# Patent

## Title
Small molecule conjugates for intracellular delivery of biologically active compounds

## Abstract

The invention provides novel compounds and conjugates of these compounds useful for the delivery of biologically active substances. Further novel design criteria for chemically stabilized siRNA particular useful when covalently attached to a delivery polymer to achieve in vivo mRNA knock down are disclosed therein.

## Background
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
This application is a continuation of International Patent Application No. PCT/EP2011/073718, filed Dec. 22, 2011, which claims priority benefit to U.S. Provisional Patent Application No. 61/427,845 filed Dec. 29, 2010, the content of each of which is hereby incorporated herein by reference in its entirety.


SEQUENCE LISTING
The instant application contains a Sequence Listing submitted via EFS-Web and hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 26, 2013, is named P5647R1_SL.txt and is 449,820 bytes in size.
FIELD OF THE INVENTION
The present invention relates to novel small molecule conjugates useful for the delivery of biologically active substances, such as nucleic acids, peptides and proteins. The delivery of nucleic acids and other substantially cell membrane impermeable compounds into a living cell is highly restricted by the complex membrane system of the cell.
One means that has been used to deliver a biologically active substance such as nucleic acids in vivo has been to attach the biologically active substance to either a small targeting molecule or a hydrophobic molecule such as a lipid or sterol. While some delivery and activity has been observed with these conjugates, the biologically active substance dose required with these methods has been prohibitively large, resulting often in undesired toxicity effects in vivo. Provided herein are small molecule compounds that can be conjugated to a biologically active substance and mediate successful delivery of said biologically active substance into a cell. Surprisingly it has been found that significantly decreased doses of the biologically active substance are now sufficient for successful delivery when using the novel compounds provided herein. Thus, the novel compounds provide a powerful tool for the delivery of biologically active substances with considerably limited toxicity in vivo.
BRIEF SUMMARY OF THE INVENTION
In one embodiment, the present invention is directed to the compounds of formula




wherein

    
    
        Y is a linker group selected from —(CH2)3— or —C(O)—N—(CH2—CH2—O)p—CH2—CH2—;
        R1 is —(C1-6) alkyl;
        
            —(CH2)-naphthyl; or
            —(CH2)m-phenyl, which phenyl is unsubstituted or up to four times substituted with a substituent independently selected from
            
                —NO2,
                —CN,
                Halogen,
                —O—(CH2)-phenyl,
                —O—(C1-6) alkyl, or
                —C(O)—NH2;
            
            
        
        
        R2 is hydrogen;
        
            —(CH2)k—N—C(Ph)3, which phenyl rings are unsubstituted or independently substituted with —O—(C1-4)alkyl;
            —(CH2)k—C(O)—NH2;
            —(CH2)k-phenyl;
            —(C1-6) alkyl, which is unsubstituted or once substituted with —S—CH3;
        
        
        R3 is —NH-phenyl, which phenyl group is further substituted with a substituent independently selected from
        
            —(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
        p is an integer from 1 to 20.
    
    


In another embodiment, the compounds of formula (I) may have the specific conformation as shown in formula (Ia),




wherein all substituents R1, R2, R3 and Y as well as the variables k, m, n, and p have the meaning given above.

In yet another embodiment, the present invention is directed to compounds of formula (I) or (Ia), wherein Y is —(CH2)3—; and all remaining substituent groups have the meaning given above.
In yet another embodiment, the present invention is directed to compounds of formula (I) or (Ia), wherein Y is —C(O)—N—(CH2—CH2—O)p—CH2—CH2—; and all substituent groups have the meaning given above.
In yet another embodiment, there are provided the compounds of formulae (I) or (Ia), wherein

    
    
        Y is —(CH2)3—;
        R2 is —(CH2)k—N—C(Ph)3, which phenyl rings are unsubstituted or independently substituted with —O—(C1-4)alkyl; and
        R3 is —NH-phenyl, which phenyl group is further substituted with
        
            —(CH2)—O—C(O)—O-(4-nitro-phenyl);
        
        
        n is 0; and
        R1 and k have the meanings given above.
    
    


In yet another embodiment, there are provided the compounds of formulae (I) or (Ia), wherein

    Y is —C(O)—N—(CH2—CH2—O)p—CH2—CH2—;
    R2 is —(CH2)k—N—C(Ph)3, which phenyl rings are unsubstituted or independently substituted with —O—(C1-4)alkyl; and
    
        R3 is —NH-phenyl, which phenyl group is further substituted with —(CH2)—O—C(O)—O-(4-nitro-phenyl);
    
    
    n is 0; and
    R1, k and p have the meanings given above.


DETAILED DESCRIPTION OF THE INVENTION
The term “(C1-6) alkyl” as used herein means a linear or branched, saturated hydrocarbon containing from 1 to 6 carbon atoms. Preferred C1-6 alkyl groups include methyl, ethyl, propyl, iso-propyl, butyl, 2-butyl and the like.
The term “halogen” as used herein means fluorine, chlorine, bromine, or iodine with fluorine and chlorine being preferred.
The compounds according to the present invention can be generally obtained using methods known to the person of ordinary skill in the art of organic- or medicinal chemistry. More particularly the compounds of formula (Ia), wherein Y is —(CH2)3— and n=0, can be obtained using compound (A) as starting material.




The synthesis of (A) is described inter alia in WO2001/070415.
The compound of formula (A) is further reacted in the presence of Huenig's base and ethyl acetate (AcOET), followed by the addition of dihydrofuran-2,5-dione in THF, to give the compounds of formula (B)




The compounds of formula (B) are further reacted with an amine of formula (C),




to give the compounds of formula (Ia).

The compounds of formula (I) or (Ia) are useful as ligands on biologically active substances, such as nucleic acids, peptides or proteins, to which they are covalently attached. Preferably, the covalent bond is created by the reaction of a suitable functional group, such as i.e. a primary amine group, in the biologically active substance with the activated carbonyl group in the —O—C(O)—O— moiety of R3 as defined herein before. Hence provided herein is a conjugate comprising the compounds of formula (I) or (Ia) and a biologically active substance.
The term “biologically active substance” as used herein refers to an inorganic or organic molecule including a small molecule, peptide (e.g. cell penetrating peptides), protein, carbohydrate (including monosaccharides, oligosaccharides, and polysaccharides), nucleoprotein, mucoprotein, lipoprotein, synthetic polypeptide or protein, or a small molecule linked to a protein, glycoprotein, steroid, nucleic acid (any form of DNA, including cDNA, or RNA, or a fragment thereof), nucleotide, nucleoside, oligonucleotides (including antisense oligonucleotides, LNA and siRNA), gene, lipid, hormone, or combination thereof, that causes a biological effect when administered in vivo to an animal, including but not limited to birds and mammals, including humans. Preferably, said biologically active substance is a peptide or a nucleic acid. Preferred nucleic acids used herein are siRNAs.
The conjugate comprising the present compounds covalently attached to a biologically active substance shows an improved ability to be taken up by cells compared to said biologically active substance alone. Once the conjugate is delivered into the cell and trafficking to the lysosome, the corresponding biologically active substance is released by enzymatic cleavage. This cleavage preferably takes place when a di-peptide motif, preferably consisting of the sequence α- or β-(phenyl)alanine and lysine as present in the compounds of formula (I) or (Ia) is incorporated in the conjugate (see scheme 1). Most preferably the conjugate contains the di-peptide motif and a spacer such as the p-aminobenzylcarbamate spacer (Bioconjugate Chem. 2002, 13, 855) that spontaneously fragments once the amide bond C-terminal of the di-peptide motif is cleaved as exemplified for siRNAs in scheme 2. Hence the conjugates comprising compounds of formula (I) or (Ia) are also referred to as dipeptide containing cholesterol conjugates. Enzymatic cleavage of the biologically active substance from the dipeptide containing cholesterol conjugates of this invention is catalyzed by innate proteases of the cell. One example of an innate protease capable of cleaving the di-peptide motif present in the compounds of formula (I) or (Ia) is Cathepsin B. Cathepsin B is a known ubiquitous cysteine protease located in the lysosomes of mammalian cells (Bioconjugate Chem. 2002, 13, 855; J. Med. Chem. 2005, 48, 1344; Nat. Biotechnology 2003, 21, 778). Thus, the di-peptide motif described above is also referred to as Cathepsin-cleavable dipeptide-motif.
The present invention therefore also provides a method for delivery of a biologically active substance, into cells wherein said biologically active substance may subsequently be cleaved off the conjugate to unfold a therapeutic activity.




In a further embodiment of the present invention, there is provided a conjugate of the compounds of formula (I) or (Ia) covalently attached to a biologically active compound, preferably siRNA or a peptide moiety. Preferably said peptide moiety is a peptide that exhibits membrane perturbing properties like cell penetrating peptides or amphiphilic peptides.
Conjugates of formula (I) or (Ia) covalently attached to a biologically active substance are designated herein as formula (II) or (IIa), respectively.
Therefore, in a further embodiment, the present invention provides a compound of formula




wherein

Ra is —(CH2)k—NH2;

R1 and k have the meanings given for formula (I) above; and

the biologically active substance is a nucleic acid, a protein or a peptide.

In a more specific embodiment, the present invention provides compounds of formula




wherein

Ra is —(CH2)k—NH2;

R1 and k have the meanings given for formula (I) above; and

the biologically active substance is a nucleic acid, a protein or a peptide.

