of dihydrofuran-2, 5-dione (4.77g,47.7mmol, eq: 1.00) in THF (42ml) was added and the reaction mixture was stirred at ambient temperature overnight = > white suspension. All volatiles were removed in vacuo, the residue was dissolved in CH2Cl2, and the organic layer was washed with NH4Cl and brine, dried over Na2SO4, and evaporated to dryness. The crude product was dissolved in CH3CN/H2O and lyophilized to give 29.8g of the title compound as a loose powder.

MS(ISP):(M-H)542.5。

Step 3N1- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a)]Phenanthren-3-yloxy) propyl) -N4- ((S) -1- ((S) -1- (4- (hydroxymethyl) phenylamino) -6- ((4-methoxyphenyl) diphenylmethylamino) -1-oxohex-2-ylamino) -3- (4-nitrophenyl) -1-oxoprop-2-yl) succinamide

In a 10mL round bottom flask, 4- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propylamino) -4-oxobutanoic acid (106mg,184 μmol, equivalents: 1.00), (S) -2- ((S) -2-amino-3- (4-nitrophenyl) propanamido) -N- (4- (hydroxymethyl) phenyl) -6- ((4-methoxyphenyl) diphenylmethylamino) hexanamide prepared above (132mg, 184. mu. mol, equivalent weight: 1.00), HOAt (25.0mg,184 μmol, equivalent: 1.00) and EDC hydrochloride (35.3mg,184 μmol, eq: 1.00) were mixed together in CH2Cl2(1.8ml) to give a yellow solution. Huenig base (47.5mg, 64.2. mu.l, 368. mu. mol, eq: 2.00) was added and the reaction was stirred at ambient temperature overnight. TLC indicated the consumption of starting material. All volatiles were removed in vacuo and the crude product was purified by flash chromatography in SiO2/7% MeOH/0.1% NEt3 in CH2Cl2 to give 128mg of the title compound as a pale yellow solid.

MS expected mass: 1240.7552, measured mass: 1240.7518.

step 4:

N1- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propyl) -N4- ((S) -1- ((S) -1- (4- (hydroxymethyl) phenylamino) -6- ((4-methoxyphenyl) diphenylmethylamino) -1-oxohex-2-ylamino) -3- (4-nitrophenyl) -1-oxoprop-2-yl) succinamide prepared above was placed in a 10mL round bottom flask (126mg, 101. mu. mol, eq: 1.00) and Huenig base (39.3mg, 53.2. mu.l, 304. mu. mol, eq: 3.00) were combined with CH2Cl2(1.4ml) and DMF (1.0ml) to give a yellow suspension; bis (4-nitrophenyl) carbonate (46.3mg, 152. mu. mol, eq: 1.50) was added and the reaction was allowed to proceed overnight. The mixture was poured onto crashed ice, extracted twice with AcOEt, washed with H2O, dried over Na2SO4, and evaporated to dryness. Trituration with-10 ml of diethyl ether afforded 99mg of the title product as an off-white solid.

MS expected mass: 1405.7614, measured mass: 1405.7518.

the necessary dipeptide building block for step 3 was prepared as follows:

step a(S) -2- [ (S) -2- (9H-fluoren-9-ylmethoxycarbonylamino) -3- (4-nitro-phenyl) -propionylamino]-6- { [ (4-methoxy-phenyl) -diphenyl-methyl]-amino } -hexanoic acid

In a 25mL round-bottomed flask, (S) -2-amino-6- ((4-methoxyphenyl) diphenylmethyl-amino) hexanoic acid (bioconjugateCHEm.2002,13,855) 869,968mg,2.31mmol, eq: 1.00) was dissolved in CH2Cl2(20mL) to give a pale yellow solution. Huenig base (897mg,1.21ml,6.94mmol, eq: 3.00) and chlorotrimethylsilane (528mg, 621. mu.l, 4.86mmol, eq: 2.10) were added and the reaction mixture was stirred for 15 min.

In a second 50mL round bottom flask, (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -3- (4-nitrophenyl) propionic acid (1g,2.31mmol, eq: 1.00) was dissolved in DMF (20mL) to give a colorless solution. Huenig base (359mg, 485. mu.l, 2.78mmol, eq.: 1.20) and TPTU [125700-71-2 ] were added](687mg,2.31mmol, eq: 1.00) and the reaction mixture was stirred for 20'. The solution in the first flask containing the corresponding silyl ester monosilylamine was added and the reaction was stirred for an additional 3 hours. Pouring the mixture into crushed ice/NH₄Over Cl, extracted twice with AcOEt, washed with H2O and brine, dried over Na2SO4, and evaporated to dryness. Flash chromatography of a solution of SiO2/10% MeOH/0.1% NEt3 in CH2Cl2 afforded 1.38g of the title compound as a brown foam.

MS(ISP):(M+H)833.5,(M+Na)855.4。

Step b[ (S) -1- ((S) -1- (4-hydroxymethyl-phenylcarbamoyl) -5- { [ (4-methoxy-phenyl) -diphenyl-methyl]-amino } -pentylcarbamoyl) -2- (4-nitro-phenyl) -ethyl]9H-fluoren-9-ylmethyl (carbamic acid) ester

In a 250mL pear-shaped flask, (S) -2- ((S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -3- (4-nitrophenyl) propionamido) -6- ((4-methoxyphenyl) diphenyl-methylamino) hexanoic acid (1.38g,1.66mmol, equivalent: 1.00), (4-aminophenyl) methanol (204mg,1.66mmol, equivalent: 1.00), HOAt (226mg,1.66mmol, equivalent: 1.00), and EDC hydrochloride (318mg,1.66mmol, equivalent: 1.00) synthesized above were dissolved in CH2Cl2(16.6mL) to give a yellow solution. Huenig base (428mg, 579. mu.l, 3.31mmol, eq.: 2.00) was added and the reaction was allowed to proceed overnight. Pouring the mixture into crushed ice/NH₄Over Cl (pH 7), extracted 2 times with AcOEt, washed with H2O, dried over Na2SO4, and evaporated to dryness. The crude product was triturated with diethyl ether (1 × 50 mL); the resulting solid was filtered off and dried to give 1.214g of the title compound as a beige solid.

MS(ISP):(M+H)938.7。

Step c(S) -2- [ (S) -2-amino-3- (4-nitro-phenyl) -propionylamino]-6- { [ (4-methoxy-phenyl) -diphenyl-methyl]-amino } -hexanoic acid (4-hydroxymethyl-phenyl) -amide

In a 50mL round bottom flask, [ (S) -1- ((S) -1- (4-hydroxymethyl-phenylcarbamoyl) -5- { [ (4-methoxy-phenyl) -diphenyl-methyl ] -amino } -pentylcarbamoyl) -2- (4-nitro-phenyl) -ethyl ] -carbamic acid 9H-fluoren-9-ylmethyl ester (1.214g,1.29mmol, eq: 1.001) prepared above was combined with THF (19mL) to give a brown solution. At 0 deg.C, diethylamine (1.77g,2.49ml,24.2mmol, eq: 18.70) was added. The reaction was stirred at ambient temperature for 3h until MS indicated disappearance of starting material. All volatiles were evaporated in vacuo; flash chromatography of a solution of SiO2/0.1% NEt3 in CH2Cl2 = >10% MeOH/0.1% NEt3 in CH2Cl2 followed by a second flash chromatography of a solution of SiO2/5% MeOH/0.1% NEt3 in CH2Cl2 gave 502mg of the title compound as a light brown foam.

MS expected mass: 715.337, measured mass: 715.3362.

example 2

O-benzyl-N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-tyrosyl-N-6- [ (4-methoxyphenyl) (diphenyl) methyl ] -N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-lysylamide

Prepared in analogy to example 1, but using in step 3 (S) -2- [ (S) -2-amino-3- (4-benzyloxy-phenyl) -propionylamino ] -6- { [ (4-methoxy-phenyl) -diphenyl-methyl ] -amino } -hexanoic acid (4-hydroxymethyl-phenyl) -amide instead of (S) -2- ((S) -2-amino-3- (4-nitrophenyl) propanamido) -N- (4- (hydroxymethyl) phenyl) -6- ((4-methoxyphenyl) diphenyl-methylamino) hexanamide as coupling partner. The former was prepared from (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -3- (4- (benzyloxy) phenyl) propionic acid as described in steps a ] -c ] above.

MS expected mass: 1466.8182, measured mass: 1466.8136.

example 3

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -4-cyano-L-phenylalanyl-N-6- [ (4-methoxyphenyl) (diphenyl) methyl ] -N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-lysyl amine

Prepared in analogy to example 1, but using in step 3 (S) -2- [ (S) -2-amino-3- (4-cyano-phenyl) -propionylamino ] -6- { [ (4-methoxy-phenyl) -diphenyl-methyl ] -amino } -hexanoic acid (4-hydroxymethyl-phenyl) -amide instead of (S) -2- ((S) -2-amino-3- (4-nitrophenyl) -propanamido) -N- (4- (hydroxymethyl) phenyl) -6- ((4-methoxyphenyl) diphenyl-methylamino) hexanamide as coupling partner. The former was prepared from (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -3- (4-cyanophenyl) propionic acid as described in steps a ] -c ] above.

MS expected mass: 1385.7716, measured mass: 1385.7696.

example 4

3, 4-dichloro-N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-phenylalanyl-N-6- [ (4-methoxyphenyl) (diphenyl) methyl ] -N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-lysyl-amine

Prepared in analogy to example 1, but using in step 3 (S) -2- [ (S) -2-amino-3- (3, 4-dichloro-phenyl) -propionylamino ] -6- { [ (4-methoxy-phenyl) -diphenyl-methyl ] -amino } -hexanoic acid (4-hydroxymethyl-phenyl) -amide instead of (S) -2- ((S) -2-amino-3- (4-nitrophenyl) -propanamido) -N- (4- (hydroxymethyl) phenyl) -6- ((4-methoxyphenyl) diphenyl-methylamino) hexanamide as coupling partner. The former was prepared from (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -3- (3, 4-dichlorophenyl) propionic acid as described in steps a ] -c above.

MS expected mass: 1428.6984, measured mass: 1428.695.

example 5

4-chloro-N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-phenylalanyl-N-6- [ (4-methoxyphenyl) (diphenyl) methyl ] -N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-lysyl amine

Prepared in analogy to example 1, but using in step 3 (S) -2- ((S) -2-amino-3- (4-chlorophenyl) propanamido) -N- (4- (hydroxymethyl) phenyl) -6- ((4-methoxyphenyl) diphenyl-methylamino) hexanamide instead of (S) -2- ((S) -2-amino-3- (4-nitrophenyl) -propanamido) -N- (4- (hydroxymethyl) phenyl) -6- ((4-methoxyphenyl) diphenyl-methylamino) hexanamide as coupling partner. The former was prepared from (S) -2- (((9H-fluoren-9-yl) methoxy) -carbonylamino) -3- (4-chlorophenyl) propanoic acid as described in steps a ] -c ] above.

MS expected mass: 1394.7373, measured mass: 1394.7342.

example 6

4- { [ (2S) -2- { [ (2S) -2- [ (4- { [3- ({ (3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- [ (2R) -6-methylhept-2-yl ] -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yl } oxy) propyl ] amino } -4-oxobutanoyl) amino ] -3- (naphthalen-1-yl) propanoyl ] amino } -6- { [ (4-methoxyphenyl) (diphenyl) methyl ] amino } hexanoyl ] amino } benzyl 4-nitrophenyl carbonate (non-preferred nomenclature).

Prepared in analogy to example 1, but using in step 3 (S) -2- ((S) -2-amino-3-naphthalen-1-yl-propionylamino) -6- { [ (4-methoxy-phenyl) -diphenyl-methyl ] -amino } -hexanoic acid (4-hydroxymethyl-phenyl) -amide instead of (S) -2- ((S) -2-amino-3- (4-nitrophenyl) -propanamido) -N- (4- (hydroxymethyl) phenyl) -6- ((4-methoxyphenyl) diphenyl-methylamino) hexanamide as coupling partner. The former was prepared from (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-1-yl) propionic acid as described in steps a ] -c ] above.

MS expected mass: 1410.792, measured mass: 1410.7918.

example 7

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -4-fluoro-L-phenylalanyl-N-6- [ (4-methoxyphenyl) (diphenyl) methyl ] -N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-lysyl amine

Prepared in analogy to example 1, but using in step 3 (S) -2- [ (S) -2-amino-3- (4-fluoro-phenyl) -propionylamino ] -6- { [ (4-methoxy-phenyl) -diphenyl-methyl ] -amino } -hexanoic acid (4-hydroxymethyl-phenyl) -amide instead of (S) -2- ((S) -2-amino-3- (4-nitrophenyl) -propanamido) -N- (4- (hydroxymethyl) phenyl) -6- ((4-methoxyphenyl) diphenyl-methylamino) -hexanamide as coupling partner. The former was prepared from (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -3- (4-fluorophenyl) propionic acid as described in steps a ] -c ] above.

MS expected mass: 1378.7669, measured mass: 1378.7609.

example 8

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -2-fluoro-L-phenylalanyl-N-6- [ (4-methoxyphenyl) (diphenyl) methyl ] -N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-lysyl amine

Prepared in analogy to example 1, but using in step 3 (S) -2- [ (S) -2-amino-3- (2-fluoro-phenyl) -propionylamino ] -6- { [ (4-methoxy-phenyl) -diphenyl-methyl ] -amino } -hexanoic acid (4-hydroxymethyl-phenyl) -amide instead of (S) -2- ((S) -2-amino-3- (4-nitrophenyl) -propanamido) -N- (4- (hydroxymethyl) phenyl) -6- ((4-methoxyphenyl) diphenyl-methylamino) -hexanamide as coupling partner. The former was prepared from (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -3- (2-fluorophenyl) propionic acid as described in steps a ] -c ] above.