In a preferred embodiment, the biologically active substance in formula (II) or (IIa) is a nucleic acid, most preferably a siRNA.
In another preferred embodiment, the biologically active substance in formula (II) or (IIa) is a protein or a peptide.
The compounds of formula (II) or (IIa) may have valuable properties in therapy. Therefore, in a further embodiment, there are provided the compounds of formula (II) or (IIa) for use as medicaments.
Another embodiment of the invention is a pharmaceutical composition comprising the conjugates of the compounds of formula (I) or (Ia) covalently attached to a biologically active substance.
In still another embodiment of the invention there is provided a pharmaceutical composition comprising the compounds of formula (IIa) together with pharmaceutically acceptable excipients.
Below embodiments are exemplified for conjugates of the compounds of formula (I) or (Ia) covalently attached to siRNA, thus the compounds of formula (II) or (IIa) wherein the biological active substance is siRNA. It is understood that these embodiments are also applicable for other biologically active substances such as peptides and proteins.
The covalent attachment of the siRNA to the compounds of formula (I) or (Ia) is achieved via reaction of a suitable nucleophilic group, i.e. a primary amine group, in the siRNA with the activated —C(O)— group in R3 of said compounds of formula (I) or (Ia). The activation of that —C(O)— group is obtained by a p-nitrophenoxy carbonate as shown in scheme 2 below.




The p-nitrophenyl activated carbonate may for example be reacted with the siRNA equipped with a hexylamino-linker to generate a carbamate linkage to yield the siRNA conjugate. Once the siRNA is taken up intracellularly and transfected to the lysosome the compounds of formula (II) or (IIa) wherein the biological active substance is siRNA are cleaved by the protease activity releasing the siRNA as also shown in scheme 2. The cholesterol moiety of the conjugate of the compounds of formula (II) or (IIa) modifies the PK properties of siRNA in such a way that systemic administration enables gene silencing in vivo.




In one embodiment the compounds of formula (II) or (IIa) wherein the biological active substance is siRNA is co-administered with a delivery polymer. Delivery polymers provide a means of disrupting cell membranes and mediate endosomal release. In another embodiment, said delivery polymer and the siRNA conjugate of the invention are not covalently attached and synthesized separately and may be supplied in separate containers or 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 demonstrated a means to reversibly regulate membrane disruptive activity of a membrane active polyamine. Reversible regulation provided a means to limit activity to the endosomes of target cells, thus limiting toxicity. Their method relied on reaction of amines on the polyamine with 2-propionic-3-methylmaleic anhydride. This modification converted the polycation to a polyanion via conversion of primary amines to carboxyl-containing groups and reversibly inhibited membrane activity of the polyamine. To enable co-delivery of the nucleic acid with the delivery vehicle, the nucleic acid was covalently linked to the delivery polymer. In U.S. provisional patent application 61/307,490 a new generation of delivery polymers is described. Therein, membrane active polyamine comprising an amphipathic terpolymer formed by random polymerization of amine-containing monomers, lower hydrophobic monomers, and higher hydrophobic monomers are provided. This new generation of delivery polymers removed the requirement that polynucleotide and polymer are associated either by covalent linkage or by charge-charge interaction.
Non-limiting examples of delivery polymers used for co-administration with the siRNA conjugates of the invention are membrane active polyamines and poly(vinyl ether) (PBAVE), Dynamic PolyConjugates (DPC; Rozema et al. 2007) and improved DPCs as disclosed in U.S. provisional patent application 61/307,490.
In a further embodiment, a new chemical siRNA modification pattern for functional in vivo delivery is provided. This new chemical siRNA modification pattern is especially useful with delivery vehicles which display a relatively strong endosomal/lysosomal retention.
It was found that siRNA stabilization against degradation by endosomal/lysosomal-localized nucleases such as DNAse II strongly improves target knock down. Such stabilization may directly effect the amount of siRNA released into the cytoplasm where the cellular RNAi machinery is located. Only the siRNA portion available in the cytoplasm will trigger the RNAi effect.
In addition to poor pharmacokinetic characteristics, siRNAs are susceptible to nucleases in the biological environment when administered as such into the circulation without a protecting delivery vehicle. Accordingly, many siRNAs are rapidly degraded either extracellularly in the tissue and blood stream or after intracellular uptake (endosome).
One well known nuclease localized in the endosomal/lysosomal compartment is DNase II. This enzyme is active at a pH below 6-6.5 with maximum activity in the pH-range of 4.5-5, reflecting conditions present in the acidified environment of the endosomal/lysosomal compartment. The following RNA degradation pathways induced by DNase II were identified in vitro and are disclosed in this invention:
A. RNA strands containing at least one 2′-OH nucleotide are rapidly degraded via a cyclic pentavalent phosphorus intermediate, leading to 2′-3′ cyclic phosphates at the 5′-cleavage product. The formation of the pentavalent intermediate can be inhibited by nucleotides lacking a 2′-OH group such as 2′-deoxy, 2′-O-methyl (2′-OMe) or 2′-deoxy-2′-fluoro (2′-F) nucleotides.

B. Additionally, RNA is degraded in a 5′-exonucleolytic pathway independent of the 2′-modification on the 5′-terminal nucleotides. This degradation pathway can be inhibited by 5′-terminal non-nucleotide moieties, like e.g. cholesterol, aminoalkyl-linker or a phosphothioate at the first internucleotide linkage.

C. A 5′-phosphate also protects and slows down the exonucleolytic cleavage kinetics, but can not fully block this pathway. This is most probably due to the cleavage of the 5′-phosphate by phosphatases or an inherent phosphatase activity of the DNase II enzyme preparation used in the stability assay.

D. The best protection was achieved with oligonucleotides lacking any 2′-OH nucleotide within the strand, starting with a 2′-OMe nucleotide at the 5′-end connected by a phosphorothioate (PTO) linkage to the second nucleotide. Other terminal non-2′-OH nucleotides also protect against the 5′-exo degradation, but to a lower extent compared to the 2′-OMe modification.

Hence the inventors of the present invention found that siRNAs can be significantly stabilized when using the following design, wherein an oligonucleotide is provided with an antisense strand with the modification pattern: 5′-(w)-(Z1)-(Z2)-(Z3)na-3′ and a sense strand with the modification pattern 5′-(Z3)ns-3′, wherein
w is independently a 5′-phosphate or 5′-phosphothioate or H,
Z1 is independently a 2′-modified nucleoside.
Z2 is independently a 2′-deoxy nucleoside or 2′-Fluoro-modified nucleoside,
Z3 is independently a 2′-modified nucleoside,
na is 8-23 and ns is 8-25.
In one preferred embodiment an oligonucleotide is provided with an antisense strand with the modification pattern: 5′-(w)-(Z1)-(Z2)-(Z3)na-3′ and a sense strand with the modification pattern 5′-(Z3)ns-3′, wherein Z1 is a 2′-Fluoro-modified nucleoside or a 2deoxy-nucleoside and all remaining substituents as well as the variables na and ns have the meaning given above.
In one preferred embodiment an oligonucleotide is provided with an antisense strand with the modification pattern: 5′-(w)-(Z1)-(Z2)-(Z3)na-3′ and a sense strand with the modification pattern 5′-(Z3)ns-3′, wherein Z3 is a 2′-O-Methyl modified nucleoside, a 2′-Fluoro-modified nucleoside or a 2deoxy-nucleoside and all remaining substituents as well as the variables na and ns have the meaning given above.
In one preferred embodiment an oligonucleotide is provided with an antisense strand with the modification pattern: 5′-(w)-(Z1)-(Z2)-(Z3)na-3′ and a sense strand with the modification pattern 5′-(Z3)ns-3′, wherein Z1 is a 2′-Fluoro-modified nucleoside or a 2deoxy-nucleoside and Z3 is a 2′-O-Methyl modified nucleoside, a 2′-Fluoro-modified nucleoside or a 2deoxy-nucleoside and all remaining substituents as well as the variables na and ns have the meaning given above.
The nucleosides in the nucleic acid sequence of the oligonucleotide with the novel modification pattern can either be linked by 5′-3′ phosphodiesters or 5′-3′ phosphorothioates.
As used herein, the “anti-sense” strand is the siRNA strand that is complementary to the target mRNA and that will be binding to the mRNA once the siRNA is unwound.
The sense strand of said siRNA comprising the novel modification pattern is complimentary to the antisense strand.
Said siRNA comprising the novel modification pattern proofed to be particularly advantageous when covalently attached to a delivery polymer as exemplified by Rozema et al. (Dynamic PolyConjugates (DPC; Rozema et al. 2007). Potency and duration of effect can be significantly enhanced employing the siRNA modification strategy outlined in this invention.
In another embodiment, said siRNA comprising the novel modification pattern are especially useful when conjugated to small molecules that alter the pharmacokinetic properties of siRNA such as cholesterol or the compounds of formula (I) and (Ia) provided herein. In one embodiment a conjugate of a small molecule and an oligonucleotide is provided wherein the oligonucleotide has the following modification pattern: the antisense strand with the modification pattern: 5′-(w)-(Z1)-(Z2)-(Z3)na-3′ and a sense strand with the modification pattern 5′-(Z3)ns-, wherein the substituents as well as the variables na and ns have the meaning given above. In one embodiment said small molecule is cholesterol. In another embodiment said small molecule is a compound of formula (I) or (I a), resulting in compounds of formula (II) or (IIa).
Preferably, said siRNAs conjugates are co-administered with a delivery polymer. Suitable delivery polymers are described above.
In one embodiment, said siRNA comprising the novel modification pattern are especially useful when conjugated to a ligand that is known to bind to a specific receptor which internalizes the conjugate into a cell. Particularly, the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes is a well-known receptor enabling the clearance (endocytosis and lysosomal degradation) of desialylated proteins from circulation. It has been shown that the N-Acetyl-D-galactosamine has a high binding affinity for the receptor, especially when presented multivalent and when the galactose residues are properly spaced (J Biol Chem, 2001, 276, 37577). In order to utilize this high capacity receptor for receptor mediated endocytosis of the biologically active substance, the synthetic ligand shown below was prepared to be covalently attached to the siRNAs comprising the novel modification pattern. Since this type of endocytosis leads to lysosomal degradation of the internalized material the siRNA must be prepared in such a way that it is stable in the lysosome, which is now solved by the novel modification pattern outlined above.
The ligand for the ASGPR is attached via an amide bond to the biologically active substance. The amide bond formation can be established with the aid of NHS chemistry. The ligand employed in the conjugation reaction is shown below (formula III). For interaction with the ASGPR the O-acetate groups need to be removed as shown in (formula IV) for an siRNA.







In one embodiment of the invention, a conjugate of a compound of formula IV and an oligonucleotide is provided, wherein the oligonucleotide has the following modification pattern: the antisense strand with the modification pattern 5′-(w)-(Z1)-(Z2)-(Z3)na-3′ and a sense strand with the modification pattern 5′-(Z3)ns-, wherein the substituents as well as the variables na and ns have the meaning given above. Said conjugate is also referred to as GalNAc palmitoyl conjugate. Preferably, said GalNAc palmitoyl conjugate are co-administered with a delivery polymer. Suitable delivery polymers are described above.
It was found that for these modification patterns cleavable linkers proofed to be advantageous compared to stably linked small molecule ligands. Possible cleavable linkers are a di-peptide motif as exemplified in scheme 1 or a cleavable RNA-linker comprising 2′-OH containing nucleotides. The cleavable RNA-linker is especially useful in connection with the siRNAs having the novel modification pattern (fully 2′-modified siRNA) described above.
In principle a nuclease cleavage site can be introduced by 3′- or 5′-overhangs containing at least one 2′-OH nucleotide at either the sense or the antisense strand. The final active siRNA species is generated by intracellular nuclease processing. Also, the use of defined cleavage sites implemented by 2′-OH nucleotides within the base paired region is possible. This can be done using at least one 2′-OH nucleotide complementary to the opposite strand or by introduction of either at least one mismatched 2′-OH nucleotide or a hairpin/bulge containing at least one 2′-OH nucleotide.
In contrast to other cleavable linker chemistries the use of defined cleavage sites by introduction of 2′-OH nucleotides lead to a more versatile conjugation approach. By introducing selective cleavage sites on one or on both strands of the siRNA either at the 3′ and/or the 5′-end or within the duplex structure, multiple conjugation is possible.
Accordingly, in one embodiment, a conjugate of a small molecule and an oligonucleotide is provided wherein

    
    
        a) the small molecule comprises a nucleotide linker comprising 1-10 preferably 1-5, most preferably 1-3 2′OH-nucleotides;
        b) the oligonucleotide has the following modification pattern: the antisense strand with the modification pattern 5′-(w)-(Z1)-(Z2)-(Z3)na-3′ and a sense strand with the modification pattern 5′-(Z3)ns-, wherein the substituents as well as the variables na and ns have the meaning given above; and
        c) the oligonucleotide is covalently attached to the nucleotide linker of the small molecule.
    
    


The nucleotide linker is cleaved by intracellular nucleases such as DNAse II after internalization of the conjugate into the endosome, thus releasing the siRNA.
Preferably, said conjugate is co-administered with a delivery polymer. Suitable delivery polymers are described above.
In another embodiment of the invention a compound of formula (V) is provided. This compound comprises a cholesterol moiety, and a nucleotide linker comprising 1-10 preferably 1-5, most preferably 1-3 2′OH-nucleotides. This nucleotide linker is useful for covalently attaching an oligonucleotide such as a siRNA to the compound of formula (V). Preferably, said oligonucleotide has the novel modification pattern outlined above. Hence in another embodiment a conjugate of a compound of formula (V) and an oligonucleotide is provided, wherein the oligonucleotide is covalently attached to the nucleotide linker of the compound of formula (V).
The nucleotide linker is cleaved by intracellular nucleases such as DNAse II after internalization of the conjugate of a compound of formula (V) and an oligonucleotide into the endosome, thus releasing the siRNA.




Preferably, said conjugate of a compound of formula (V) and an oligonucleotide is co-administered with a delivery polymer. Suitable delivery polymers are described above.
In another embodiment, said delivery polymer and the conjugate of a compound of formula (V) and an oligonucleotide of the invention are not covalently attached and synthesized separately and may be supplied in separate containers or a single container.
DEFINITIONS
The term “small molecule” as used herein, refers to organic or inorganic molecules either synthesized or found in nature, generally having a molecular weight less than 10,000 grams per mole, optionally less than 5,000 grams per mole, and optionally less than 2,000 grams per mole.
The term “peptide” as used herein refers to any polymer compound produced by amide bond formation between an .alpha.-carboxyl group of one D- or L-amino acid and an .alpha.-amino group of another D- or L-amino acid. The term “protein” as used herein refers to polypeptides of specific sequence of more than about 50 residues.
The term “di-peptide motif” as used herein refers to any motif comprising an amide bond formed by either the D- or L-alpha or beta amino group of a first amino acid with the alpha-carboxyl group of a second D- or L-amino acid.
As used herein, the term “amino acid” refers to any molecule that contains both amine and carboxyl functional groups. Thus the term “amino acid” refers to both natural, non-natural and synthetic amino acids. Any natural amino acids used in the present invention are referred to herein by their common abbreviations.
The term “ligand” as used herein refers to a moiety that is capable of covalently or otherwise chemically binding a biologically active substance. The term “ligand” in the context of the invention is preferably a compound of formula (I) or (Ia) covalently attached to a biologically active substance.
The term “biologically active substance” as used herein refers to an inorganic or organic molecule including a small molecule, peptide (e.g. cell penetrating peptides), protein, carbohydrate (including monosaccharides, oligosaccharides, and polysaccharides), nucleoprotein, mucoprotein, lipoprotein, synthetic polypeptide or protein, or a small molecule linked to a protein, glycoprotein, steroid, nucleic acid (any form of DNA, including cDNA, or RNA, or a fragment thereof), nucleotide, nucleoside, oligonucleotides (including antisense oligonucleotides, LNA and siRNA), gene, lipid, hormone, or combination thereof, that causes a biological effect when administered in vivo to an animal, including but not limited to birds and mammals, including humans. Preferably, said biologically active substance is a peptide or a nucleic acid. Preferred nucleic acids used herein are siRNAs.
The term “nucleic acid” as used herein means an oligomer or polymer composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, or compounds produced synthetically (e.g., PNA as described in U.S. Pat. No. 5,948,902 and the references cited therein) which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions. Non-naturally occurring nucleic acids are oligomers or polymers which contain nucleobase sequences which do not occur in nature, or species which contain functional equivalents of naturally occurring nucleobases, sugars, or inter-sugar linkages, like peptide nucleic acids (PNA), threose nucleic acids (TNA), locked nucleic acids (LNA), or glycerol nucleic acids (GNA). This term includes oligomers that contain the naturally occurring nucleic acid nucleobases adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U), as well as oligomers that contain base analogs or modified nucleobases. Nucleic acids can derive from a variety of natural sources such as viral, bacterial and eukaryotic DNAs and RNAs. Other nucleic acids can be derived from synthetic sources, and include any of the multiple oligonucleotides that are being manufactured for use as research reagents, diagnostic agents or potential and definite therapeutic agents. The term includes oligomers comprising of a single strand nucleic acid or a double strand nucleic acid.
The term “2′-modified” as used herein refers to a β-D-ribonucleoside or β-D-ribonucleotide comprising of naturally occurring nucleobases having the 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 β-D-ribonucleotide comprising of naturally occurring nucleobases having a 2′-OH group.
The term “5′-phosphate” as used herein refers to the formula —O—P(═O)(OH)OH. In another aspect the phosphate is modified that one of the O or OH groups is replaced by S and termed herein as “5′-phosphothioate”
The term “phosphorothioate” as used herein refers to a internucleotide linkage in which one of the non-bridging oxygens is replaced by sulfur.
The term “delivery polymer” as used herein refers to polymers suitable for functional delivery of a biologically active substance. In the context of the present invention the delivery polymer is either covalently attached to or coadministered with the biologically substance conjugated to the compounds described herein and mediates endosomal escape after internalization into the cell and uptake into the endosome. The term “polymer” in this context means any compound that is made up of two or more monomeric units covalently bonded to each other, where the monomeric units may be the same or different, such that the polymer may be a homopolymer or a heteropolymer. Representative polymers include peptides, polysaccharides, nucleic acids and the like, where the polymers may be naturally occurring or synthetic. Non-limiting examples of delivery polymers are for example reviewed in INTERNATIONAL JOURNAL OF PHARMACEUTICAL RESEARCH AND DEVELOPMENT, October—2010/Volume—2/Issue—8/Article No—2. Non-limiting examples of delivery polymers useful for delivery of nucleic acids are disclosed in EP applications 10165502.5 and 10191030.5, PCT publication WO 2008/0022309 and U.S. provisional application 61/307,490 and references cited herein; which are all included by reference.
As used herein, “pharmaceutical composition” includes the conjugates of the invention, a pharmaceutical carrier or diluent and any other media or agent 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 the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).
A conjugate of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. To administer a conjugate of the invention by certain routes of administration, it may be necessary to coat the conjugate with, or co-administer the conjugate with, a material to prevent its inactivation. For example, the conjugate may be administered to a subject in an appropriate carrier or a 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 dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.
The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
These carriers may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. 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.
Regardless of the route of administration selected, the conjugates of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
The pharmaceutical composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier preferably is an isotonic buffered saline solution.
Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.