MS expected mass: 1378.7669, measured mass: 1378.7689.

example 9

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -3-fluoro-L-phenylalanyl-N-6- [ (4-methoxyphenyl) (diphenyl) methyl ] -N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-lysyl amine

Prepared in analogy to example 1, but using in step 3 (S) -2- [ (S) -2-amino-3- (3-fluoro-phenyl) -propionylamino ] -6- { [ (4-methoxy-phenyl) -diphenyl-methyl ] -amino } -hexanoic acid (4-hydroxymethyl-phenyl) -amide instead of (S) -2- ((S) -2-amino-3- (4-nitrophenyl) -propanamido) -N- (4- (hydroxymethyl) phenyl) -6- ((4-methoxyphenyl) diphenyl-methylamino) -hexanamide as coupling partner. The former was prepared from (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -3- (3-fluorophenyl) propionic acid as described in steps a ] -c ] above.

MS expected mass: 1378.7669, measured mass: 1378.7659.

example 10

Step 1: n1- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylhept-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propyl) -N4- ((S) -1- (4-fluorophenyl) -4- ((S) -1- (4- (hydroxymethyl) phenylamino) -6- ((4-methoxyphenyl) diphenylmethylamino) -1-oxohex-2-ylamino) -4-oxobutan-2-yl) succinamide.

4- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propylamino) -4-oxobutanoic acid (109mg, 188. mu. mol, equivalents: 1.00), (S) -2- [ (S) -3-amino-4- (4-fluoro-phenyl) -butyrylamino ] -6- { [ (4-methoxy-phenyl) -diphenyl-methyl ] -amino } -hexanoic acid (4-hydroxymethylmethyle-nohexanoic acid), prepared above, was placed in a 10mL round-bottomed flask -phenyl) -amide (132mg,188 μmol, equivalents: 1.00), HOAt (25.6mg,188 μmol, equivalent: 1.00) and EDC hydrochloride (36.1mg,188 μmol, eq: 1.00) in CH2Cl2(2ml) were mixed together to give a yellow solution. Huenig base (48.7mg, 64.1. mu.l, 377. mu. mol, eq: 2.00) was added and the reaction stirred at ambient temperature overnight. TLC indicated the consumption of starting material. All volatiles were removed in vacuo and the crude product was purified by flash chromatography on a solution of SiO2/5% MeOH/0.1% NEt3 in CH2Cl2 to give 197mg of the title compound as an off-white solid.

MS expected mass: 1227.7763, measured mass: 1227.7714.

step 2- ((S) -3- (4- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propylamino) -4-oxobutanamido) -4- (4-fluorophenyl) butanamido) -6- ((4-methoxyphenyl) diphenylmethylamino) hexanamido) -benzyl 4-nitrophenyl carbonate.

In a 10mL round bottom flask, N1- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propyl) -N4- ((S) -1- (4-fluorophenyl) -4- ((S) -1- (4- (hydroxymethyl) phenylamino) -6- ((4-methoxyphenyl) diphenylmethylamino) -1-oxohex-2-ylamino) -4-oxobutan-2-yl) succinamide prepared above (3- ((3S,8S,9S,10R,13R,14S,17R) -1, 13-dimethyl-17- ((R) -6-methylheptan-3-yloxy) propyl) -N4- ((S) -1 196mg, 160. mu. mol, equivalent weight: 1.00) and Huenig base (61.9mg,81.4 μ l,479 μmol, equivalents: 3.00) was combined with CH2Cl2(1.6ml) and DMF (0.8ml) to give a yellow suspension; bis (4-nitrophenyl) carbonate (72.8mg,239 μmol, eq: 1.50) was added and the reaction was allowed to proceed overnight at ambient temperature. The mixture was poured onto crashed ice/NH 4Cl (pH 6), extracted 2 times with AcOEt, washed with H2O and brine, dried over Na2SO4, and evaporated to dryness. After trituration with AcOEt/heptane, 123mg of the title compound were obtained as a pale yellow solid.

MS expected mass: 1392.7825, measured mass: 1392.7819.

the necessary dipeptide building blocks for step 1 are prepared as follows:

step a (S) -2- [ (S) -3- (9H-fluoren-9-ylmethoxycarbonylamino) -4- (4-fluoro-phenyl) -butyrylamino ] -6- { [ (4-methoxy-phenyl) -diphenyl-methyl ] -amino } -hexanoic acid

In a 25mL round-bottomed flask, (S) -2-amino-6- ((4-methoxyphenyl) diphenylmethyl-amino) hexanoic acid (bioconjugateCHEm.2002,13,855-869,1040mg,2.48mmol, equivalent: 1.00) was dissolved in CH2Cl2(12.5mL) to give a pale yellow solution. Huenig base (961mg,1.27ml,7.44mmol, equiv: 3.00) and chlorotrimethylsilane (566mg, 621. mu.l, 5.21mmol, equiv: 2.10) were added and the reaction mixture was stirred at ambient temperature for 20 min.

In a second 50mL round bottom flask, (S) -3- (((9H-fluoren-9-yl) methoxy) carbonyl-amino) -4- (4-fluorophenyl) butanoic acid (1040mg,2.48mmol, eq: 1.00) was dissolved in DMF (12.5mL) to give a colorless solution. Huenig base (385mg, 506. mu.l, 2.98mmol, eq.: 1.20) and TPTU [125700-71-2 ] were added](737mg,2.48mmol, eq: 1.00) and the reaction mixture was stirred for 15 minutes. The solution in the first flask containing the corresponding silyl ester monosilylamine was added and the reaction was stirred at ambient temperature for an additional 3 hours. Pouring the mixture into crushed ice/NH₄Over Cl, extracted twice with AcOEt, washed with H2O and brine, dried over Na2SO4, and evaporated to dryness. Flash chromatography of a solution of SiO2/5% MeOH/0.1% NEt3 in CH2Cl2 afforded 2.10g of the title compound as a yellow foam.

MS(ISP):(M+H)820.6。

Step b { (S) -2- (4-fluoro-phenyl) -1- [ ((S) -1- (4-hydroxymethyl-phenylcarbamoyl) -5- { [ (4-methoxy-phenyl) -diphenyl-methyl ] -amino } -pentylcarbamoyl) -methyl ] -ethyl } -carbamic acid 9H-fluoren-9-ylmethyl ester

In a 250mL pear-shaped flask, { (S) -2- (4-fluoro-phenyl) -1- [ ((S) -1- (4-hydroxymethyl-phenylcarbamoyl) -5- { [ (4-methoxy-phenyl) -diphenyl-methyl-synthesized above]-amino } -pentylcarbamoyl) -methyl]-Ethyl } -carbamic acid 9H-fluoren-9-ylmethyl ester (2.10g,2.56mmol, eq: 1.00), (4-aminophenyl) methanol(315mg,2.55mmol, eq: 1.00), HOAt (349mg,2.56mmol, eq: 1.00) and EDC hydrochloride (491mg,2.56mmol, eq: 1.00) were dissolved in CH2Cl2(12.5 ml). Huenig base (662mg, 871. mu.l, 5.21mmol, eq: 2.00) was added and the reaction was allowed to proceed overnight. Pouring the mixture into crushed ice/NH₄Over Cl (pH 7), extracted 2 times with AcOEt, washed with H2O and brine, dried over Na2SO4, and evaporated to dryness. The crude product was triturated with diethyl ether (1 × 50 mL); the resulting solid was filtered off and dried to give 0.796g of the title compound as a beige solid.

MS(ISP):(M+H)925.6。

Step c (S) -2- [ (S) -3-amino-4- (4-fluoro-phenyl) -butyrylamino ] -6- { [ (4-methoxy-phenyl) -diphenyl-methyl ] -amino } -hexanoic acid (4-hydroxymethyl-phenyl) -amide

In a 50mL round bottom flask, { (S) -2- (4-fluoro-phenyl) -1- [ ((S) -1- (4-hydroxymethyl-phenylcarbamoyl) -5- { [ (4-methoxy-phenyl) -diphenyl-methyl-prepared above]-amino } -pentylcarbamoyl) -methyl]-Ethyl } -carbamic acid 9H-fluoren-9-ylmethyl ester (793mg,857 mol, eq: 1.001) was combined with THF (12ml) to give a brown solution. At 0 deg.C, diethylamine (1.13g,1.59ml,15.4mmol, eq: 18) was added. The reaction was stirred at ambient temperature overnight. Pouring the mixture into crushed ice/NH₄Over Cl (pH 7), extracted 2 times with AcOEt, washed with H2O and brine, dried over Na2SO4, and evaporated to dryness. Flash chromatography of a solution of SiO2/10% MeOH/0.1% NEt3 in CH2Cl2 afforded 500mg of the title compound as an off-white solid.

MS expected mass: 702.3581, measured mass: 702.3578.

example 11

4- ((S) -2- ((S) -3- (4- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propylamino) -4-oxobutanamido) -4-phenylbutanamido) -6- ((4-methoxyphenyl) diphenylmethylamino) hexanamido) benzyl 4-nitrophenyl carbonate

Prepared in analogy to example 10, but using in step 1(S) -2- ((S) -3-amino-4-phenylbutanoylamino) -N- (4- (hydroxymethyl) phenyl) -6- ((4-methoxyphenyl) diphenyl-methylamino) hexanamide instead of (S) -2- [ (S) -3-amino-4- (4-fluoro-phenyl) -butyrylamino ] -6- { [ (4-methoxy-phenyl) -diphenyl-methyl ] -amino } -hexanoic acid (4-hydroxymethyl-phenyl) -amide as coupling partner. The former was prepared from (S) -3- (((9H-fluoren-9-yl) methoxy) carbonylamino) -4-phenylbutyric acid as described in steps a ] -c ] above.

MS expected mass: 1374.792, measured mass: 1374.7877.

example 12

4- ({ N-2- [ (3S) -4- (4-chlorophenyl) -3- { [4- ({3- [ (3. beta) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] amino } butanoyl ] -N-6- [ (4-methoxyphenyl) (diphenyl) methyl ] -L-lysyl } amino) benzyl 4-nitrophenyl carbonate

Prepared in analogy to example 10, but using in step 1(S) -2- ((S) -3-amino-4- (4-chlorophenyl) butanamido) -N- (4- (hydroxymethyl) phenyl) -6- ((4-methoxyphenyl) -diphenylmethylamino) hexanamide instead of (S) -2- [ (S) -3-amino-4- (4-fluoro-phenyl) -butyrylamino ] -6- { [ (4-methoxy-phenyl) -diphenyl-methyl ] -amino } -hexanoic acid (4-hydroxymethyl-phenyl) -amide as coupling partner. The former was prepared from (S) -3- (((9H-fluoren-9-yl) methoxy) carbonylamino) -4- (4-chlorophenyl) -butanoic acid as described in steps a ] -c ] above.

MS(ISP):(M+H)1409.9。

Example 13

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -O-methyl-L-tyrosyl-N-6- [ (4-methoxyphenyl) (diphenyl) methyl ] -N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-lysylamide

Prepared in analogy to example 1, but using in step 3 (S) -2- ((S) -2-amino-3- (4-methoxyphenyl) propanamido) -N- (4- (hydroxymethyl) phenyl) -6- ((4-methoxyphenyl) -diphenylmethylamino) hexanamide instead of (S) -2- ((S) -2-amino-3- (4-nitrophenyl) -propanamido) -N- (4- (hydroxymethyl) phenyl) -6- ((4-methoxyphenyl) diphenyl-methylamino) hexanamide as coupling partner. The former was prepared from (S) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -3- (4-methoxyphenyl) propionic acid as described above in example 1, steps a ] -c ].

MS(ISP):(M+H)1391.9。

Example 14

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -D-phenylalanyl-N-6- [ (4-methoxyphenyl) (diphenyl) methyl ] -N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -D-lysylamide

Prepared in analogy to example 1, but using in step 3 (R) -2- ((R) -2-amino-3-phenyl-propionamido) -N- (4- (hydroxymethyl) phenyl) -6- ((4-methoxyphenyl) diphenylmethylamino) -hexanamide instead of (S) -2- ((S) -2-amino-3- (4-nitrophenyl) -propionamido) -N- (4- (hydroxymethyl) phenyl) -6- ((4-methoxyphenyl) diphenyl-methylamino) hexanamide as coupling partner. This building block was synthesized from (R) -2- (((9H-fluoren-9-yl) methoxy) carbonylamino) -6-aminocaproic acid and (R) -2-amino-6- ((4-methoxyphenyl) -diphenylmethylamino) hexanoic acid (cf. bioconjugateCHEm.2002,13,885-869) as described in steps a ] -c ] above.

MS expected mass: 1360.7763, measured mass: 1360.7774.

example 15

4- ({ N-2- [ [ (3S) -3- { [4- ({3- [ (3. beta) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] amino } -4- (4-cyanophenyl) butanoyl ] -N-6- [ [ (4-methoxyphenyl) (diphenyl) methyl ] -L-lysyl } amino) benzyl 4-nitrophenyl carbonate

Prepared in analogy to example 10, but using in step 1(S) -2- ((S) -3-amino-4- (4-cyanophenyl) butanamido) -N- (4- (hydroxymethyl) phenyl) -6- ((4-methoxyphenyl) diphenyl-methylamino) hexanamide instead of (S) -2- [ (S) -3-amino-4- (4-fluoro-phenyl) -butyrylamino ] -6- { [ (4-methoxy-phenyl) -diphenyl-methyl ] -amino } -hexanoic acid (4-hydroxymethyl-phenyl) -amide as coupling partner. The former was prepared from (S) -3- (((9H-fluoren-9-yl) methoxy) carbonylamino) -4- (4-cyanophenyl) butanoic acid as described in steps a ] -c ] above.

MS expected mass: 1399.7872, measured mass: 1399.7857.

example 16

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-phenylalanyl-N-6- [ (4-methoxyphenyl) (diphenyl) methyl ] -N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-lysyl-amine

Step 1:

(S) -2- ((S) -2-amino-3-phenyl-propionylamino) -6- { [ (4-methoxy-phenyl) -diphenyl-methyl ] -amino } -hexanoic acid (4-hydroxymethyl-phenyl) -amide

The structural unit (S) -2- ((S) -2-amino-3-phenyl-propionylamino) -6- { [ (4-methoxy-phenyl) -diphenyl-methyl ] -amino } -hexanoic acid (4-hydroxymethyl-phenyl) -amide is prepared analogously to the process described in bioconjugateCHEm, Vol.13, No.4,2002, 855-.