SHORT DESCRIPTION OF THE FIGURES
FIG. 1 shows Co-Administration of siRNA-conjugates comprising the compounds of formula (I) or (Ia) and a delivery polymer in vivo.
FIG. 2 shows Co-Administration of siRNA-conjugates comprising the compounds of formula (I) or (Ia) and a delivery polymer in vivo.
FIG. 3 shows Co-Administration of siRNA-conjugates comprising the compounds of formula (I) or (Ia) and a delivery polymer in vivo.
FIG. 4 shows Co-Administration of siRNA-conjugates comprising the compounds of formula (I) or (Ia) and a delivery polymer in vivo.
FIG. 5a shows antisense strand mediated gene silencing with fully 2′-modified siRNAs. COS7 cells were cotransfected with EGFP-directed siRNAs at 3 nM and psiCHECK2-AT. The knockdown activity of the siRNAs was assessed by measuring renilla versus firefly luciferase activity from the reporter construct. siRNAs were sorted by knockdown activity of unmodified (2-19-2) reference siRNAs.
FIG. 5b shows sense strand mediated gene silencing with fully 2′-modified siRNAs. COS7 cells were cotransfected with EGFP-directed siRNAs at 3 nM and psiCHECK2-ST. The knockdown activity of the siRNAs was assessed by measuring luciferase expression from the reporter construct. siRNAs were sorted by knockdown activity of unmodified (2-19-2) reference siRNAs.
FIG. 6a shows reduction of serum FVII activity in non-human primates upon intravenous injection of various 2′-modified siRNAs covalently attached to a delivery polymer.
FIG. 6b shows the development of the prothrombin time in non-human primates upon treatment with 2′-modified siRNAs covalently conjugated to a delivery polymer.



The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention.
EXAMPLES
Example 1
Step 1: 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]-propylamine




The title amine was prepared from its nitrile precursor according to a literature protocol [Lollo et al, WO2001/070415].
Step 2: 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




In a 2 μL round-bottomed 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 (21.15 g, 47.7 mmol, Eq: 1.00) and Huenig's base (12.3 g, 16.6 ml, 95.3 mmol, Eq: 2.00) were combined with AcOEt (845 ml) to give a colorless solution. Dihydrofuran-2,5-dione (4.77 g, 47.7 mmol, Eq: 1.00) in THF (42 ml) was added and the reaction mixture was stirred at ambient temperature over night=>white suspension. All volatiles were removed i. v., the residue dissolved in CH2Cl2, the organic layer washed with NH4Cl and brine, dried over Na2SO4, and evaporated to dryness. The crude product was dissolved in CH3CN/H2O and lyophilized to yield 29.8 g of the title compound as fluffy powder. MS (ISP): (M−H) 542.5.
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)-6-((4-methoxyphenyl)diphenylmethylamino)-1-oxohexan-2-ylamino)-3-(4-nitrophenyl)-1-oxopropan-2-yl)succinamide




In a 10 mL round-bottomed flask, the above prepared 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 (106 mg, 184 μmol, Eq: 1.00), (S)-2-((S)-2-amino-3-(4-nitrophenyl)propanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)diphenylmethylamino)hexanamide (132 mg, 184 μmol, Eq: 1.00), HOAt (25.0 mg, 184 μmol, Eq: 1.00) and EDC hydrochloride (35.3 mg, 184 μmol, Eq: 1.00) were mixed together in CH2Cl2 (1.8 ml) to give a yellow solution. Huenig's Base (47.5 mg, 64.2 μl, 368 μmol, Eq: 2.00) was added and the reaction stirred at ambient temperature over night. TLC indicated the consumption of starting material. All volatiles were removed i. V. and the crude product purified by flash chromatography SiO2/7% MeOH/0.1% NEt3 in CH2Cl2 to produce 128 mg of the title compound as light yellow solid. MS: expected mass: 1240.7552, found mass: 1240. 7518.
Step 4




In a 10 mL round-bottomed flask, the above prepared 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-oxohexan-2-ylamino)-3-(4-nitrophenyl)-1-oxopropan-2-yl)succinamide (126 mg, 101 μmol, Eq: 1.00) and Huenig's base (39.3 mg, 53.2 μl, 304 μmol, Eq: 3.00) were combined with CH2Cl2 (1.4 ml) and DMF (1.0 ml) to give a yellow suspension; bis(4-nitrophenyl) carbonate (46.3 mg, 152 μmol, Eq: 1.50) was added and the reaction allowed to proceed over night. The mixture was poured onto crashed ice, extracted 2× with AcOEt, washed with H2O, dried over Na2SO4, and evaporated to dryness. After trituration with  ̃10 ml of diethyl ether, 99 mg of the title product was obtained as an off-white solid. MS: expected mass: 1405.7614, found 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 25 mL round-bottomed flask, (S)-2-amino-6-((4-methoxyphenyl)diphenylmethyl-amino)hexanoic acid (Bioconjugate Chem. 2002, 13, 855-869, 968 mg, 2.31 mmol, Eq: 1.00) was dissolved in CH2Cl2 (20 ml) to give a light yellow solution. Huenig's base (897 mg, 1.21 ml, 6.94 mmol, Eq: 3.00) and trimethylchlorosilane (528 mg, 621 μl, 4.86 mmol, Eq: 2.10) were added and the reaction mixture was stirred for 15 min.
In a second 50 mL round-bottomed flask, (S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(4-nitrophenyl)propanoic acid (1 g, 2.31 mmol, Eq: 1.00) was dissolved in DMF (20 ml) to give a colorless solution. Huenig's base (359 mg, 485 μl, 2.78 mmol, Eq: 1.20) and TPTU [125700-71-2](687 mg, 2.31 mmol, Eq: 1.00) were added and the reaction mixture was stirred for 20′. The solution from the first flask containing the corresponding silyl ester monosilylamine was added and the reaction was stirred for another 3 hours. The mixture was poured onto crashed ice/NH4Cl, extracted 2× with AcOEt, washed with H2O and brine, dried over Na2SO4, and evaporated to dryness. Flash chromatography SiO2/10% MeOH/0.1% NEt3 in CH2Cl2 afforded 1.38 g of the title compound as brownish 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]-carbamic acid 9H-fluoren-9-ylmethyl ester




In a 250 mL pear-shaped flask, the above synthesized (S)-2-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-3-(4-nitrophenyl)propanamido)-6-((4-methoxyphenyl)diphenyl-methylamino)hexanoic acid (1.38 g, 1.66 mmol, Eq: 1.00), (4-aminophenyl)methanol (204 mg, 1.66 mmol, Eq: 1.00), HOAt (226 mg, 1.66 mmol, Eq: 1.00) and EDC hydrochloride (318 mg, 1.66 mmol, Eq: 1.00) were dissolved in CH2Cl2 (16.6 ml) to give a yellow solution. Huenig's base (428 mg, 579 μl, 3.31 mmol, Eq: 2.00) was added and the reaction allowed to proceed over night. The mixture was poured onto crashed ice/NH4Cl (pH  ̃7), extracted 2× with AcOEt, washed with H2O, dried over Na2SO4, and evaporated to dryness. The crude product was triturated with diethyl ether (1×50 mL); the resultant solid was filtered off and dryed to yield 1.214 g of the title compound as light-brown 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 50 mL round-bottomed flask, the above prepared [(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 (1.214 g, 1.29 mmol, Eq: 1.001) was combined with THF (19 ml) to give a brown solution. At 0°, diethylamine (1.77 g, 2.49 ml, 24.2 mmol, Eq: 18.70) was added. The reaction was stirred at ambient temperature for 3 h when MS indicated the disappearance of the starting material. All volatiles were evaporated i. V.; ensuing flash chromatography SiO2/0.1% NEt3 in CH2Cl2=>10% MeOH/0.1% NEt3 in CH2Cl2, followed by a second flash chromatography SiO2/5% MeOH/0.1% NEt3 in CH2Cl2 afforded 502 mg of the title compound as light brown foam. MS: expected mass: 715.337, found mass: 715.3362.
Example 2
O-Benzyl-N-[4-({3-[(3beta)-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-lysinamide




Was 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)propanoic acid as described above in steps a]-c]. MS: expected mass: 1466.8182, found mass: 1466.8136.
Example 3
N-[4-({3-[(3Beta)-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-lysinamide




Was 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)propanoic acid as described above in steps a]-c]. MS: expected mass: 1385.7716, found mass: 1385.7696.
Example 4
3,4-Dichloro-N-[4-({3-[(3beta)-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-lysinamide




Was 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)propanoic acid as described above in steps a]-c]. MS: expected mass: 1428.6984, found mass: 1428.695.
Example 5
4-Chloro-N-[4-({3-[(3beta)-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-lysinamide




Was 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 above in steps a]-c]. MS: expected mass: 1394.7373, found 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-methylheptan-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 name)




Was 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)propanoic acid as described above in steps a]-c]. MS: expected mass: 1410.792, found mass: 1410.7918.
Example 7
N-[4-({3-[(3Beta)-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-lysinamide




Was 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)propanoic acid as described above in steps a]-c]. MS: expected mass: 1378.7669, found mass: 1378.7609.
Example 8
N-[4-({3-[(3Beta)-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-lysinamide




Was 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)propanoic acid as described above in steps a]-c]. MS: expected mass: 1378.7669, found mass: 1378.7689.
Example 9
N-[4-({3-[(3Beta)-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-lysinamide




Was 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)propanoic acid as described above in steps a]-c]. MS: expected mass: 1378.7669, found mass: 1378.7659.
Example 10
Step 1: 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-oxohexan-2-ylamino)-4-oxobutan-2-yl)succinamide




In a 10 mL round-bottomed flask, the above prepared 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 (109 mg, 188 μmol, Eq: 1.00), (S)-2-[(S)-3-amino-4-(4-fluoro-phenyl)-butyrylamino]-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoic acid (4-hydroxymethyl-phenyl)-amide (132 mg, 188 μmol, Eq: 1.00), HOAt (25.6 mg, 188 μmol, Eq: 1.00) and EDC hydrochloride (36.1 mg, 188 μmol, Eq: 1.00) were mixed together in CH2Cl2 (2 ml) to give a yellow solution. Huenig's Base (48.7 mg, 64.1 μl, 377 μmol, Eq: 2.00) was added and the reaction stirred at ambient temperature over night. TLC indicated the consumption of starting material. All volatiles were removed i. V. and the crude product purified by flash chromatography SiO2/5% MeOH/0.1% NEt3 in CH2Cl2 to yield 197 mg of the title compound as off-white solid. MS: expected mass: 1227.7763, found mass: 1227.7714.
Step 2: 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-(4-fluorophenyl)butanamido)-6-((4-methoxyphenyl)diphenylmethylamino)hexanamido)-benzyl 4-nitrophenyl carbonate




In a 10 mL round-bottomed flask, the above prepared 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-oxohexan-2-ylamino)-4-oxobutan-2-yl)succinamide (196 mg, 160 μmol, Eq: 1.00) and Huenig's base (61.9 mg, 81.4 μl, 479 μmol, Eq: 3.00) were combined with CH2Cl2 (1.6 ml) and DMF (0.8 ml) to give a yellow suspension; bis(4-nitrophenyl) carbonate (72.8 mg, 239 μmol, Eq: 1.50) was added and the reaction allowed to proceed at ambient temperature over night. The mixture was poured onto crashed ice/NH4Cl (pH  ̃6), extracted 2× with AcOEt, washed with H2O and brine, dried over Na2SO4, and evaporated to dryness. After trituration with AcOEt/heptane one obtained 123 mg of the title compound as light yellow solid. MS: expected mass: 1392.7825, found mass: 1392.7819.
The necessary dipeptidic building block for step 1 was 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 25 mL round-bottomed flask, (S)-2-amino-6-((4-methoxyphenyl)diphenylmethyl-amino)hexanoic acid (Bioconjugate Chem. 2002, 13, 855-869, 1040 mg, 2.48 mmol, Eq: 1.00) was dissolved in CH2Cl2 (12.5 ml) to give a pale yellow solution. Huenig's base (961 mg, 1.27 ml, 7.44 mmol, Eq: 3.00) and trimethylchlorosilane (566 mg, 621 μl, 5.21 mmol, Eq: 2.10) were added and the reaction mixture was stirred at ambient temperature for 20 min.
In a second 50 mL round-bottomed flask, (S)-3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(4-fluorophenyl)butanoic acid (1040 mg, 2.48 mmol, Eq: 1.00) was dissolved in DMF (12.5 ml) to give a colorless solution. Huenig's base (385 mg, 506 μl, 2.98 mmol, Eq: 1.20) and TPTU [125700-71-2] (737 mg, 2.48 mmol, Eq: 1.00) were added and the reaction mixture was stirred for 15 min. The solution from the first flask containing the corresponding silyl ester monosilylamine was added and the reaction was stirred for another 3 hours at ambient temperature. The mixture was poured onto crashed ice/NH4Cl, extracted 2× with AcOEt, washed with H2O and brine, dried over Na2SO4, and evaporated to dryness. Flash chromatography SiO2/5% MeOH/0.1% NEt3 in CH2Cl2 afforded 2.10 g of the title compound as 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 250 mL pear-shaped flask, the above synthesized {(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 (2.10 g, 2.56 mmol, Eq: 1.00), (4-aminophenyl)methanol (315 mg, 2.55 mmol, Eq: 1.00), HOAt (349 mg, 2.56 mmol, Eq: 1.00) and EDC hydrochloride (491 mg, 2.56 mmol, Eq: 1.00) were dissolved in CH2Cl2 (12.5 ml). Huenig's base (662 mg, 871 μl, 5.21 mmol, Eq: 2.00) was added and the reaction allowed to proceed over night. The mixture was poured onto crashed ice/NH4Cl (pH  ̃7), extracted 2× 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 resultant solid was filtered off and dryed to yield 0.796 g of the title compound as light-brown 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 50 mL round-bottomed flask, the above prepared {(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 (793 mg, 857 mol, Eq: 1.001) was combined with THF (12 ml) to give a brownish solution. At 0°, diethylamine (1.13 g, 1.59 ml, 15.4 mmol, Eq: 18) was added. The reaction was stirred at ambient temperature over night. The mixture was poured onto crashed ice/NH4Cl (pH  ̃7), extracted 2× with AcOEt, washed with H2O and brine, dried over Na2SO4, and evaporated to dryness. Flash chromatography SiO2/10% MeOH/0.1% NEt3 in CH2Cl2 yielded 500 mg of the title compound as off-white solid. MS: expected mass: 702.3581, found 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




Was prepared in analogy to Example 10, but using in step 1 (S)-2-((S)-3-amino-4-phenylbutanamido)-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-phenylbutanoic acid as described above in steps a]-c]. MS: expected mass: 1374.792, found mass: 1374.7877.
Example 12
4-({N ̃2 ̃-[(3S)-4-(4-chlorophenyl)-3-{[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]amino}butanoyl]-N ̃6 ̃-[(4-methoxyphenyl)(diphenyl)methyl]-L-lysyl}amino)benzyl 4-nitrophenyl carbonate




Was 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 above in steps a]-c]. MS (ISP): (M+H) 1409.9.
Example 13
N-[4-({3-[(3Beta)-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-lysinamide




Was 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)propanoic acid as described above in steps a]-c] of example 1. MS (ISP): (M+H) 1391.9.
Example 14
N-[4-({3-[(3Beta)-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-lysinamide




Was prepared in analogy to example 1, but using in step 3 (R)-2-((R)-2-amino-3-phenyl-propanamido)-N-(4-(hydroxymethyl)phenyl)-6-((4-methoxyphenyl)diphenylmethyl)amino)-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. This building block was synthesized from (R)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-6-aminohexanoic acid and (R)-2-amino-6-((4-methoxyphenyl)-diphenylmethylamino)hexanoic acid (see Bioconjugate Chem. 2002, 13, 885-869) as described above in steps a]-c]. MS: expected mass: 1360.7763, found mass: 1360.7774.
Example 15
4-({N ̃2 ̃-[(3S)-3-{[4-({3-[(3Beta)-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




Was 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 above in steps a]-c]. MS: expected mass: 1399.7872, found mass: 1399.7857.
Example 16
N-[4-({3-[(3beta)-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-lysinamide




Step 1: (S)-2-((S)-2-Amino-3-phenyl-propionylamino)-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoic acid (4-hydroxymethyl-phenyl)-amide




The building block (S)-2-((S)-2-amino-3-phenyl-propionylamino)-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoic acid (4-hydroxymethyl-phenyl)-amide was prepared in analogy to the procedure described in Bioconjugate Chem., Vol. 13, No. 4, 2002, 855-869 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-((S)-1-(4-(hydroxymethyl)phenylamino)-6-((4-methoxyphenyl)diphenylmethylamino)-1-oxohexan-2-ylamino)-1-oxo-3-phenylpropan-2-yl)succinamide




TPTU [125700-71-2] (233 mg, 784 μmol, Eq: 1.00) was added to a solution of 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) (426 mg, 0.784 mmol, Eq: 1.00) and Huenig's base (304 mg, 411 μl, 2.35 mmol, Eq: 3) in DMF (10 ml). After 3 minutes (S)-2-((S)-2-amino-3-phenyl-propionylamino)-6-{[(4-methoxy-phenyl)-diphenyl-methyl]-amino}-hexanoic acid (4-hydroxymethyl-phenyl)-amide (step 1) was added TLC at t=1 h showed the reaction was complete. The solvent was removed under reduced pressure. The remaining residue was taken up in ethyl acetate and extracted with NaHCO3 half sat. solution (1×), potassium hydrogen phthalate solution 0.05M (2×), water (1×) and brine (1×). The organic extract was dried over MgSO4 and concentrated under reduced pressure. The crude material was purified by flash chromatography to obtain the titled product (682 mg, 513, μmol) as a light brown solid. MS (ISP): (M+H) 1196.8
Step 3: Hünig's base (465 mg, 629 μl, 3.6 mmol, Eq: 6) was added to a solution of the previous alcohol (718 mg, 600 μmol, Eq: 1.00) and bis(4-nitrophenyl) carbonate (548 mg, 1.8 mmol, Eq: 3) in THF (20 ml). The yellow solution was stirred overnight at room temperature. The solvent was removed under reduced pressure. The remaining residue was triturated with diethyl ether. The solid was collected by filtration, washed with ether and dried under reduced pressure to obtain the title compound (800 mg, 529 μmol) as a light 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 (0.969 g, 2.00 mmol, Eq: 1.00) was suspended in a 1:1 v/v mixture of 1,2-dimethoxyethane and water (17 ml) and treated at 0° C. with L-norleucine (0.275 g, 2.10 mmoll, Eq: 1.05) and NaHCO3 (0.185 g, 2.20 mmol, Eq: 1.10). The cooling bath was removed and the reaction allowed to proceed at ambient temperature for 14 h. The mixture was poured onto crashed ice/citric acid (pH  ̃3), extracted 2× with ethyl acetate, washed with H2O and brine, dried over Na2SO4, and evaporated to dryness. Flash chromatography SiO2/AcOEt yielded 0.870 mg of the title compound as 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