MS(ISP):(M+H)671.5

Step 2:

n1- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propyl) -N4- ((S) -1- (4- (hydroxymethyl) phenylamino) -6- ((4-methoxyphenyl) diphenylmethylamino) -1-oxohex-2-ylamino) -1-oxo-3-phenylpropan-2-yl) succinamide.

Mixing TPTU [125700-71-2](233mg, 784. mu. mol, equivalent: 1.00) was added N- {3- [ (3S,8S,9S,10R,13R,14S,17R) -17- ((R) -1, 5-dimethyl-hexyl) -10, 13-dimethyl-2, 3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ]]Phenanthren-3-yloxy]-propyl } -succinamic acid (see example 1, step 2) (426mg,0.784mmol, eq: 1.00) and Huenig base (304mg, 411. mu.l, 2.35mmol, eq: 3) in DMF (10 ml). After 3 min, (S) -2- ((S) -2-amino-3-phenyl-propionylamino) -6- { [ (4-methoxy-phenyl) -diphenyl-methyl-was added]-amino } -hexanoic acid (4-hydroxymethyl-phenyl) -amide (step 1), TLC showed complete reaction at t =1 h. The solvent was removed under reduced pressure. The remaining residue was dissolved in ethyl acetate and taken up with NaHCO₃Extraction with half-saturated solution (1X), 0.05M (2X) potassium hydrogen phthalate solution, water (1X) and brine (1X). The organic extract was extracted with MgSO₄Dried and concentrated under reduced pressure. The crude material was purified by flash chromatography to give the title product (682mg,513, μmol) as a light brown solid.

MS(ISP):(M+H)1196.8

And step 3:

huenig base (465mg, 629. mu.l, 3.6mmol, eq: 6) was added to a solution of the aforementioned alcohol (718mg, 600. mu. mol, eq: 1.00) and bis (4-nitrophenyl) carbonate (548mg,1.8mmol, eq: 3) in THF (20 ml). The yellow solution was stirred at room temperature overnight. The solvent was removed under reduced pressure. The remaining residue was triturated with ether. The solid was collected by filtration, washed with ether and dried under reduced pressure to give the title compound (800mg, 529. mu. mol) as a pale brown solid.

MS(ISP):(M+H)1361.9

Example 17

Step 1(S) -2- [ (S) -2- (9H-fluoren-9-ylmethoxycarbonylamino) -3-phenyl-propionylamino ] -hexanoic acid

Commercially available L-Fmoc-Phe-OSu (0.969g,2.00mmol, eq: 1.00) was suspended in a 1:1v/v mixture of 1, 2-dimethoxyethane and water (17ml) and treated with L-norleucine (0.275g,2.10mmol, eq: 1.05) and NaHCO at 0 deg.C₃(0.185g,2.20mmol, eq: 1.10). The cooling bath was removed and the reaction was allowed to proceed at ambient temperature for 14 hours. The mixture was poured onto crushed ice/citric acid (pH 3), extracted 2 times with ethyl acetate, washed with H2O and brine, dried over Na2SO4, and evaporated to dryness. Flash chromatography SiO2/AcOEt purification afforded 0.870mg of the title compound as a white solid.

MS(ISP):(M+H)501.2。

Step 2 { (S) -1- [ (S) -1- (4-hydroxymethyl-phenylcarbamoyl) -pentylcarbamoyl ] -2-phenyl-ethyl } -carbamic acid 9H-fluoren-9-ylmethyl ester

(S) -2- [ (S) -2- (9H-fluoren-9-ylmethoxy-carbonylamino) -3-phenyl-propionylamino) -synthesized above was placed in a pear-shaped flask]Hexanoic acid (10.72g,21mmol, eq: 1.00), (4-aminophenyl) methanol (2.717g,22mmol, eq: 1.03) and 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (EEDQ) (7.994g,32mmol, eq: 1.50) were dissolved in CH2Cl2(320ml) and stirred under argon overnight. Pouring the mixture into crushed ice/NH₄Over Cl, extracted 2 times with AcOEt, washed with H2O, dried over Na2SO4, and the volume concentrated to-300 ml. The precipitate was filtered off and dried to yield 5.25g of the title compound as a beige solid.

MS(ISP):(M+H)606.3。

Step 3 (S) -2- ((S) -2-amino-3-phenyl-propionylamino) -hexanoic acid (4-hydroxymethyl-phenyl) -amide

In a round-bottomed flask, { (S) -1- [ (S) -1- (4-hydroxymethyl-phenylcarbamoyl) -pentylcarbamoyl prepared above]-2-phenyl-ethyl } -carbamic acid 9H-fluoren-9-ylmethyl ester (4.738g,7.822mmol, eq: 1.0) dissolved in CH₂Cl₂(28 ml). At 0 deg.C, diethylamine (28ml,19.80g,271mmol, eq: 35) was added and the reaction mixture was stirred at ambient temperature overnight. All volatiles were removed by vacuum evaporation; flash chromatography purification with SiO2/CH2Cl2/10% MeOH followed by crystallization from AcOEt gave 2.116g of the title compound as light brown crystals.

MS(ISP):(M+H)384.2。

Step 4

N1- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propyl) -N4- ((S) -1- ((S) -1- (4- (hydroxymethyl) phenylamino) -1-oxohex-2-ylamino) -1-oxo-3-phenylpropan-2-yl) succinamide

Prepared in a similar manner to step 2 of example 16.

MS(ISP):(M+H)909.7(M+Na)931.8。

Step 5

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-phenylalanyl-N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-N-lysine amide

Prepared in a similar manner to step 3 of example 16.

MS expected mass: 1073.6453 found 1073.642

Example 18

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-alanyl-N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] glycinamide

Step 1:

FMOC-4-aminobenzyl alcohol was added to 2-chlorotrityl resin

2-Chlorotrityl chloride resin (Novabiochem01-64-0114,100-200 mesh), 1% DVB (18g,21.6mmol, equivalent: 1.00) was swollen in DCM/DMF =1/1(300mL) for 10 min. The resin was drained and a solution of FMOC-4-aminobenzyl alcohol (14.9g,43.2mmol, eq: 2) and pyridine (6.83g,6.99mL,86.4mmol, eq: 4) in DCM/DMF =1/1(300mL) was added. The mixture was shaken overnight. The resin was drained and capped with a 10% solution of Huenig base in methanol (300 mL). The resin was washed with DMF and DCM and dried overnight with HV to give 21.7g of resin. The loading was determined to be 0.41 mmoL/g.

Step 2:

n1- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propyl) -N4- ((S) -1- (2- (4- (hydroxymethyl) phenylamino) -2-oxoethylamino) -1-oxopropan-2-yl) succinamide

The resin obtained from step 1 (1g,410 μmol, eq: 1.00) was pre-washed with DMF (2X) and treated with piperidine/DMF =1/4(10mL) for 5 and 10 min. The resin was washed with DMF and IPA (3X10mL) in turn. A solution of Fmoc-Gly-OH (488mg,1.64mmol, eq: 4), TPTU (487mg,1.64mmol, eq: 4) and Huenig base (636mg, 859. mu.l, 4.92mmol, eq: 12) in DMF (10mL) was stirred for 5 min and then shaken with resin for one hour. The resin was washed with DMF and isopropyl alcohol (3X) in turn.

The following Fmoc cleavage and subsequent coupling of Fmoc-Ala-OH (511mg,1.64mmol, eq: 4) and N- {3- [ (3S,8S,9S,10R,13R,14S,17R) -17- ((R) -1, 5-dimethyl-hexyl) -10, 13-dimethyl-2, 3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy ] -propyl } -succinamic acid (example 1, step 2) (892mg,1.64mmol, eq: 4) were then carried out. The dried peptide resin was stirred in TFA1%/DCM (2X20mL) for approximately 2X30 min. The reaction mixture was filtered and the resin was washed with DCM. The filtrates were combined and the solvent was evaporated in vacuo. The crude material was triturated with ether (2 ×). After purification by flash chromatography, the product was obtained (84mg,97.3 μmol) as a white solid.

MS expected mass: 776.5452 found 776.5455

And step 3:

the alcohol N1- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propyl) -N4- ((S) -1- (2- (4- (hydroxymethyl) phenylamino) -2-oxoethylamino) -1-oxoprop-2-yl) succinamide [ RO5545270] (70mg,90.1 μmol, equivalent: 1.00) and bis (4-nitrophenyl) carbonate (137mg, 450. mu. mol, equivalent weight: 5) dissolved in DMF (4ml) at room temperature and purified with Huenig base (34.9mg,47.2 μ l,270 μmol, eq: 3) work up and the mixture was allowed to react overnight. The solvent was removed in vacuo. The resulting solid was triturated with ether. The solid was collected by filtration and washed with diethyl ether. The product was dried in vacuo to give the title compound (84mg, 80.2. mu. mol) as a light brown solid.

MS expected mass: 941.5514 found 941.5518

Example 19

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-leucyl-N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-methionine amide (methioninamide)

Step 1:

addition of FMOC-4-aminobenzyl alcohol to 2-chlorotrityl resin

Prepared in a similar manner to step 1 of example 18.

Step 2

N1- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propyl) -N4- ((S) -1- ((S) -1- (4- (hydroxymethyl) phenylamino) -4- (methylthio) -1-oxobutan-2-ylamino) -4-methyl-1-oxopent-2-yl) succinamide

Prepared in analogy to step 2 of example 18, using Fmoc-Met-OH (609mg,1.64mmol, eq: 4) and Fmoc-Leu-OH (580mg,1.64mmol, eq: 4) as amino acids.

The product was obtained (208mg, 210. mu. mol) as a pale yellow solid.

MS(ISP):(M+H)893.6183

Step 3

Prepared in a similar manner to step 3 of example 18. After purification on silica gel, the title compound (161mg,137 μmol) was obtained as a light brown solid.

MS expected mass: 1057.6174 found 1057.6184

Example 20

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-leucyl-N-1- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-asparagine

Step 1:

addition of FMOC-4-aminobenzyl alcohol to 2-chlorotrityl resin

Prepared in a similar manner to step 1 of example 18.

Step 2

(S) -2- ((S) -2- (4- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propylamino) -4-oxobutanamide) -4-methylpentamamido) -N1- (4- (hydroxymethyl) phenyl) succinamide

Prepared according to a similar method to step 2 in example 18, using Fmoc-Asn-OH (581mg,1.64mmol, eq: 4) and Fmoc-Leu-OH (580mg,1.64mmol, eq: 4) as amino acids.

The product was obtained (87mg, 89.4. mu. mol) as a pale yellow solid.

MS expected mass: 875.6136 found 875.6133

Step 3

The title compound was prepared in analogy to example 18, step 3. After purification on silica gel the title compound (87mg, 89.4. mu. mol) was obtained as a light brown solid.

MS expected mass: 1040.6198 found 1040.6188

Example 21

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-alanyl-N-1- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-asparagine

Step 1:

addition of FMOC-4-aminobenzyl alcohol to 2-chlorotrityl resin

Prepared in a similar manner to step 1 of example 18.

Step 2

(S) -2- ((S) -2- (4- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propylamino) -4-oxobutanoylamino) propanamido) -N1- (4- (hydroxymethyl) phenyl) succinamide

Prepared according to a similar method to step 2 of example 18, using Fmoc-Asn-OH (581mg,1.64mmol, eq: 4) and Fmoc-Ala-OH (511mg,1.64mmol, eq: 4) as amino acids.

The product was obtained (140mg, 159. mu. mol) as a pale yellow solid.

MS(ISP):(M+H)834.8(M+Na)856.7

Step 3

The title compound was prepared in analogy to example 18, step 3. After purification on silica gel the title compound (169mg, 152. mu. mol) was obtained as a light brown solid.

MS expected mass: 998.5729 found 998.5739

Example 22

N-2- [4- ({3- [ (3. beta) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-asparaginyl-N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] glycinamide

Step 1:

addition of FMOC-4-aminobenzyl alcohol to 2-chlorotrityl resin

Prepared in a similar manner to step 1 of example 18

Step 2

(S) -2- (4- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propylamino) -4-oxobutyramido) -N1- (2- (4- (hydroxymethyl) phenylamino) -2-oxoethyl) succinamide

Prepared according to a similar method to step 2 in example 18, using Fmoc-Gly-OH (488mg,1.64mmol, eq: 4) and Fmoc-Asn-OH (581mg,1.64mmol, eq: 4) as amino acids.

The product was obtained (140mg, 162. mu. mol) as a white solid.

MS expected mass: 819.551 found 819.5503

Step 3 the title compound (176mg, 161. mu. mol) was prepared as a light brown solid in analogy to step 3 of example 18.

MS expected mass: 984.5572 found 984.5489

Example 23

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-phenylalanyl-N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] glycinamide

Step 1:

addition of FMOC-4-aminobenzyl alcohol to 2-chlorotrityl resin

Prepared in a similar manner to step 1 of example 18.

Step 2:

n1- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propyl) -N4- ((S) -1- (2- (4- (hydroxymethyl) phenylamino) -2-oxoethylamino) -1-oxo-3-phenylpropan-2-yl) succinamide

Prepared according to a similar method to step 2 in example 18, using Fmoc-Gly-OH (488mg,1.64mmol, eq: 4) and Fmoc-Phe-OH (635mg,1.64mmol, eq: 4) as amino acids.

The product was obtained (259mg, 288. mu. mol) as a white solid.

MS expected mass: 852.5765 found 852.5754

Step 3

The title compound (280mg,247 μmol) was obtained as a pale brown solid in a similar manner to step 3 in example 18.

MS expected mass: 1017.5827 found 1017.5775

Example 24

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-leucinyl-N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] glycinamide

Step 1:

addition of FMOC-4-aminobenzyl alcohol to 2-chlorotrityl resin

Prepared in a similar manner to step 1 of example 18.