In a pear-shaped flask, the above synthesized (S)-2-[(S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-phenyl-propionylamino]-hexanoic acid (10.72 g, 21 mmol, Eq: 1.00), (4-aminophenyl)methanol (2.717 g, 22 mmol, Eq: 1.03), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) (7.994 g, 32 mmol, Eq: 1.50) were dissolved in CH2Cl2 (320 ml) and stirred overnight under a balloon of Ar. The mixture was poured onto crashed ice/NH4Cl, extracted 2× with AcOEt, washed with H2O, dried over Na2SO4, and the volume reduced to  ̃300 ml. The precipitate was filtered off and dryed to give 5.25 g of the title compound as light-brown 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, the above prepared {(S)-1-[(S)-1-(4-hydroxymethyl-phenylcarbamoyl)-pentylcarbamoyl]-2-phenyl-ethyl}-carbamic acid 9H-fluoren-9-ylmethyl ester (4.738 g, 7.822 mmol, Eq: 1.0) was dissolved in CH2Cl2 (28 ml). At 0°, diethylamine (28 ml, 19.80 g, 271 mmol, Eq: 35) was added and the reaction mixture stirred at ambient temperature overnight. All volatiles were evaporated i. V.; ensuing flash chromatography SiO2/CH2Cl2/10% MeOH, followed by crystallization from AcOEt, yielded 2.116 g 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-oxohexan-2-ylamino)-1-oxo-3-phenylpropan-2-yl)succinamide




was prepared therewith in analogy to example 16 step 2. MS (ISP): (M+H) 909.7 (M+Na) 931.8.

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




was prepared therewith in analogy to example 16 step 3. MS expected mass: 1073.6453, found mass 1073.642
Example 18
N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-alanyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]glycinamide




Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-Chlorotrityl Resin




2-Chlorotrityl chloride resin (Novabiochem 01-64-0114, 100-200 mesh), 1% DVB (18 g, 21.6 mmol, Eq: 1.00) was swollen in DCM/DMF=1/1 (300 mL) for ten minutes. The resin was drained and a solution of FMOC-4-aminobenzyl alcohol (14.9 g, 43.2 mmol, Eq: 2) and pyridine (6.83 g, 6.99 ml, 86.4 mmol, Eq: 4) in DCM/DMF=1/1 (300 mL) was added. The mixture was shaken overnight. The resin was drained and capped with a solution of 10% Hünig's Base in methanol (300 mL). The resin was washed with DMF and DCM and dried over night with HV to obtain 21.7 g resin. Determination of the load resulted in 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 from step 1 (1 g, 410 μmol, Eq: 1.00) was prewashed with DMF (2×) and treated with piperidine/DMF=1/4 (10 mL) for 5 and 10 minutes. The resin was washed alternately with DMF and IPA (3×10 mL). A solution of Fmoc-Gly-OH (488 mg, 1.64 mmol, Eq: 4), TPTU (487 mg, 1.64 mmol, Eq: 4) and Huenig's base (636 mg, 859 μl, 4.92 mmol, Eq: 12) in DMF (10 mL) was stirred for 5 minutes and then shaken with the resin for one hour. The resin was washed alternately with DMF and isopropyl alcohol (3×).
The following Fmoc cleavages and subsequent couplings of Fmoc-Ala-OH (511 mg, 1.64 mmol, 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) (892 mg, 1.64 mmol, Eq: 4) were performed accordingly. The dried peptide resin was stirred for about 2×30 min in TFA 1%/DCM (2×20 mL). The reaction mixture was filtered and the resin was washed with DCM. The filtrates were pooled and the solvents evaporated under vacuum. The crude material was triturated with diethyl ether (2×). After purification by flash chromatography, the product (84 mg, 97.3 μmol) was obtained as a white solid. MS expected mass: 776.5452, found mass 776.5455
Step 3: The above prepared 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-oxopropan-2-yl)succinamide [RO5545270](70 mg, 90.1 μmol, Eq: 1.00) and bis(4-nitrophenyl) carbonate (137 mg, 450 μmol, Eq: 5) under Argon at room temperature were dissolved in DMF (4 ml) and treated with Huenig's base (34.9 mg, 47.2 μl, 270 μmol, Eq: 3). and the mixture was allowed to react overnight. The solvent was removed in vacuo. The resulting solid was triturated with diethylether. The solid was collected by filtration and washed with diethyl ether. The product was dried in vacuo to obtain the title compound (84 mg, 80.2 μmol) as a light brown solid. MS expected mass: 941.5514, found mass 941.5518
Example 19
N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-leucyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-methioninamide




Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-Chlorotrityl Resin




was prepared in analogy to example 18, step 1

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-oxopentan-2-yl)succinamide




was prepared in analogy to example 18, step 2, using Fmoc-Met-OH (609 mg, 1.64 mmol, Eq: 4) and Fmoc-Leu-OH (580 mg, 1.64 mmol, Eq: 4) as amino acids. The product (208 mg, 210 μmol) was obtained as a light yellow solid. MS (ISP): (M+H) 893.6183
Step 3: was prepared in analogy to example 18, step 3. After purification on silica gel, the title compound (161 mg, 137 μmol) was obtained as light brown solid. MS expected mass: 1057.6174, found mass 1057.6184
Example 20
N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-leucyl-N ̃1 ̃-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-aspartamide




Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-Chlorotrityl Resin




was performed in analogy to example 18, step 1

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-oxobutanamido)-4-methylpentanamido)-N1-(4-(hydroxymethyl)phenyl)succinamide




Was prepared in analogy to example 18, step 2, using Fmoc-Asn-OH (581 mg, 1.64 mmol, Eq: 4) and Fmoc-Leu-OH (580 mg, 1.64 mmol, Eq: 4) as amino acids. The product (87 mg, 89.4 μmol) was obtained as a light yellow solid. MS expected mass: 875.6136, found mass 875.6133
Step 3: The titled compound was prepared in analogy to example 18, step 3. After purification on silica gel (87 mg, 89.4 μmol) the title compound was obtained as light brown solid. MS expected mass: 1040.6198, found mass 1040.6188
Example 21
N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-alanyl-N ̃1 ̃-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-aspartamide




Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-Chlorotrityl Resin




was performed in analogy to example 18, step 1
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-oxobutanamido)propanamido)-N1-(4-(hydroxymethyl)phenyl)succinamide




Was prepared in analogy to example 18, step 2, using Fmoc-Asn-OH (581 mg, 1.64 mmol, Eq: 4) and Fmoc-Ala-OH (511 mg, 1.64 mmol, Eq: 4) as amino acids. The product (140 mg, 159 μmol) was obtained as light 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 (169 mg, 152 μmol) it was obtained as light brown solid. MS expected mass: 998.5729, found mass 998.5739
Example 22
N ̃2 ̃-[4-({3-[(3beta)-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-Aminobenzylalcohol to the 2-Chlorotrityl Resin




Was performed in analogy to example 18, step 1
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-oxobutanamido)-N1-(2-(4-(hydroxymethyl)phenylamino)-2-oxoethyl)succinamide




Was prepared in analogy to example 18, step 2, using Fmoc-Gly-OH (488 mg, 1.64 mmol, Eq: 4) and Fmoc-Asn-OH (581 mg, 1.64 mmol, Eq: 4) as amino acids. The product (140 mg, 162 μmol) was obtained as white solid. MS expected mass: 819.551, found mass 819.5503
Step 3: The title compound was obtained in analogy to example 18, step 3 (176 mg, 161 μmol) as light brown solid. MS expected mass: 984.5572, found mass 984.5489
Example 23
N-[4-({3-[(3beta)-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-Aminobenzylalcohol to the 2-Chlorotrityl Resin




Was performed in analogy to example 18, step 1
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




Was prepared in analogy to example 18, step 2, using Fmoc-Gly-OH (488 mg, 1.64 mmol, Eq: 4) and Fmoc-Phe-OH (635 mg, 1.64 mmol, Eq: 4) as amino acids. The product (259 mg, 288 μmol) was obtained as white solid. MS expected mass: 852.5765, found mass 852.5754
Step 3: The title compound was obtained in analogy to example 18, step 3. (280 mg, 247 μmol) as light brown solid. MS expected mass: 1017.5827, found mass 1017.5775
Example 24
N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-leucyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]glycinamide




Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-Chlorotrityl Resin




Was performed in analogy to example 18, step 1
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-oxopentan-2-yl)succinamide




Was prepared in analogy to example 18, step 2, using Fmoc-Gly-OH (488 mg, 1.64 mmol, Eq: 4) and Fmoc-Leu-OH (580 mg, 1.64 mmol, Eq: 4) as amino acids. The product (240 mg, 278 μmol) was obtained as a light yellow solid. MS expected mass: 818.5921, found mass 818.5921
Step 3: The title compound was prepared in analogy to example 18, step 3. After purification on silica gel, it (194 mg, 177 μmol) was obtained as light yellow solid. MS expected mass: 983.5983 found mass 983.6004
Example 25
N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-leucyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-phenylalaninamide




Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-Chlorotrityl Resin




Was performed in analogy to example 18, step 1
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-oxopentan-2-yl)succinamide