Step 2

N1- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propyl) -N4- ((S) -1- (2- (4- (hydroxymethyl) phenylamino) -2-oxoethylamino) -4-methyl-1-oxopent-2-yl) succinamide

Prepared according to a similar method to step 2 in example 18, using Fmoc-Gly-OH (488mg,1.64mmol, eq: 4) and Fmoc-Leu-OH (580mg,1.64mmol, eq: 4) as amino acids.

The product was obtained (240mg, 278. mu. mol) as a pale yellow solid.

MS expected mass: 818.5921 found 818.5921

Step 3

The title compound was prepared in analogy to example 18, step 3. Purification on silica gel gave the product (194mg, 177. mu. mol) as a pale yellow solid.

MS expected mass: 983.5983 found 983.6004

Example 25

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-leucinyl-N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-phenylpropanamide

Step 1:

addition of FMOC-4-aminobenzyl alcohol to 2-chlorotrityl resin

Prepared in a similar manner to step 1 of example 18.

Step 2

N1- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propyl) -N4- ((S) -1- ((S) -1- (4- (hydroxymethyl) phenylamino) -1-oxo-3-phenylpropan-2-ylamino) -4-methyl-1-oxopent-2-yl) succinamide

Prepared according to a similar method to step 2 in example 18, using Fmoc-Phe-OH (635mg,1.64mmol, eq: 4) and Fmoc-Leu-OH (580mg,1.64mmol, eq: 4) as amino acids.

The product was obtained (153mg, 151. mu. mol) as a pale yellow solid.

MS expected mass: 908.6391 found 908.637

And step 3:

the title compound was prepared in analogy to example 18, step 3. After purification on silica gel the product was obtained (117mg, 98. mu. mol) as a white solid.

MS expected mass: 1073.6453 found 1073.646

Example 26

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-phenylalanyl-N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-phenylpropanamide

Step 1:

addition of FMOC-4-aminobenzyl alcohol to 2-chlorotrityl resin

Prepared in a similar manner to step 1 of example 18.

Step 2

N1- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propyl) -N4- ((S) -1- ((S) -1- (4- (hydroxymethyl) phenylamino) -1-oxo-3-phenylpropan-2-ylamino) -1-oxo-3-phenylpropan-2-yl) succinamide

Prepared in analogy to example 18, step 2, using Fmoc-Phe-OH (635mg,1.64mmol, eq: 4) as amino acid.

The product was obtained (240mg, 204. mu. mol) as a pale yellow solid.

MS expected mass: 942.6234 found 942.6218

And step 3:

the title compound was prepared in analogy to example 18, step 3. After purification on silica gel the product was obtained (190mg, 154. mu. mol) as a white solid.

MS expected mass: 1107.6296 found 1107.6287

Example 27

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-leucinyl-N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-leucinamide

Step 1:

addition of FMOC-4-aminobenzyl alcohol to 2-chlorotrityl resin

The procedure was carried out analogously to step 1 in example 18.

Step 2

N1- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propyl) -N4- ((S) -1- ((S) -1- (4- (hydroxymethyl) phenylamino) -4-methyl-1-oxopent-2-ylamino) -4-methyl-1-oxopent-2-yl) succinamide

Prepared in analogy to example 18, step 2, using Fmoc-Leu-OH (1.59g,4.5mmol, eq: 3) as amino acid.

The product was obtained (254mg, 284. mu. mol) as a white solid.

MS expected mass: 874.6547 found 874.6527

Step 3

The title compound was prepared in analogy to example 18, step 3. After purification on silica gel the product was obtained (178mg, 168. mu. mol) as a white solid.

MS expected mass: 1039.6609 found 1039.6588

Example 28

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-alanyl-N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-alaninamide

Step 1

{ (S) -1- [ (S) -1- (4-hydroxymethyl-phenylcarbamoyl) -ethylcarbamoyl ] -ethyl } -carbamic acid 9H-fluoren-9-ylmethyl ester

A solution of Fmoc-Ala-Ala-OH (1g,2.61mmol, eq: 1.00) and (4-aminophenyl) methanol (483mg,3.92mmol, eq: 1.5) in THF (20ml) was treated with EEDQ (970mg,3.92mmol, eq: 1.5). The solution was stirred at room temperature overnight. The mixture was diluted with 10% 2-propanol/ethyl acetate (100mL) and the solution was washed with KHSO45%/K2SO410% (2X), water (1X) and brine (1X), dried over MgSO4 and evaporated in vacuo. The residue was sonicated in ether for a few minutes and the solid was collected by filtration to give the product (1.27g,1.2mmol) as a light brown solid.

MS(ISP):(M+H)488.3

Step 2:

(S) -2-amino-N- [ (S) -1- (4-hydroxymethyl-phenylcarbamoyl) -ethyl ] -propionamide

This compound was prepared in analogy to example 1, step c to give the product (245mg,877 μmol) as light yellow solid.

MS(ISP):(M+H)266.3,(M+Na)288.2(2M+H)531.3

And step 3:

n1- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propyl) -N4- ((S) -1- ((S) -1- (4- (hydroxymethyl) phenylamino) -1-oxoprop-2-ylamino) -1-oxoprop-2-yl) succinamide

This compound (165mg,198 μmol) was prepared as a light brown solid in analogy to example 16, step 2.

MS expected mass: 790.5608 found 790.5587

Step 4

The title compound was prepared in analogy to example 18, step 3. After purification on silica gel the product was obtained (99mg, 98.4. mu. mol) as a white solid.

MS expected mass: 955.567 found 955.5651

Example 29

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-isoleucyl-N-1- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-asparagine

Step 1

(S) -2- [ (2S,3S) -2- (9H-fluoren-9-ylmethoxycarbonylamino) -3-methyl-pentanoylamino ] -succinamic acid

The 2-chlorotrityl chloride resin (5g,7.5mmol, eq: 1.00) was swollen in DCM and subsequently treated overnight with a solution of Fmoc-Asn (Trt) -OH (8.95g,15.0mmol, eq: 2) and Huenig base (3.88g,5.1ml,30.0mmol, eq: 4) in DCM. The resin was washed with DCM and capped with 10% Huenig base in methanol. Coupling of Fmoc-Ile-OH (5.3g,15.0mmol, eq: 2) with TPTU (4.46g,15.0mmol, eq: 2) and Huenig base (3.88g,5.1ml,30.0mmol, eq: 4) was performed according to standard solid phase peptide synthesis methods. The product was cleaved from the resin with a combination of TFA/water/triisopropylsilane (95/2.5/2.5v/v/v) reagents at room temperature for two hours. The resin was filtered off and the filtrate was concentrated at least volumetrically under reduced pressure. After trituration with ether, the product was filtered and dried in vacuo to give the product (2.85g,5.79mmol) as a white solid.

MS expected mass: 467.2056 found 467.2056

Step 2

{ (1S,2S) -1- [ 2-carbamoyl-1- ((S) -4-hydroxymethyl-phenylcarbamoyl) -ethylcarbamoyl ] -2-methyl-butyl } -carbamic acid 9H-fluoren-9-ylmethyl ester

This compound (620mg,336 μmol) was prepared as a pale yellow solid in a similar manner to step 1 of example 28.

Step 3

(S) -2- ((2S,3S) -2-amino-3-methyl-pentanoylamino) -N x 1- (4-hydroxymethyl-phenyl) -succinamide

This compound (100mg, 228. mu. mol) was prepared as a pale yellow solid in analogy to example 1, step c.

Step 4

(S) -2- ((2S,3S) -2- (4- (3- ((3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- ((R) -6-methylheptan-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxy) propylamino) -4-oxobutanamido) -3-methylpentamamido) -N1- (4- (hydroxymethyl) phenyl) succinamide

This compound (89mg, 91.4. mu. mol) was prepared as a pale yellow solid in a similar manner to step 2 in example 16.

Step 5

The title compound was obtained from the compound obtained in the above step by following a reaction similar to step 3 in example 18. After purification on silica gel the product was obtained (42mg, 36.3. mu. mol) as a light brown solid.

MS expected mass: 1040.6198 found 1040.6177

Example 30

N- [4- ({3- [ (3 β) -cholest-5-en-3-yloxy ] propyl } amino) -4-oxobutanoyl ] -L-phenylalanyl-N-6- [ (4-methoxyphenyl) (diphenyl) methyl ] -N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -D-lysylamide

This compound (158mg,116 μmol) was prepared as a light brown solid starting from Fmoc-D-Lys (Boc) -OH according to a similar procedure as in step 1 of example 16.

MS(ISP):(M+H)1362.8(M+Na)1383.8

Example 31

N- {15- [ (3 β) -cholest-5-en-3-yloxy ] -4, 15-dioxo-8, 11-dioxa-5, 14-diazepin-1-acyl } -L-phenylalanyl-N.6- [ (4-methoxyphenyl) (diphenyl) methyl ] -N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-lysyl amine

The title compound was prepared in analogy to example 16, using the cholesterol-oligo-PEG derivative in step 2 of the synthesis method.

MS(ISP):(M+H)1479.8

The cholesterol-PEG intermediate N- [2- (2- {2- [ (3S,8S,9S,10R,13R,14S,17R) -17- ((R) -1, 5-dimethyl-hexyl) -10, 13-dimethyl-2, 3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] phenanthren-3-yloxycarbonylamino ] -ethoxy } -ethoxy) -ethyl ] -succinamic acid necessary in step 2 was prepared as follows:

step a{2- [2- (2-amino-ethoxy) -ethoxy ] -ethyl]-ethyl } -carbamic acid (3S,8S,9S,10R,13R,14S,17R) -17- ((R) -1, 5-dimethyl-hexyl) -10, 13-dimethyl-2, 3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ] a]Phenanthren-3-yl esters

A solution of cholesteryl chloroformate (1g,2.23mmol) in 25mL of dichloromethane was added dropwise, with stirring, to a solution of 2, 2' - (ethylenedioxy) di- (ethylamine) (495mg,3.34mmol) in 75mL of dichloromethane. The reaction was stirred at room temperature overnight. The reaction was diluted with dichloromethane and extracted with water. The organic extracts were dried over anhydrous MgSO4 dihydrate, filtered and evaporated. After purification on amino-modified silica gel (eluent: MeCl2- > MeCl2/MeOH =975:25v/v) the product was obtained (615mg) as a white waxy solid.

MS(ISP):(M+H)561.5

Step bN- [2- (2- {2- [ (3S,8S,9S,10R,13R,14S,17R) -17- ((R) -1, 5-dimethyl-hexyl) -10, 13-dimethyl-2, 3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ]]Phenanthrene-3-yloxycarbonylamino]-ethoxy } -ethoxy) -ethyl]-succinamic acid

Will come fromStep aThe amine (480mg,0.856mmol) and triethylamine (0.13mL,0.94mmol) were dissolved in 5mL of dichloromethane. Succinic anhydride (90mg,0.9mmol) was added to the solution, which was then stirred at room temperature overnight. TLC detection showed still some starting material. More succinic anhydride (20mg,0.2mmol) was added. After stirring the reaction at room temperature for a further 3 hours, it was diluted with dichloromethane and washed with a 5% KHSO4/10% K2SO4 mixture. The organic extract was dried over anhydrous MgSO4 dihydrate, filtered and evaporated in vacuo to give the desired acid (490mg,0.667 mmol).

MS(ISP):(M+H)661.5

Example 32

N- {30- [ (3 beta) -cholest-5-en-3-yloxy ] -4, 30-dioxo-8, 11,14,17,20,23, 26-heptaoxa-5, 29-diazatridecan-1-yl } -L-phenylalanyl-N-6- [ (4-methoxyphenyl) (diphenyl) methyl ] -N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-lysyl-amine

The title compound was prepared in analogy to example 16, using the cholesterol-PEG derivative in step 2 of the synthesis.

MS(ISP):(M+H)1699.9

The cholesterol-PEG intermediate 1- [ (3 β) -cholest-5-en-3-yloxy ] -1, 27-dioxo-5, 8,11,14,17,20, 23-heptaoxa-2, 26-diazatritriacontane-30-oic acid necessary in step 2 was prepared according to the following procedure:

step a[25- ({ (3S,8S,9S,10R,13R,14S,17R) -10, 13-dimethyl-17- [ (2R) -6-methylhept-2-yl group ]]2,3,4,7,8,9,10,11,12,13,14,15,16, 17-tetradecahydro-1H-cyclopenta [ a ]]Phenanthren-3-yl } oxy) -25-oxo-3, 6,9,12,15,18, 21-heptaoxa-24-azapentacosan-1-yl]Carbamic acid tert-butyl ester

Cholesterol chloroformate (476mg,1.06mmol) and triethylamine (155uL,1.113mmol) were dissolved in 5mL of dichloromethane. Then a solution of α -amino- ω -boc-amino-octa (ethylene glycol) (497mg,1.06mmol) dissolved in 1mL of dichloromethane was added. The solution was stirred at room temperature overnight, diluted with dichloromethane and extracted with an aqueous mixture of KHSO45%/K2SO 410%. The organic extracts were dried over anhydrous MgSO4, filtered and evaporated in vacuo. Purification on silica gel (eluent: MeCl2/MeOH =975:25- >95:5v/v) gave the product (530mg,0.571mmol) as a colorless oil.

MS(ISP):(M+NH4)898.7

Step b1- [ (3. beta) -cholest-5-en-3-yloxy]-1, 27-dioxo-5, 8,11,14,17,20, 23-heptaoxa-2, 26-diazatritriacontane-30-oic acid

The Boc derivative described above (450mg,0.511mmol) was dissolved in a solution of HCl4M in dioxane (10.2mL,40.9 mmol). The solution was stirred at room temperature for 40 minutes. The solvent was removed in vacuo and the remaining white solid was dissolved in 5mL of dichloromethane and treated with triethylamine (32uL,0.229mmol) and succinic anhydride (11.5mg,0.114mmol) overnight. More succinic anhydride (11mg,0.11mmol,0.2 equiv.) was added and after 60 min the reaction was diluted with dichloromethane and washed with KHSO45%/K2SO410% buffer. The organic extracts were dried over anhydrous MgSO4, filtered and evaporated to give 390mg of the desired product.