Was prepared in analogy to example 18, step 2, using Fmoc-Phe-OH (635 mg, 1.64 mmol, Eq: 4) and Fmoc-Leu-OH (580 mg, 1.64 mmol, Eq: 4) as amino acids. The product (153 mg, 151 μmol) was obtained as light yellow solid. MS expected mass: 908.6391 found mass 908.637.
Step 3: The title compound was prepared in analogy to example 18, step 3. After purification on silica gel, it (117 mg, 98 μmol) was obtained as white solid. MS expected mass: 1073.6453 found mass 1073.646
Example 26
N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-phenylalanyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-phenylalaninamide




Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-Chlorotrityl Resin




Was performed in analogy to example 18, step 1
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




Was prepared in analogy to example 18, step 2, with Fmoc-Phe-OH (635 mg, 1.64 mmol, Eq: 4) as amino acid. The product (240 mg, 204 μmol) was obtained as light yellow solid. MS expected mass: 942.6234 found mass 942.6218

Step 3: The title compound was prepared analogously to example 18, step 3. After purification on silica gel, it (190 mg, 154 μmol) was obtained as white solid. MS expected mass: 1107.6296 found mass 1107.6287
Example 27
N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-leucyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-leucinamide




Step 1: Addition of FMOC-4-Aminobenzylalcohol to the 2-Chlorotrityl Resin




Was performed analogously to example 18, step 1
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-oxopentan-2-ylamino)-4-methyl-1-oxopentan-2-yl)succinamide




Was prepared in analogy to example 18, step 2, with Fmoc-Leu-OH (1.59 g, 4.5 mmol, Eq: 3) as amino acid. The product (254 mg, 284 μmol) was obtained as white solid. MS expected mass: 874.6547 found mass 874.6527.
Step 3: The title compound was prepared in analogy to example 18, step 3. After purification on silica gel it was obtained as white solid (178 mg, 168 μmol). MS expected mass: 1039.6609 found mass 1039.6588
Example 28
N-[4-({3-[(3beta)-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 (1 g, 2.61 mmol, Eq: 1.00) and (4-aminophenyl)methanol (483 mg, 3.92 mmol, Eq: 1.5) in THF (20 ml) was treated with EEDQ (970 mg, 3.92 mmol, Eq: 1.5). The solution was stirred over night at room temperature. The mixture was diluted with 10% 2-propanol/ethyl acetate (100 mL) and the solution was washed with KHSO4 5%/K2SO4 10% (2×), water (1×) and brine (1×), dried over MgSO4 and evaporated in vacuo. The residue was sonicated in diethyl ether for several minutes and the solid was collected by filtration to obtain the product (1.27 g, 1.2 mmol) as light brown solid. MS (ISP): (M+H) 488.3
Step 2: (S)-2-Amino-N—[(S)-1-(4-hydroxymethyl-phenylcarbamoyl)-ethyl]-propionamide




The compound was prepared in analogy to example 1 step c to obtain the product (245 mg, 877 μmol) as light yellow solid. MS (ISP): (M+H) 266.3, (M+Na) 288.2 (2M+H) 531.3
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-oxopropan-2-ylamino)-1-oxopropan-2-yl)succinamide




The compound was prepared in analogy to example 16 step 2 (165 mg, 198 μmol) as light brown solid. MS expected mass: 790.5608, found mass 790.5587.
Step 4: The title compound was prepared in analogy to example 18, step 3. After purification on silica gel it was obtained as white solid (99 mg, 98.4 μmol). MS expected mass: 955.567, found mass 955.5651.
Example 29
N-[4-({3-[(3beta)-cholest-5-en-3-yloxy]propyl}amino)-4-oxobutanoyl]-L-isoleucyl-N ̃1 ̃-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-aspartamide




Step 1: (S)-2-[(2S,3S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-methyl-pentanoylamino]-succinamic acid




2-Chlorotrityl chloride resin (5 g, 7.5 mmol, Eq: 1.00) was swollen in DCM and then treated with a solution of Fmoc-Asn(Trt)-OH (8.95 g, 15.0 mmol, Eq: 2) and Huenig's base (3.88 g, 5.1 ml, 30.0 mmol, Eq: 4) in DCM overnight. The resin was washed with DCM and capped with a solution of 10% Huenig's base in methanol. Coupling of Fmoc-Ile-OH (5.3 g, 15.0 mmol, Eq: 2) with TPTU (4.46 g, 15.0 mmol, Eq: 2) and Huenig's base (3.88 g, 5.1 ml, 30.0 mmol, Eq: 4) according to standard solid phase peptide synthesis. The product was cleaved from the resin with a cocktail of TFA/Water/triisopropylsilane (95/2.5/2.5 v/v/v) for two hours at room temperature. The resin was filtered and the filtrate was concentrated under reduced pressure to a small volume. After trituration with diethyl ether, the product was filtered and dried in vacuum to obtain the product (2.85 g, 5.79 mmol) as white solid. MS expected mass: 467.2056, found mass 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




The compound was prepared in analogy to example 28 step 1 (620 mg, 336 μmol) as light yellow solid.
Step 3: (S)-2-((2S,3S)-2-Amino-3-methyl-pentanoylamino)-N*1*-(4-hydroxymethyl-phenyl)-succinamide




The compound was prepared in analogy to example 1 step c (100 mg, 228 μmol) as light yellow solid.

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-methylpentanamido)-N1-(4-(hydroxymethyl)phenyl)succinamide




The compound was prepared in analogy to example 16 step 2 (89 mg, 91.4 μmol) as light yellow solid.
Step 5: The compound from the previous step was reacted to the title compound analogously to example 18, step 3. After purification on silica gel, it (42 mg, 36.3 μmol) was obtained as a light brown solid. MS expected mass: 1040.6198, found mass 1040.6177
Example 30
N-[4-({3-[(3beta)-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-lysinamide




The compound was prepared in analogy to example 16 step 1, starting with Fmoc-D-Lys(Boc)-OH, (158 mg, 116 μmol) as light brown solid. MS (ISP): (M+H) 1362.8 (M+Na) 1383.8
Example 31
N-{15-[(3beta)-cholest-5-en-3-yloxy]-4,15-dioxo-8,11-dioxa-5,14-diazapentadecan-1-oyl}-L-phenylalanyl-N ̃6 ̃-[(4-methoxyphenyl)(diphenyl)methyl]-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-lysinamide




The title compound was prepared analogous to example 16 using a cholesterol-oligo-PEG derivative in step 2 of the synthesis. MS (ISP): (M+H) 1479.8
The necessary 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 for step 2 was prepared as follows:
Step a: {2-[2-(2-Amino-ethoxy)-ethoxy]-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]phenanthren-3-yl ester




A solution of cholesteryl chloroformate (1 g, 2.23 mmol) in 25 mL dichloromethane was added dropwise under stirring to a solution of 2,2′-(ethylenedioxy)bis-(ethylamine) (495 mg, 3.34 mmol) in 75 mL dichloromethane. The reaction was stirred overnight at room temperature. The reaction was diluted with dichloromethane and extracted with water. The organic extract was dried over anhydrous MgSO4 dihydrate, filtered and evaporated. After purification on amino-modified silica gel (eluent: MeCl2→MeCl2/MeOH=975:25 v/v) the product (615 mg) was obtained as a white, waxy solid. MS (ISP): (M+H) 561.5
Step b: 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




The amine from step a (480 mg, 0.856 mmol) and triethylamine (0.13 mL, 0.94 mmol) were dissolved in 5 mL dichloromethane. After adding succinic anhydride (90 mg, 0.9 mmol) the solution was stirred overnight at room temperature. TLC check showed still some starting material. More succinic anhydride (20 mg, 0.2 mmol) was added. After stirring the reaction for another 3 hours at room temperature, 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 obtain the desired acid (490 mg, 0.667 mmol). MS (ISP): (M+H) 661.5
Example 32
N-{30-[(3beta)-cholest-5-en-3-yloxy]-4,30-dioxo-8,11,14,17,20,23,26-heptaoxa-5,29-diazatriacontan-1-oyl}-L-phenylalanyl-N ̃6 ̃-[(4-methoxyphenyl)(diphenyl)methyl]-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-lysinamide




The title compound was prepared analogous to example 16 using a cholesterol-PEG derivative in step 2 of the synthesis. MS (ISP): (M+H) 1699.9
The necessary cholesterol-PEG intermediate 1-[(3beta)-cholest-5-en-3-yloxy]-1,27-dioxo-5,8,11,14,17,20,23-heptaoxa-2,26-diazatriacontan-30-oic acid for step 2 was prepared as follows:
Step a: tert-butyl [25-({(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-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-azapentacos-1-yl]carbamate




Cholesteryl chloroformate (476 mg, 1.06 mmol) and triethylamine (155 uL, 1.113 mmol) were dissolved in 5 mL dichloromethane. Then a solution of alpha-amino-omega-boc-amino-octa(ethylene glycol) (497 mg, 1.06 mmol) dissolved in 1 mL dichloromethane was added. The solution was stirred over night at room temperature and diluted with dichloromethane and extracted with a KHSO4 5%/K2SO4 10% aqueous mixture. The organic extract was dried over anhydrous MgSO4, filtered and evaporated in vacuo. After purification on silica gel (eluent: MeCl2/MeOH=975:25→95:5 v/v) the product (530 mg, 0.571 mmol) was obtained as a colorless oil. MS (ISP): (M+NH4) 898.7
Step b: 1-[(3beta)-cholest-5-en-3-yloxy]-1,27-dioxo-5,8,11,14,17,20,23-heptaoxa-2,26-diazatriacontan-30-oic acid