MS(ISP):(M+H)881.7

Example 33

N- {66- [ (3 β) -cholest-5-en-3-yloxy ] -4, 66-dioxo-8, 11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59, 62-nonaoxa-5, 65-diazahexadecane-1-acyl } -L-phenylalanyl-N-6- [ (4-methoxyphenyl) (diphenyl) methyl ] -N- [4- ({ [ (4-nitrophenoxy) carbonyl ] oxy } methyl) phenyl ] -L-lysyl-amine

The title compound was prepared in analogy to example 16, using the cholesterol-PEG derivative in step 2 of the synthesis.

MS(ISP):(M+H)2228.1

The cholesterol-PEG intermediate 1- [ (3 β) -cholest-5-en-3-yloxy ] -1, 63-dioxo-5, 8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56, 59-nonaoxa-2, 62-diazahexadecane-66-oic acid necessary in step 2 was prepared according to the following procedure:

step a(3. beta) -cholest-5-en-3-yl) carbamate (59-amino-3, 6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57 nonadecaoxapentadecan-1-yl) carbamate

α, ω -di-amino-20 (ethylene glycol) (538mg,0.6mmol) and triethylamine (92uL,0.66mmol) were dissolved in 15mL of anhydrous dichloromethane. A solution of cholesteryl chloroformate (270mg,0.6mmol) in 2mL of anhydrous dichloromethane was added dropwise at room temperature. The solution was stirred overnight, then at least the volume was concentrated in vacuo and purified directly on silica gel (eluent: MeCl2/MeOH =95:5- >9:4- >4:1v/v) to give the product (350mg,0.254mmol) as a waxy solid.

MS(ISP):(M+H)1309.9

Step b1- [ (3. beta) -cholest-5-en-3-yloxy]-1, 63-dioxo-5, 8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56, 59-nonadecaoxa-2, 62-diazahexadecane-66-oic acid

Will come fromStep aWas dissolved in 5mL of anhydrous dichloromethane, and was dissolved in (329mg,0.251mmol) succinic anhydride (26.4mg,0.264mmol) and triethylamine (40uL,0.286 mmol). After addition of more triethylamine (40uL,0.286mmol), the solution was washed (pH)>8) Stir at room temperature overnight. The reaction was diluted with dichloromethane and washed twice with an aqueous mixture of KHSO45%/K2SO 410%. The organic extracts were dried over anhydrous MgSO4, filtered and evaporated to give the product (260mg,0.175mmol) as a colourless waxy solid.

MS(ISP):(M+NH4)1408.9

The following working examples serve to illustrate the invention:

example 34 general procedure for preparation of RNA conjugates

Material

Dimethyl sulfoxide (DMSO), N-Diisopropylethylamine (DIPEA) and sodium acetate solution (3M, pH5.2) were purchased from Sigma Aldrich Chemie GmbH (Traufkirchen, Germany).

Triethylammonium acetate (TEAA) (2.0M, pH7.0) and acetonitrile (ACN, HPLC quality) for RP-HPLC were purchased from Biosolve (Valkenswaard, the Netherlands).

Ethanol (EtOH, analytical grade) was purchased from Merck (Darmstadt, germany). Pure water was obtained from the optilab hf (MembraPure, germany) system used.

ResourceRPC3mL column (10X0,64 cm; 15 μm particle size) was purchased from GEHealthcare (Freiburg, Germany).

HPLC purification is carried out usingExplorer100 system (GEHealthcare) was completed.

Synthesis of amino-modified RNA

RNA with hexylamino linker at 5' -end of sense strand was used on 1215. mu. mol scale according to standard phosphoramidite solid phase synthesis methodOligopilot100(GEHealthcare, Freiburg, Germany) and controlled pore glass as solid support (PrimeSynthesis, Aston, Pa., USA). RNA containing 2 '-O-methyl nucleotides was generated using the corresponding phosphoramidite, 2' -O-methyl phosphoramidite and TFA-hexylamino linker phosphoramidite (Sigma-Aldrich, SAFC, Hamburg, Germany). Cleavage and deprotection, as well as purification, are accomplished by methods known in the art (WincottF., et al, NAR1995,23,14, 2677-84).

The amino-modified RNA was identified by anion exchange HPLC (purity: 96.1%) and the identity was verified by ESI-MS ([ M + H ]1+ calculated: 6937.4; [ M + H ]1+ measured mass: 6939.0.

The sequence is as follows: 5 '- (NH2C6) GGAAUCuAuuAuuGAUCcAsA-3'; u, c are 2' -O-methyl nucleotides corresponding to RNA nucleotides and s is phosphorothioate.

General Experimental conjugation methods

The title compounds of examples 1-33 were coupled to amino-modified RNA according to the following method:

RNA with a C-6 amino linker at the 5' -end (16.5mg,1 eq) was dissolved in 500. mu.L LDMSO and 150. mu.L water. A solution of the p-nitrophenyl carbonate derivative (10 equivalents) in 1mL of LDMSO was added, followed by 8 μ of LDIPEA. The reaction mixture was shaken in the dark at 35 ℃ and monitored by RP-HPLC (ResourceRPC3mL, buffer: A:0.1 aqueous MTEAA, B:0.1 aqueous MTEAA in 95% ACN, gradient: from 3% B to 100% B over 20 column volumes). When the reaction was complete, the RNA conjugate was precipitated with a solution of sodium acetate (3M) in EtOH at-20 ℃. For example, lacking MMT protecting groups in the dipeptide motif, the corresponding conjugate is purified using the conditions described above. The purified fractions were combined and the material was precipitated with sodium sulfate/EtOH to give the desired RNA conjugate.

The RNA conjugates comprising MMT protecting groups in the dipeptide sequence were further processed according to the method described below.

General procedure for MMT cleavage

The crude RNA conjugate pellet was dissolved in 500. mu.L of water and 1.5mL of sodium acetate buffer (3M, pH5.2 or 0.1M, pH 4.0). The solution was shaken at 30 ℃ for 2 days. The reaction mixture was monitored by RP-HPLC (resource RPC3mL, buffer: A:0.1 aqueous MTEAA, B:0.1 aqueous MTEAA in 95% ACN, gradient: 3% B-100% B, 20 column volumes). After complete cleavage of the MMT protecting group, the RNA conjugate is directly purified using the conditions described above. The purified fractions were combined and the desired conjugate was precipitated with sodium sulfate/EtOH.

An RNA conjugate lacking the dipeptide motif was synthesized as a control. For this purpose, cholesterol is linked to the 5' -end via a linker as described in the literature (NatureBiotech,2007,25, 1149). This conjugate is referred to as "non-cleavable".

All RNA conjugates were purity analyzed by RPHPLC and verified by ESIMS (negative ion mode). Briefly, RP-HPLC isIn the presence of XBridgeC₁₈The column (2.5X50mm,2.5 μm particle size, Waters, Eschborn, Germany) was carried out at a column temperature of 65 ℃ on a Dionex Ultimate system (Dionex, Idstein, Germany). The gradient elution was carried out with 100mM Hexafluoroisopropanol (HFIP) and 16mM triethylamine in 1% methanol as eluent A and its solution in 95% methanol as eluent B (1% B to 18% B over 30 min). UV detection was recorded at 260 nm. For mass spectrometry a ThermoFinniganlcQDecaXPESI-MS system with a microjet source and an ion trap detector were coupled on-line to an HPLC system.

Examples of specific compounds of formula (IIa) are disclosed in table 1. The resulting compound is referred to as "dipeptide-containing cholesterol siRNA conjugate", wherein a specific dipeptide-containing cholesterol siRNA conjugate is also referred to as "title compound example X- (NHC6) - (siRNA sequence)" and "siRNA with the title compound of example X".

SiRNA preparation

Antisense sequence of 5 '-uuGGAUcAAAuAuAAGAuUCcscsu-3'

u, c 2' -O-methyl nucleotides corresponding to RNA nucleotides, s phosphorothioate

The cholesterol siRNA conjugate containing a dipeptide against apolipoprotein BmRNA is produced by mixing equimolar solutions of the complementary strands in an annealing buffer (20mM sodium phosphate, pH 6.8; 100mM sodium chloride), heating at 80-85 ℃ for 3 minutes in a water bath, and cooling to room temperature over a period of 3-4 hours. The formation of the double strand was confirmed by non-denaturing gel electrophoresis.

All prepared cholesterol siRNA conjugates comprising the dipeptide are listed in table 2.

TABLE 1 Cholesterol siRNA conjugates comprising dipeptides (5 '-3') and analytical data. Note that: the lower case letters a, c, g, u are 2' -O-methyl nucleotides; phosphorothioate linkages are indicated by the lower case "s" symbol. (NHC6) is an aminohexyl linker introduced at the 5' -end of the sense strand.

Note: TitlecompoundEx in the above table refers to the title compound, example No.

TABLE 2 Cholesterol siRNA conjugates comprising a dipeptide. The last bar (seq id no pair 266/154) represents an siRNA conjugate lacking a dipeptide motif. Note that: the lower case letters a, c, g, u are 2' -O-methyl nucleotides; phosphorothioate linkages are indicated by the lower case "s" symbol. (NHC6) is an aminohexyl linker introduced at the 5' -end of the sense strand.

Example 35 in vivo assay

In vivo combined administration of cholesterol siRNA conjugates comprising a dipeptide and a delivery polymer

Six to eight weeks old mice (strain C57BL/6 or ICR, 18-20g each) were obtained from Harlan Sprague Dawley (Indianapolisin). Mice were housed for at least 2 days prior to injection. Chow ad libitum with Harlan teklad rodent diet (Harlan, MadisonWI).

Mice (n =3 per group) were injected with 0.2mL of a solution of delivery polymer and 0.2mL of cholesterol siRNA conjugate comprising a dipeptide. Unless otherwise indicated, the injected dose was 15mg/kg delivery polymer and 0.1mg/kg cholesterol siRNA conjugate comprising dipeptide. The solution was injected into the tail vein by infusion. 48 hours after injection, ApoB levels were measured and compared to animals treated with isotonic glucose as described below.

Serum ApoB level determination

Mice were fasted for 4 hours before collecting serum with mandibular bleeding. Serum ApoB protein levels were determined by standard sandwich ELISA methods. Briefly, polyclonal goat anti-mouse ApoB antibody and rabbit anti-mouse ApoB antibody (biodesign international) were used as capture antibody and detection antibody, respectively. HRP-conjugated goat anti-rabbit IgG antibody (Sigma) was then used to bind the ApoB/antibody complex. The change in absorbance of the tetramethyl-benzidine (TMB, Sigma) colorimetric assay was then detected at 450nm using a TecanSafire2 (Austria, Europe) full-automatic quantitative-plotter microplate reader.

In fig. 1, a number of dipeptide-containing cholesterol siRNA conjugates were benchmark tested in this section with the same siRNA previously prepared and conjugated to cholesterol but lacking a cleavable motif. The effect of the siRNA conjugate (pair of seq id no266/154, "non-cleavable control") on serum ApoB levels was set to 1 to evaluate the effect of the conjugate comprising the dipeptide relative to the non-cleavable control. Replacing the initially used Phe-Lys motif (siRNA with the title compound of example 16) with the corresponding D-amino acid (siRNA with the title compound of example 14) or replacing Lys with the unnatural enantiomer (siRNA with the title compound of example 30) yielded sirnas with ApoB levels that were not significantly reduced or equivalent to non-cleavable controls. Replacement of Lys with Gly (siRNA with the title compound of example 23) or Phe with p-methoxyphenylalanine (siRNA with the title compound of example 13) decreased potency compared to siRNA with the title compound of example 16. Other siRNA conjugates containing dipeptide motifs were shown to have the same potency as the original Phe-Lys containing conjugate.

FIG. 2 summarizes dipeptide-containing cholesterol siRNA conjugates with equal or improved potency as compared to siRNAs containing a Phe-Lys motif with the title compound of example 16. All these conjugates were significantly more active than the "non-cleavable" cholesterol siRNA conjugate seqind no266/154 pair. The best-effective cholesterol siRNA conjugates containing dipeptides had fluorine-modified benzene rings (siRNA with the title compound of example 8, siRNA with the title compound of example 9) or phenylalanine substituted with β -phenylalanine (siRNA with the title compound of example 11) or derivatives thereof (siRNA with the title compound of example 10) in the Phy-Lys motif.

Since the cholesterol siRNA conjugates comprising a dipeptide sequence consisting of D-amino acids showed the same potency as the non-cleavable control conjugate, it is believed that the other dipeptide sequence is actually cleaved by the protease activity in vivo. However, given the wide acceptance of different amino acids and their analogous derivatives, it is possible that more than one enzyme may be involved in the cleavage reaction, as proposed in the literature (bioconjugatechem.2002,13,855).

As shown in figure 3, the introduction of a cathepsin-cleavable dipeptide motif (in this case Phe-Lys, siRNA with the title compound of example 16) between siRNA and small molecule ligand cholesterol significantly improved the potency of the siRNA conjugate compared to a direct cholesterol siRNA conjugate (seq id no266/154 pair). The cholesterol ligand is further spaced from the dipeptide motif by a PEG-based linker, which decreases potency proportionally with the length of the PEG linker.

In FIG. 4, the dose of polymer was kept constant at 15 mg/kg. The siRNA dose was titrated and the effect on serum ApoB content was examined. The cholesterol siRNA conjugates containing the dipeptide containing the Phe-Lys (F-K) motif were significantly more effective than the control conjugate lacking the dipeptide sequence.

Example 36 Synthesis of 2' -modified oligoribonucleotides

Oligoribonucleotides were synthesized according to the phosphoramidite technique on solid phase. On a scale basis, ABI394 synthesizers (applied biosystems) or AKTAoligopilot100 (GE) were usedHealthcare, Freiburg, Germany). The synthesis was carried out using controlled pore glass (CPG,loading 75 μmol/g, obtained from prime synthesis, Aston, PA, usa). All 2' -modified RNA phosphoramidites and auxiliary reagents were purchased from SAFC (Hamburg, Germany). Specifically, the following 2' -O-methylphosphorous acid amides were used: (5' -O-dimethoxytrityl-N⁶- (benzoyl) -2 ' -O-methyl-adenosine-3 ' -O- (2-cyanoethyl-N, N-diisopropylamino) phosphoramidite, 5 ' -O-dimethoxytrityl-N⁴- (acetyl) -2 ' -O-methyl-cytidine-3 ' -O- (2-cyanoethyl-N, N-diisopropylamino) phosphoramidite, (5 ' -O-dimethoxytrityl-N²- (isobutyryl) -2 ' -O-methyl-guanosine-3 ' -O- (2-cyanoethyl-N, N-diisopropylamino) phosphoramidite and 5 ' -O-dimethoxytrityl-2 ' -O-methyl-uridine-3 ' -O- (2-cyanoethyl-N, N-diisopropylamino) phosphoramidite. 2 ' -deoxy-2 ' -fluoro-phosphoramidites with the same protecting groups as 2 ' -O-methyl RNA phosphoramidites. All phosphoramidites were dissolved in anhydrous acetonitrile (100mM) and molecular sieves were addedTo form the 5 '-phosphate ester, 2- [2- (4, 4' -dimethoxytrityloxy) ethylsulfonyl from GlenResearch (Sterling, Virginia, USA) was used]Ethyl- (2-cyanoethyl) - (N, N-diisopropyl) -phosphoramidite. To introduce the C-6 amino linker at the 5' -end of the oligomer, 6- (trifluoroacetylamino) -hexyl- (2-cyanoethyl) - (N, N-diisopropyl) -phosphoramidite from ThermoFisher scientific (Milwaukee, Wisconsin, USA) was used. The introduction of the 5' -modification was not modified by any synthesis cycles. 5-Ethylthiotetrazole (ETT,500mM in acetonitrile) was used as the activator solution. The coupling time was 6 minutes. To introduce phosphorothioate linkages, 50mM of 3- ((dimethylamino-methylene) amino) -3H-1,2, 4-dithiazole-3-thione (DDTT, available from AMChemicals, Oceanside, Calif., USA) in anhydrous acetonitrile/pyridine (1:1v/v) was usedAnd (4) liquid.

Example 37 cleavage and deprotection of an oligomer bound to a vector.

After the solid phase synthesis is complete, the dried solid support is transferred to a 15mL tube and treated with concentrated ammonia (Aldrich) for 18 hours at 40 ℃. After centrifugation, the supernatant was transferred to a new tube and the CPG was washed with ammonia. The combined solutions were evaporated and the solid residue was reconstituted in buffer a (see below).

Example 38 purification of oligoribonucleotides

The crude oligomer was purified by anion exchange HPLC using a column packed with SourceQ15 (gehelthiocarbare) and an AKTAExplorer system (gehelthiocarbare). Buffer a was 10mM sodium perchlorate, 20mM tris, 1mM edta, ph7.4(Fluka, Buchs, switzerland) and contained 20% acetonitrile, while buffer B contained 500mM sodium perchlorate as identical to buffer a. Elution was performed using a gradient of 22% B to 42% B with 32 Column Volumes (CV). The UV trace at 280nm was recorded. Appropriate fractions were combined and precipitated with 3m naoac, pH =5.2 and 70% ethanol. Finally, the precipitate was washed with 70% ethanol.

Example 39 annealing of oligoribonucleotides to generate siRNA

The complementary strands are mixed by combining equimolar solutions of RNA. The mixture was lyophilized and reconstituted with the appropriate volume of annealing buffer (100mM NaCl,20mM sodium phosphate, pH6.8) to achieve the desired concentration. The solution was placed in a water bath at 85 ℃ and cooled to room temperature over 3 hours.

Example 40 in vitro Activity of siRNA lacking 2' -OH residue

To investigate whether siRNA lacking any 2' -OH residue showed the presence in vitroEfficient knock-down deactivation, we examined a panel of sirnas targeting EGFPmRNA with different 2' -modification chemistries (seq id pairs 31/32 to 149/150, and see e.g. table 3). Use of psiCHECK2 vector (Promega) with Dual-Glo in COS7 cells (DSMZ, Braunschweig, Germany, Cat. No. ACC-60)The luciferaseassaysystem (promega) screens the siRNA for sense and antisense activity. To satisfy the silencing activity conferred by the sense and antisense strands, we cloned the target site sequence of each corresponding 19mer as a different psiCHECK2 construct (psiCHECK2-AT for antisense activity, psiCHECK2-ST for sense activity) into the polyclonal region located 3' to the translation stop codon of the synthetic anthlemia luciferase. COS7 cells were co-transfected with the vector construct and 3nM of the corresponding siRNA complementary to the cloned target site by using Lipofectamine2000(Invitrogen GmbH, Karlsruhe, Germany, Cat. No. 11668-019). Successful siRNA mediated silencing was determined 24 hours post transfection by the florist luciferase activity, normalized to firefly luciferase levels, to investigate its transfection efficiency (antisense activity see figure 5a, sense activity see figure 5 b).

TABLE 3 exemplary siRNA sequences and chemical modifications used to determine in vitro knockdown activity dependent on 2' -modification. Examples of reference to double strands and selection of corresponding modified variants were used in this study. Xf refers to the 2 '-fluoro modification of nucleotide X, lower case letters refer to the 2' -O-methyl modification, underlined letters refer to DNA nucleotides, and all other capital letters refer to ribonucleotides. The letter "p" refers to 5' -phosphate.

It has been found that the 5 most effective modified siRNA (. gtoreq.60% knockdown) are designed in a selected 2 '-fluoro/2' -O-methyl (2 'F/2' -OMe) pattern. While conferring antisense strand activity, the chemical modification completely abolished the activity of the corresponding sense strand, as indicated by the loss or minimization of the florida luciferase activity for all of the 2 'F/2' -OMe variants tested.

We conclude that this 2 'F/2' -OMe pattern enhances the expected antisense strand activity of siRNA, and completely inhibits the undesired off-target effects from the sense strand.

Example 41 detection of DNaseII sensitive sites by in vitro assay

Methods based on ion-pair (IP) Reverse Phase (RP) High Performance Liquid Chromatography (HPLC) coupled with electrospray ionization (ESI) Mass Spectrometry (MS) or Anion Exchange (AEX) -HPLC were established to detect the in vitro stability of selected single-stranded or double-stranded RNAs.

Description of the method: for stability analysis, 10. mu.M solutions of single-or double-stranded RNA were incubated at 37 ℃ in 5mM sodium acetate buffer solution (pH4.5) containing 0.8 or 8 units of DNaseII (from bovine spleen, type V, SigmaAldrich). The incubation reaction was terminated by adding a 100mM solution of triethylammonium acetate (TEAA), the pH was adjusted to 7, and the dnase ii enzyme was inactivated. The analysis was done by LC/MS connected to a UV detector or by AEX-HPLC with a UV detector. UV detection at 260nm was followed for quantitative analysis, and MS data was used to determine cleavage sites in RNA sequences.

A. Use of Waters XBridgeC at a column temperature of 65 ℃₁₈Column (2.5X50mm,2.5 μm particle size) was subjected to IP-RP-HPLC. Gradient elution was performed using a solution of 100mM Hexafluoroisopropanol (HFIP) and 16mM triethylamine in 1% methanol as eluent A and a solution of composition A in 95% methanol as eluent B. The gradient used was from 1% B to 18% B over 30 minutes.

B. AEX-HPLC was performed at 50 ℃ on a DionexDNAPac200 column (4x250mM) using 20mM phosphate buffer pH =11 containing 10% ACN. Eluent B comprises a solution of 1m nabr in eluent a. Gradient from 25 to 62% B over 18 min.

TABLE 4 evaluation of stability of double stranded and other intact strands against DNaseII. Note that: the lower case letters a, c, g, u are 2' -O-methyl nucleotides; the capital A, C, G, U followed by "f" refers to a 2' -fluoro nucleotide. The lower case letter "p" refers to 5' -phosphate. (invdT) represents inverted deoxythymidine (3 '-3' -linkage). Phosphorothioate linkages are indicated by the lower case symbol "s". dT is deoxythymidine. (NHC6) is an aminohexyl linker introduced at the 5' end of the sense strand.

And (4) conclusion:

A. the RNA strand comprising at least one 2 '-OH nucleotide (e.g., both strands of seq id no pair 157/158) is rapidly degraded by the cyclic pentavalent intermediate, resulting in a 2' -3 'cyclic phosphate at the 5' -cleavage product. The formation of the pentavalent intermediate can be inhibited by using nucleotides lacking a2 '-OH group (e.g., 2' -deoxy, 2 '-OMe or 2' -F).

B. In addition, RNA is degraded via the 5 ' -exonucleolytic pathway, which is independent of 2 ' -modifications of the 5 ' -terminal nucleotide. This degradation pathway can use a 5' -terminal non-nucleotide moiety to inhibit, for example, a C6-amino linker (e.g., seq id no160 in seq id No. pair 160/159 or seq id no165 in seq id No. pair 165/166) or a phosphorothioate at the first internucleotide linkage (e.g., seq id no160 in seq id No. pair 160/159).

The c.5' -phosphate group slows the cleavage kinetics of exonucleolytic cleavage, but does not completely prevent degradation from this end (e.g., seq id no160 in seq id no pair 160/159). This is most likely due to cleavage of the 5' -phosphate by the intrinsic phosphatase activity of the phosphatase or DNaseII enzyme.

D. The best protection of the RNA strand is performed with oligonucleotides that do not contain a2 ' -OH nucleotide, starting from the 2 ' -OMe nucleotide at the 5 ' -end linked to the second nucleotide via a phosphorothioate bond (e.g. seq id no173 in seq id no pair 173/174). Other nucleotides with terminal non-2 ' -OH groups also protected against 5 ' -exo-degradation, but to a lesser extent with respect to 2 ' -OMe modification (see Table 9).

Example 42 knockdown Activity of siRNA lacking 2' -OH residue in vivo assay

In vivo experiments were performed with mice injected with sirnas targeting factor vii (fvii) (seq id no pair 179/166 and 180/168, see table 5) in combination with administration of DPC-GalNac.

TABLE 5a in vivo assay of siRNA sequences. Note that: the lower case letters a, c, g, u are 2' -O-methyl nucleotides; the capital A, C, G, U followed by "f" refers to a 2' -fluoro nucleotide. The lower case letter "p" refers to 5' -phosphate. (invdT) represents inverted deoxythymidine (3 '-3' -linkage). Phosphorothioate linkages are indicated by the lower case "s" symbol. dT is deoxythymidine. (NHC6) is an aminohexyl linker introduced at the 5' end of the sense strand. GalNAc refers to the structure of formula (IV).

FVIIsiRNA with optional 2 '-OMe/2' -F patterns on the sense and antisense strands were generated with the 5 '-terminal 2' -OMe nucleotide on the antisense strand and the 5 '-terminal 2' -F strand on the sense strand. Both strands are protected at the 3' -end overhang by inv (dT). The antisense strand carries a 5' -phosphate group to retain the activity of the siRNA. Conjugated to the GalNAc-palmitoyl structure at the 5' end of the sense strand, enables it to be targeted to hepatocytes by sialyl glycoprotein receptors expressed in these cells. siRNA (2.5mg/kg) was administered in combination with GalNAc-targeted PBAVE delivery polymer (15mg/kg) to mice.

Detection of fviia mrna from liver homogenates was accomplished using quantigene1.0 branchedsdna (bDNA) AssayKit (Panomics, Fremont, calif., USA, catalog No. QG 0004).

1-2g of liver tissue from autopsy was snap frozen in liquid nitrogen. The frozen tissue was powdered with a mill and evaporated on dry ice. 15-25mg of tissue was transferred to a cooled 1.5mL reaction tube, 1mL1:3 lysis mix pre-diluted in MilliQ water and 3.3. mu.L proteinase K (50. mu.g/. mu.L) were added, and the tissue was sonicated at 30-50% power (HD2070, Bandelin, Berlin, Germany) for a few seconds for lysis. The lysate was stored at-80 ℃ until analysis. For mRNA analysis, the lysates were thawed and digested with proteinase K in a thermal stirrer (Thermomixercomfort, Eppendorf, Hamburg, Germany) at 65 ℃ for 15 min at 1000 rpm. FVII and GAPDH mRNA levels were determined using QuantiGene1.0 bDNAssaykit reagent according to manufacturer's recommendations. Fviia mrna expression was analyzed with 20 μ L lysate and mouse probe set. Gapdh mrna expression was analyzed using 40 μ L of lysate and norwegian (rattunnorwegicus) probe set, showing cross-reactivity with mice (probe set sequence see above). As an experimental reading, the chemiluminescent signal was detected at the end of the experiment as Relative Light Units (RLU) using a Victor2Light luminescence counter (PerkinElmer, wissbaden, germany). Fviia mrna signal from the same lysate was divided by GAPDH mrna signal, and values are reported for fviia mrna expression normalized to GAPDH.

The results showed that 79% of fviia mrna was knocked down 48 hours after 179/166 was dosed with seq id no. In contrast, the 2' -OH nucleotide with sirnaseqidn showed no significant knockdown (<25%) for 180/168, as shown in table 5.

TABLE 5b results of in vivo knockdown studies

	Pair of SEQ ID NO 179/166	Pair of SEQ ID NO 180/168
			Time [ hour]	Residual mRNA [% ]]	Residual mRNA [% ]]
1	84	92
			6	83	88
24	53	100
			48	21	76

Example 43 tissue distribution of siRNA lacking 2' -OH residues

The concentration of siRNA in liver tissue samples was determined using the proprietary oligonucleotide detection method described in WO 2010043512. siRNA quantitation was based on hybridization of a fluorescently (Atto-425) labeled PNA probe (Atto425-OO-GCAAAGGCGTGCCAACT, available from Panagene, Korea) complementary to the antisense strand of the siRNA duplex, followed by AEX-HPLC isolation. Quantification was done by fluorescence detection by reference to an external standard curve generated by serial dilutions of two fviia sirnas used in vivo assays (see example 42). For plasma samples, 0.2-2 μ L was injected into the HPLC system, and for tissues, approximately 1mg of sample was injected into the HPLC system.

Liver tissue analysis of the stabilized siRNA lacking 2 '-OH nucleotides showed high concentrations of the intact antisense strand in the ug/g range in the liver, but 95% was present in the 5' -dephosphorylated, inactivated form (see table 6). The resulting RNA with terminal 2' -OMe nucleotides does not tend to be phosphorylated again in the cytoplasm by the phosphokinase hClp1 (see below). In contrast, siRNA containing the antisense strand of 2' -OH was completely degraded in tissues within the first 6 hours after administration.

TABLE 6 liver tissue analysis of stabilized siRNA not containing 2' -OH nucleotides

^*BDL = detection Limit or less

Example 44 in vitro knock-Down of siRNA with optimized 5' -end

Other in vitro screens for FVIIsiRNA were performed in order to identify siRNAs that could be (re) phosphorylated at the 5' -end of the antisense strand to give RNAi-effective substances within the cell. All sirnas screened this time are shown in table 7. The alternative 2 '-OMe/2' -F modification pattern is identical to the first generation design (without any 2 '-OH residues) except for multiple modifications of the first two nucleotides at the 5' -terminus of the antisense strand. The 5 ' -ends of the two antisense strands are 2 ' -F or2 ' -deoxy modified nucleotides, respectively, and nucleotides with or without additional 5 ' -phosphate or 5 ' -phosphorothioate in different combinations. After transfection of primary mouse hepatocytes (30000 cells per well; 96-well plate format), Lipofectamine2000 was used according to the manufacturer's instructions in a dose-response mannerAll sirnas (24nM to 0.00037nM, at 4-fold dilution) were screened for knock-down inactivation assay. According to IC₅₀Value, both sirnas were comparable in activity to the parental duplex (seq id no pair 182/168); siRNA with comparable activity, SEQ ID NO pairs 181/186 and 181/185), one 2 ' -F and phosphate group with a 5 ' -terminus, one 2 ' -deoxynucleotide and 5 ' -phosphorothioate with two 5 ' -termini (see IC50 values in Table 7). They were all about 5-6 times more active than the siRNA with terminal 2' -OMe nucleotides used in the first animal experiment (seq id No. pair 181/166).

Example 45 in vitro 5 '-phosphorylation of siRNA with optimized 5' end

All sirnas listed in table 7 without 5 '-phosphate or 5' -phosphorothioate were evaluated for phosphorylation by hClp1 in HeLaS100 cell extracts.

5' -phosphorylation was analyzed from S100HeLa extracts as described by Weitzer and Martinez (S.Weitzer and J.Martinez.hClp1: anovelkinasezelesz RNAmetabolism.CellCycle6(17): 2133. 2137. 2007). After incubation with 1. mu. MsiRNA in S100HeLa extract containing 5mMATP, the solution was directly analyzed by IP-RP-HPLC or AEX-HPLC under denaturing conditions by injecting 5. mu.L of sample solution.

A. Using Waters XBridgeC₁₈Column (2.5X50mm,2.5 μm particle size) IP-RP-HPLC was carried out at a column temperature of 65 ℃. Gradient elution was performed using a solution of 100mM Hexafluoroisopropanol (HFIP) and 16mM triethylamine in 1% methanol as eluent A and a solution of composition A in 95% methanol as eluent B. A gradient from 1% B to 18% B was used over 30 min.

aex-HPLC was performed on a DionexDNAPac200 column (4x250mM) at 50 ℃ using 20mM phosphate buffer containing 10% ACN, pH = 11. Eluent B contained 1m nabr in eluent a. A gradient from 25-62% B was used over 18 minutes.

TABLE 7 IC50 values

Note: wherein, Senssestrandsequence refers to the sense strand sequence, and Antisensestrandessendsequence refers to the antisense strand sequence.

The ratio of 5' -phosphorylation was calculated for each strand of siRNA from UV trace at 260nm using the following equation (PA is peak area):

%_{(5' -phosphorylation)}=100*PA_{[ 5' -phosphorylated chain]}/(PA_{[ 5' -phosphorylated chain]}+PA_{[ parent chain]})

As shown in Table 8, when the 2 ' -OMe nucleotide was located at the 5 ' -end (SEQ ID NO: 181/196 and SEQ ID NO: 181/195), the antisense strand of the siRNA could not be 5 ' -phosphorylated. In contrast, the antisense strand is susceptible to 5 ' -phosphorylation when 2 ' -F, 2 ' -deoxy or2 ' -OH nucleotides are incorporated at the 5 ' -terminus (seq id no pair 181/195, seq id no pair 181/192, seq id no pair 181/197, seq id no pair 181/199 and seq id no pair 182/168). Once the synthetically introduced 5 ' -phosphate/5 ' -PTO group is cleaved in vivo, e.g., by phosphatase, the two sirnas with similar activity to the parent seq id no pair 182/168(seq id no pairs 181/186 and 181/185) are susceptible to 5 ' -phosphorylation in an in vitro assay.

TABLE 8 percentage of 5' -phosphorylated strands after 4 hours incubation in S100HeLa cell extracts. Note that: the lower case letters a, c, g, u are 2' -O-methyl nucleotides; the capital A, C, G, U followed by "f" refers to a 2' -fluoro nucleotide. (invdT) represents inverted deoxythymidine (3 '-3' -linkage). Phosphorothioate linkages are indicated by the lower case "s" symbol. dT is deoxythymidine.

Example 46 in vivo DNAseII-stability of siRNA with optimized 5' end

All antisense strands were screened for DNAseII stability as described in example 41. The two antisense strands present in the siRNA that are active comparable to the parent duplex (seq id no186 and seq id no pair 185), one with the 5 ' -terminal 2 ' -F and phosphate groups and one with the two 5 ' -terminal 2 ' -deoxynucleotides and 5 ' -phosphorothioate, are stable to DNAseII cleavage (> 70% of the entire strand after 20 hours incubation).

TABLE 9 in vitro stability of siRNA to DNAseII after 20 hours incubation

Sense SEQ ID NO	Antisense SEQ ID NO	Sequence (5 '-3')	Complete%
				181	192	UfsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)	11
181	197	dTsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)	0
				181	199	dTsdGaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)	0
181	193	psUfsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)	106
				181	187	psdTsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)	96
181	194	psdTsdGaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)	101
				181	191	psUfGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)	100
181	198	psdTGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)	95
				181	186	psdTdGaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)	99
181	185	pUfsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)	71
				181	189	pdTsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)	74
181	188	pdTsdGaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)	64

Example 47 in vivo knock-down of siRNA with optimized 5' end

To assess whether the improvement by optimization of the 5' end could be transferred to an in vivo state, we performed further mouse experiments with GalNAc-palmitoyl conjugates of selected sirnas (see table 10). The siRNA was administered under the same conditions as described in the first mouse experiment (example 42, this patent application).

To measure FVII levels, plasma samples from mice were prepared according to standard procedures by collecting blood (9 volumes) from mandible by apheresis into microcentrifuge tubes containing 0.109mol/L sodium citrate anticoagulant (1 volume). FVII activity in plasma was measured chromogenically using BIOPHENVII kit (HyphenBioMed/Anaara, Mason, OH) according to the manufacturer's recommendations. The absorbance established by the colorimetric method was detected at 405nm using a TecanSafire2 microplate reader.

The siRNA studied showed improved in vitro activity, which is very relevant to the in vitro screening results. At 48 hours post-administration, both sirnas reduced serum FVII activity by more than 80%, compared to 49% reduction using the first generation siRNA design (see table 10). This result clearly demonstrates the importance of the 5 ' -terminal nucleotide of the antisense strand, which can be efficiently phosphorylated when the synthetic 5 ' -phosphate or 5 ' -phosphorothioate group is cleaved in vivo by a phosphatase. When the 5 '-terminal 2' -OMe nucleotide used in the original design or described in the literature as a more effective siRNA design was based on comparison with standard siRNAs in vitro (Allersonetal. J. Medchem.2005,48,901-904), in vivo cleavage of the synthetic phosphate could lead to a significant decrease in the potency of the corresponding siRNA.

Table 10: siRNA with optimized 5' end knockdown activity in vivo. Note that: the lower case letters a, c, g, u are 2' -O-methyl nucleotides; the capital A, C, G, U followed by "f" refers to a 2' -fluoro nucleotide. The lower case letter "p" refers to 5' -phosphate. (invdT) represents inverted deoxythymidine (3 '-3' -linkage). Phosphorothioate linkages are indicated by the lower case "s" symbol. (NHC6) is an aminohexyl linker introduced at the 5' end of the sense strand. GalNAc refers to the structure of formula (IV).

Example 48: siRNA in vitro knock-down deactivation with optimized 3' end

To further increase the activity of dnasei-stable sirnas, SAR studies on the 3' -overhang were performed. Different combinations of invdT, dTinvdT or dTsdT at the 3 '-overhang of the sense or antisense strand were applied to the Aha 1-and EGFP-targeted siRNAs (see tables 11 and 12, respectively), and the compositions of the 3' ends were compared in pairs in the most effective siRNAs. After transfection of primary mouse hepatocytes (30000 cells per well; 96-well plate format), all sirnas were screened in a dose-response format (24nM to 0.00037nM at 4-fold dilutions) for knock-down deactivation assays using Lipofectamine2000 according to the manufacturer's instructions.

Table 11: EGFP-targeted siRNAs with different 3' ends abolish activity in vitro.

Note: wherein, Senssestrandsequence refers to the sense strand sequence, and Antisensestrandessendsequence refers to the antisense strand sequence.

Table 12: aha 1-targeted siRNA with different 3' ends knockdown activity in vitro.

Note: wherein, Senssestrandsequence refers to the sense strand sequence, and Antisensestrandessendsequence refers to the antisense strand sequence.

It was found that siRNAs with two nucleotide dTsdT-overhangs on the antisense strand were more effective than siRNAs with only one invdT-overhang at the 3' -end of the antisense strand (and the sense strand was identical). More advantageously in combination with the use of a single invdT-overhang modified sense strand as the 3' overhang.

Example 49: in vivo knock-down deactivation of siRNA in non-human primates

Preparation and administration of DPC

DPC was prepared as follows: the polymer-siRNA conjugates were covalently linked by disulfide bonding by polymer "149 RAFT" with the indicated siRNA targeting factor VII (siF7) in a 4:1 weight ratio (polymer: siRNA), followed by modification of the polymer-siRNA conjugates with a 2:1 weight ratio CDM-PEG: CDM-NAG mixture in a 7x weight ratio (CDM: polymer). Macaques were administered 1mg/kg DPC (polymer weight) and 0.25mg/kg siRNA as indicated. One animal received DPC comprising siF7seq id no pair 151/152, two animals received DPC comprising siF7seq id no pair 253/254), #1 and #2), and two animals received DPC comprising seq id no pair 251/255, #1 and # 2). F7 values were normalized to the mean of the two pre-dose values. Animals receiving DPC comprising seq id no pair 253/254 or seq id no pair 251/255 had higher levels of F7 knockdown and longer PT than animals receiving seq id no pair 251/252.

DPC injection procedure

For each injection procedure, animals were given an IM injection containing a combination of ketamine (up to 7mg/kg) and dexmedetomidine (up to 0.03mg/kg) and moved to the procedure room. In the operating room, animals were placed on a water jacket hot plate and the injection sites were shaved and disinfected. An intravenous catheter (No. 20-22) was inserted into a body vein (cephalic or lesser saphenous vein) and slowly infused with DPC solution (2ml/kg) over 1-2 minutes. The heart rate and oxygen saturation are monitored using a pulse oximeter during and after the injection procedure. Each injection procedure took about 20 minutes to complete. After injection, the catheter was removed and the puncture site was gently pressed. The animals were returned to their cages and given an IM injection of the reversal drug, altimezole (0.10-0.15 mg/kg). Animals were monitored until they returned to normal activity.

Blood collection procedure

Blood samples (1-5ml) were obtained for determination of gene inhibition (F7 activity, clotting time), blood chemistry and liver injury markers (CBC, chemistry inventory, ALT, cytokines, complement). For each injection procedure, animals were given IM injections containing a combination of ketamine (up to 7mg/kg) and dexmedetomidine (up to 0.03 mg/kg). After sedation, the animals were moved to a portable operating table and blood was collected from the femoral vein using a 22-gauge needle and syringe. Immediately after the blood sample was collected, the puncture site was pressed and the blood was dispensed into appropriate sample tubes for each blood test. Animals were then given an IM injection of the reversal drug, altimezole (0.10-0.15mg/kg), and returned to their cages. No more than 20% of the total blood volume was drawn over any 30 day period (estimated blood volume =60 ml/kg). Each blood collection procedure took about 10 minutes to complete.

Factor VII (F7) Activity assay

A blood sample of a non-human primate was prepared by filling whole blood into a serum separation tube and allowing the blood to clot at room temperature for at least 20 minutes. After coagulation, the blood tubes were centrifuged at 9000rpm for 3 minutes, aliquoted into microcentrifuge tubes, and stored at-20 ℃ until use. F7 activity in serum was determined by a chromogenic method using BIOPHENVII kit (HyphenBioMed/Anira, Mason, OH) according to the manufacturer's recommendations. The absorbance established by the colorimetric method was detected at 405nm using a TecanSafire2 microplate reader.

Blood coagulation test (prothrombin time, partial prothrombin time and fibrinogen)

Blood samples of non-human primates were prepared by completely filling whole blood into sodium citrate tubes (BDVacutainer) and gently mixing to prevent clot formation. The tubes were transferred to a clinical laboratory within 1 hour and clotting experiments were performed within 4 hours after the collection time.

Table 13: fviia dsrna for NHP experiments. Note that: the lower case letters a, c, g, u are 2' -O-methyl nucleotides; the capital A, C, G, U followed by "f" refers to a 2' -fluoro nucleotide. The lower case letter "p" refers to 5' -phosphate. (invdT) represents inverted deoxythymidine (3 '-3' -linkage). Phosphorothioate linkages are indicated by the lower case "s" symbol. dT is deoxythymidine. NH2C6 was incorporated into the aminohexyl linker at the 5' -end of the sense strand.

From a single nucleotide (invdT) -3 '-overhang on both strands to an asymmetric siRNA design with a 3' - (invdT) overhang on the sense strand and a dTsdT overhang on the antisense strand, but the constant modification pattern resulted in a more effective serum FVII decline in non-human primates and a significantly extended duration of this effect (see figure 6 a). This result is supported by the expected biological consequences, i.e. a more pronounced effect on prothrombin time corresponding to the degree of reduction of factor 7 (see fig. 6 b).

Example 50: in vivo knock-down deactivation of siRNA with cleavable RNA linker

In table 14, serum-based FVII protein inhibition in vivo potency was compared using cholesterol or GalNAc-palmitoyl siRNA conjugates in mice with the same sequence background. The in vivo assay was performed as described in example 42. Cholesterol conjugated siRNA not containing 2' -OH nucleotides had greatly reduced FVII inhibition compared to GalNAc-palmitoyl conjugated product (seq id no pair 179/166 vs 179/190, seq id no pair 257/264 vs 179/262, seq id no pair 257/263 vs 179/163, and seq id no pair 257/166 vs 179/166). In contrast to siRNA comprising 2' -OH, the cholesterol conjugate resulted in higher FVII inhibition relative to GalNAc-palmitoyl derivative (seq id no pair 180/168 versus seq id no pair 258/168).

The small molecule ligands GalNAc-palmitoyl and cholesterol used in the in vivo assay were linked to the 5' -end of the sense strand by a non-cleavable linker. In the case of a sense strand with 2' -OH nucleotides, the ligand remains cleavable by a nuclease (e.g., dnasei in an endosomal or lysosomal compartment). The cleavage reaction releases free siRNA, which is then released into the cytoplasm by the delivery of a polymer that interferes with the activity of the endosome.

For sirnas lacking 2' -OH nucleotides in the sense strand, the ligand is stably attached to the double strand because there is no enzymatic (nuclease/protease/esterase, etc.) or chemical mechanism to trigger cleavage of the ligand. Thus, a very stable cholesterol conjugated siRNA can be embedded in the cell membrane due to the interaction of the lipophilic cholesterol ligand with the membrane. Even high concentrations of siRNA in tissues are not sufficient to effectively release siRNA into the cytoplasm. In contrast, GalNAc-palmitoyl conjugated siRNA, which is less lipophilic, is able to be released into the cytoplasm because it interacts significantly less with the cell membrane. For this reason, the non-cleavable GalNAc-palmitoyl siRNA conjugates were more effective than the same siRNA conjugated with cholesterol.

The development of a cleavable linker structure can help prevent the problem of membrane encapsulation of stably conjugated cholesterol sirnas. The use of disulfide linker chemistry is described as an attractive method to introduce defined cleavage sites, but cleavage is restricted to the reducing organelles of the cell (PNAS,2006,103,13872). Since cleavage is expected to slow in the endosomal/lysosomal compartment, most of the cholesterol-disulfide conjugated siRNA can still be embedded in the membrane as described for the non-cleavable cholesterol conjugates.

TABLE 14

In addition to creating disulfide cleavable linker chemistry, another possibility is to create defined cleavage sites by using 2' -OH nucleotides at certain positions. The introduction of 2' -OH nucleotides at selected positions is a novel way to achieve the removal of the conjugate from the RNA strand. The 2 ' -OH nucleotides can be implemented by adding a single-stranded overhang with at least one 2 ' -OH nucleotide at the 3 ' -or 5 ' -end of the RNA strand, or using 2 ' -OH nucleotides in the double-stranded region of the siRNA. Nuclease activity present in the endosomes/lysosomes selectively cleaves at the site. In the first generation design, cholesterol was linked to the sense strand by a single stranded overhang comprising 32 '-OH nucleotides (AUC) at the 5' -end.

A comparison of cholesterol conjugated siRNA and various cleavable linker chemistries is shown in table 15. All sirnas have the same sequence context, only the linker is changed. Cholesterol was linked to the sense strand via a single-stranded overhang consisting of 32 '-OH nucleotides (AUC) at the 5' -end. When administered in combination with a delivery polymer, this siRNA (seq id no pair 260/263) resulted in 77% downregulation of FVII in mouse serum, while the same siRNA with stably linked cholesterol (seq id no pair 257/263) was only 60%. The siRNA and cholesterol conjugate with the 5' -end connected to the sense strand (seq id no pair 261/263) by a linker according to formula Ia resulted in a 93% decrease in FVII activity in serum. All results were obtained by combined administration of 15mg/kg of delivery polymer and 2.5mg/kg cholesterol conjugated siRNA in mice.

These results demonstrate that the use of a cleavable linker improves the in vivo efficacy of siRNA that does not contain a 2' -OH nucleotide. The cleavable linker may be composed of a nucleotide comprising a 2' -OH, a dipeptide cleavage motif or a disulfide linker. All cleavable linker structures improve in vivo efficacy in administering cholesterol conjugated siRNA in combination with a delivery polymer that slows endosomal release.

Table 15: in vivo comparison of different linker chemistries for cholesterol conjugated siRNA

Example 51: in vitro serum stability of siRNA with cleavable linker

The stability of the cleavable linker was evaluated in an in vitro stability assay. The cholesterol conjugated sense strand was incubated in 90% mouse serum at 37 ℃ for various time points. The incubation reaction was stopped by adding a solution of proteinase K in a buffer containing Sodium Dodecyl Sulfate (SDS). This treatment degrades all proteins and enzymes, but does not interfere with the integrity of the RNA strand. 25 μ L of this solution was injected directly into an AEX-HPLC system connected to a UV detector (260 nm). AEX-HPLC was performed on a DionexDNAPac200 column (4x250mm) at 75 ℃ using 20mm tris buffer (pH =8) containing 50% ACN. A solution of 800mm nabr in eluent B was used for elution desalting. A gradient of 25-62% B was used over 18 minutes.

Single-stranded RNA containing cholesterol eluted from the HPLC column as the widest peak at 260 nm. After cutting off the cholesterol, a sharp symmetrical peak was observed at lower retention times. The rate of cholesterol cleavage was determined according to the following equation (PA = peak area):

%_{(free RNA)}=100*PA_{[ free RNA ]]}/(PA_{[ free RNA ]]}+PA_{[ Cholesterol-conjugated RNA]})

In vitro, 3 nucleotide (AUC) -projections were shown to be quantitatively cleaved in 90% mouse serum in less than 1 hour. The cleavage occurred 3' to the two pyrimidine nucleotides of the protrusion, resulting in two different cleaved metabolites (peak areas of the metabolites are summarized for data evaluation). In contrast, dipeptides comprising a linker according to formula Ia, a disulfide bond and stably linked cholesterol were completely stable in mouse serum.

Example 52: tissue distribution of siRNA with cleavable linker

These results demonstrate that the use of a cleavable linker improves the in vivo efficacy of siRNA that does not contain a 2' -OH nucleotide. The cleavable linker may be composed of a nucleotide comprising a 2' -OH, a dipeptide cleavage motif or a disulfide linker. All cleavable linker structures improve in vivo efficacy in administering cholesterol conjugated siRNA in combination with a delivery polymer that slows endosomal release.

Briefly, siRNA quantitation was based on hybridization of a fluorescently (Atto-425) -labeled PNA probe (Atto425-OO-TGAGTTGGCACGCCTTT, available from Panagene, Korea) complementary to the sense strand of the siRNA duplex, followed by AEX-HPLC isolation. Quantification was done by fluorescence detection by reference to an external standard curve generated by serial dilutions of two fviia sirnas used in vivo assays (see example 42). For plasma samples, 0.2-2 μ L was injected into the HPLC system, and for tissues, approximately 1mg of sample was injected into the HPLC system.

The results of the liver tissue analysis are shown in table 16. In analyzing the siRNA content, it was found that the sense strand was present in the liver tissue, and quantitatively detached from cholesterol when a dipeptide linker motif or an unmodified linker sequence AUC at the 5' -overhang of 3 nucleotides was used. In contrast, only 15% of the disulfide-linked sirnas in the liver detached from cholesterol within the first 48 hours after administration, and stably linked cholesterol did not detach from the sirnas.

When comparing the absolute amount of siRNA without cholesterol in liver tissue, it was found that the amount was similar for the disulfide linker and RNAAUC-linker, fully consistent with the same FVII serum activity 48 hours after administration. Dipeptide-linked cholesterol sirnas give lower FVII activity, fully consistent with higher amounts of cholesterol-free siRNA released.

The total amount of cholesterol siRNA conjugates with (AUC) -linker on the sense strand delivered to liver was about 6-fold lower compared to the cholesterol stably linked or disulfide linked, and about 3-fold lower compared to dipeptide conjugated cholesterol siRNA. This reduced tissue presence can be attributed to the fact that: the AUC-linker is not only a substrate for intracellular nucleases but also for nucleases present in the circulation, as shown by in vitro incubation with mouse serum. When the cholesterol ligand has been excised from the siRNA in the circulatory system, the resulting siRNA is prone to renal clearance and is rapidly excreted into the urine, rather than being delivered into the tissue.

Table 16:

example 53 in vivo knock-Down Activity of siRNA with cleavable RNA linker

In vivo experiments were performed as described in example 50, using cholesterol siRNA conjugates. In example 50, cholesterol was linked to the sense strand via a single-stranded overhang comprising 32 '-OH nucleotides at the 5' -end (AUC) (seq id no pair 260/263), which showed low serum stability as described in example 51. This resulted in significantly lower tissue concentrations compared to the serum-stable linker chemistry described in example 52. The combination of only one or two selected 2 '-OH nucleotides with 2' -OMe nucleotides in the linker results in higher serum stability, but maintains sensitivity to nucleases present in endosomes/lysosomes. The nuclease activity present in endosomes/lysosomes selectively cleaves at the 2' -OH nucleotide.

A comparison of cholesterol conjugated siRNA with different cleavable nucleotide linker motifs is summarized in table 17. All sirnas have the same sequence context, only the linker is changed. Cholesterol was ligated to the sense strand by a single-stranded overhang consisting of 3 or 4 nucleotides with different numbers of 2 ' -OH and 2 ' -OMe at the 5 ' -end. All sirnas resulted in FVII down-regulation in mouse serum when administered in combination with a delivery polymer. siRNA (SEQ ID NO vs 276/282) resulted in 87% downregulation of FVII activity in the 48 h serum and 95% downregulation at 168 h post-dose. siRNA (SEQ ID NO vs 277/282) resulted in 79% downregulation of FVII activity in the 48 h serum and 97% downregulation at 168 h post-dose. All results were obtained by combined administration of 15mg/kg of delivery polymer and 2.5mg/kg cholesterol conjugated siRNA in mice.

TABLE 17 in vivo comparison of different nucleotide linker motifs for cholesterol conjugated siRNA

These results demonstrate that the use of a serum stable and endosomal/lysosomal cleavable linker further improves the in vivo potency of siRNA compared to siRNA with a serum stable linker. All cleavable linker structures improve in vivo efficacy in the combined administration of cholesterol conjugated siRNA and a delivery polymer that slows endosomal release.

In the following table, the sirnas used in the examples are summarized:

table 18: core sequence

Table 19: list of core and modified sequences

Claims (10)

1. Use of a compound of formula (I) for delivering nucleic acids

Wherein

Y is- (CH)₂)₃-；

R¹Is- (C1-6) alkyl; or

-(CH₂)_m-phenyl, wherein phenyl is unsubstituted or independently selected fromThe substituents described above are substituted up to four times:

-NO₂、

-CN or

Halogen;

R²is hydrogen;

-(CH₂)_k-a phenyl group;

- (C1-6) alkyl;

-(CH₂)_k-C(O)-NH₂(ii) a Or

-(CH₂)_k-NH-C(Ph)₃Wherein the phenyl rings are unsubstituted or independently substituted with-O- (C1-4) alkyl;

R³is-NH-phenyl, wherein the phenyl group is further substituted with a substituent independently selected from the group consisting of:

-(CH₂) -OH; or

-(CH₂) -O-c (O) -O- (4-nitro-phenyl);

k is 1,2, 3,4, 5 or 6;

m is 1,2, 3 or 4; and is

n is 0 or 1.

2. Use according to claim 1, wherein the compound of formula (I) has the configuration shown in formula (Ia)

3. Use according to claim 1 or2, wherein the substituents of the compounds of formula (I) or (Ia)

Y is- (CH)₂)₃-；

R²Is- (CH)₂)_k-N-C(Ph)₃Wherein the phenyl rings are unsubstituted or independently substituted with-O- (C1-4) alkyl; and is

R³is-NH-phenyl, wherein the phenyl group is further substituted by- (CH)₂) -O-c (O) -O- (4-nitro-phenyl) substitution;

n is 0; and is

R¹And k has the meaning described above.

4. The use of claim 1 or2, wherein the compound is covalently linked to a nucleic acid.

5. A compound of the formula (II),

wherein

R^aIs- (CH)₂)_k-NH₂；

R¹And k has the meaning given above for formula (I).

6. The compound of claim 5, having the configuration of formula (IIa)

Wherein

R^aIs- (CH)₂)_k-NH₂；

R¹And k has the meaning given above for formula (I).

7. The compound of claim 5 or 6, wherein the nucleic acid is an oligonucleotide comprising a nucleic acid having a modification pattern of 5' - (w) - (Z1) - (Z2) - (Z3) n_a3 'antisense strand and 5' - (Z3) n with modification pattern_s-a sense strand of 3', wherein

w is independently 5 '-phosphate or 5' -phosphorothioate or H,

z1 is independently a 2' -modified nucleoside,

z2 is independently a2 '-deoxynucleoside or a 2' -fluoro-modified nucleoside,

z3 is independently a 2' -modified nucleoside,

n_ais 8-23 and n_sIs 8 to 25.

8. The compound of claim 5 or 6, wherein the nucleic acid is an siRNA.

9. The compound of claim 5 or 6, wherein the nucleic acid is administered with a delivery polymer.

10. A pharmaceutical composition comprising a compound according to any one of claims 5-9.