The previous Boc derivative (450 mg, 0.511 mmol) was dissolved in HCl 4M in dioxane (10.2 mL, 40.9 mmol). The solution was stirred at room temperature for 40 min. The solvent was removed in vacuo and the remaining white solid was dissolved in 5 mL dichloromethane and treated with triethylamine (32 uL, 0.229 mmol) and succinic anhydride (11.5 mg, 0.114 mmol) overnight. More succinic anhydride (11 mg, 0.11 mmol, 0.2 equiv.) was added and after 60 min the reaction was diluted with dichloromethane and washed with KHSO4 5%/K2SO4 10% buffer. The organic extract was dried over MgSO4 anhydrous, filtered and evaporated to obtain 390 mg of the desired product. MS (ISP): (M+H) 881.7
Example 33
N-{66-[(3beta)-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-nonadecaoxa-5,65-diazahexahexacontan-1-oyl}-L-phenylalanyl-N ̃6 ̃-[(4-methoxyphenyl)(diphenyl)methyl]-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-lysinamide




The title compound was prepared analogous to example 16 using a cholesterol-PEG derivative in step 2 of the synthesis. MS (ISP): (M+H) 2228.1
The necessary cholesterol-PEG intermediate 1-[(3beta)-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-diazahexahexacontan-66-oic acid for step 2 was prepared as follows:
Step a: (3beta)-cholest-5-en-3-yl(59-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57 nonadecaoxanonapentacont-1-yl)carbamate




Alpha, omega-bis-amino 20 (ethylene glycol) (538 mg, 0.6 mmol) and triethylamine (92 uL, 0.66 mmol) were dissolved in 15 mL dry dichloromethane. A solution of cholesteryl chloroformate (270 mg, 0.6 mmol) in 2 mL dry dichloromethane was added dropwise at room temperature. The solution was stirred overnight, then concentrated in vacuo to a small volume and purified directly on silica gel (eluent: MeCl2/MeOH=95:5→9:4→4:1 v/v) to obtain the product (350 mg, 0.254 mmol) as a waxy solid. MS (ISP): (M+H) 1309.9
Step b: 1-[(3beta)-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-diazahexahexacontan-66-oic acid




The amine from step a (329 mg, 0.251 mmol), succinic anhydride (26.4 mg, 0.264 mmol) and triethylamine (40 uL, 0.286 mmol) were dissolved in 5 mL dry dichloromethane. After adding more triethylamine (40 uL, 0.286 mmol), the solution (pH>8) was stirred overnight at room temperature. The reaction was diluted with dichloromethane and washed twice with a KHSO4 5%/K2SO4 10% aqueous mixture. The organic extract was dried over anhydrous MgSO4, filtered and evaporated to obtain the product (260 mg, 0.175 mmol) as a colorless, waxy solid. MS (ISP): (M+NH4) 1408.9
Example 34
General Procedure for the Preparation of RNA Conjugates Materials
Dimethyl sulfoxide (DMSO), N,N-Diisopropylethylamine (DIPEA) and sodium acetate solution (3 M, pH 5.2) were purchased from Sigma Aldrich Chemie GmbH (Traufkirchen, Germany).
Triethylammonium acetate (TEAA) (2.0 M, pH 7.0) and Acetonitrile (ACN, HPLC quality) for RP-HPLC were purchased from Biosolve (Valkenswaard, Netherlands).
Ethanol (EtOH, p.a.) was purchased from Merck (Darmstadt, Germany). Purified water from a Optilab HF (Membra Pure, Germany) system was used.
Resource RPC 3 mL column (10×0.64 cm; 15 μm particle size) was purchased from GE Healthcare (Freiburg, Germany).
HPLC purification was accomplished using an ÄKTA Explorer 100 system (GE Healthcare).
Synthesis of Amino-Modified RNA
RNA equipped with a hexylaminolinker at the 5′-end of the sense strand was produced by standard phosphoramidite chemistry on solid phase at a scale of 1215 μmol using an ÄKTA Oligopilot 100 (GE Healthcare, Freiburg, Germany) and controlled pore glass as solid support (Prime Synthesis, Aston, Pa., USA). RNA containing 2′-O-methyl nucleotides were generated employing the corresponding phosphoramidites, 2′-O-methyl phosphoramidites and TFA-hexylaminolinker amidite (Sigma-Aldrich, SAFC, Hamburg, Germany). Cleavage and deprotection as well as purification was achieved by methods known in the field (Wincott F., et al, NAR 1995, 23, 14, 2677-84).
The amino-modified RNA was characterized by anion exchange HPLC (purity: 96.1%) and identity was confirmed by ESI-MS ([M+H]1+ calculated: 6937.4; [M+H] 1+measured: 6939.0.
Sequence: 5′-(NH2C6)GGAAUCuuAuAuuuGAUCcAsA-3′ (SEQ ID NO: 283); u, c: 2′-O-methyl nucleotides of corresponding RNA nucleotides, s: phosphorthioate.
General Experimental Conjugation Procedure
The title compounds of examples 1-33 were coupled via the amino-modified RNA according the following procedure:
RNA equipped with a C-6 aminolinker at the 5′-end (16.5 mg, 1 equivalent) is dissolved in 500 μL DMSO and 150 μL water. The p-Nitrophenylcarbonate derivative (10 equivalents) dissolved in 1 mL DMSO is added followed by 8 μL DIPEA. The reaction mixture is shaken at 35° C. in the dark and monitored using RP-HPLC (Resource RPC 3 mL, buffer: A: 0.1M TEAA in water, B: 0.1M TEAA in 95% ACN, gradient: 3% B to 100% B in 20 CV). Once the reaction is gone to completion the RNA conjugate is precipitated using sodium acetate (3 M) in EtOH at −20° C. For examples lacking a MMT protecting group in the dipeptide motif the corresponding conjugates are purified using the conditions described above. Pure fractions are pooled and the material is precipitated using sodium acetate/EtOH to give the desired RNA conjugate.
RNA conjugates containing a MMT protecting group in the dipeptide sequence are further processed according to the procedure given below.
General Procedure for MMT Cleavage
The crude RNA conjugate pellet is dissolved in 500 μL water and 1.5 mL sodium acetate buffer (3 M, pH 5.2 or 0.1M, pH 4.0). The solution is shaken for 2 days at 30° C. The reaction mixture is monitored using RP-HPLC (Resource RPC 3 mL, buffer: A: 0.1M TEAA in water, B: 0.1M TEAA in 95% ACN, gradient: 3% B to 100% B in 20 CV). After complete cleavage of the MMT protecting group the RNA conjugate is directly purified using the conditions just mentioned above. Pure fractions are pooled and the desired conjugate is precipitated using sodium acetate/EtOH.
As a control a RNA conjugate lacking the dipeptide motif was synthesized. For this purpose cholesterol was attached to the 5′-end via a linker described in the literature (Nature Biotech, 2007, 25, 1149). This conjugate is referred to as “non-cleavable”.
All the RNA conjugates were analyzed by RP HPLC for purity and identity was confirmed by ESI MS (negative mode). Briefly, RP-HPLC was performed on a Dionex Ultimate system (Dionex, Idstein, Germany) equipped with a XBridge C18 column (2.5×50 mm, 2.5 μm particle size, Waters, Eschborn, Germany) at 65° C. column temperature. Gradient elution was performed using 100 mM hexafluoroisopropanol (HFIP) and 16 mM triethylamine in 1% methanol as eluent A and in 95% methanol as eluent B (1% B to 18% B in 30 minutes). UV detection was recorded at 260 nm. For mass spectrometric analysis a ThermoFinnigan LCQ DecaXP ESI-MS system with micro-spray source and ion trap detector was coupled online to the HPLC system.
Examples of specific compounds of formula (IIa) are disclosed in table 1. The resulting compounds are referred to “di-peptide containing cholesterol siRNA conjugates”, wherein the specific di-peptide containing cholesterol siRNA conjugates are further referred to as “Title compound example X—(NHC6)-(siRNA sequence)” and “siRNA with Title compound of Example X”.
siRNA Preparation









Antisense sequence:



(SEQ ID NO: 154)



5′-uuGGAUcAAAuAuAAGAuUCcscsU-3′






u, c: 2′-O-methyl nucleotides of corresponding RNA nucleotides, s: phosphorthioate
The di-peptide containing cholesterol siRNA conjugates directed against the apolipoprotein B mRNA were generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 80-85° C. for 3 minutes and cooled to room temperature over a period of 3-4 hours. Duplex formation was confirmed by native gel electrophoresis.
All prepared di-peptide containing cholesterol siRNA conjugates are listed in table 2.







TABLE 1







Di-peptide containing cholesterol siRNA conjugates (5′-3′) and analytical data.


Key: lower case letters a, c, g, u, are 2′-O-Methyl nucleotides; A phosphorothioate


linkages is symbolized with a lower case “s”. (NHC6) is the aminohexyl linker


incorporatd at the 5′-end of the sense strand.












Title







compound

Base





from

sequence
Mol
Mol
Purity


example

SEQ ID
mass
mass
(%)


No.

NO:
calc.
exp.
(RP)















16
(Title compound Ex 16)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7885.7
7887.5
94.4


 


31
(Title compound Ex 16)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
8003.8
8003.3
97.3


 


33
(Title compound Ex 33)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
8752.7
8752.4
97.6


 


32
(Title compound Ex 32)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
8223.1
8226.5
97.3


 


17
(Title compound Ex 17)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7870.7
7873.5
90.6


 


30
(Title compound Ex 30)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7884.8
7888.8
95.2


 


27
(Title compound Ex 27)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7840.0
7840.0
94.8


 


28
(Title compound Ex 28)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7755.4
7754.9
93.2


 


29
(Title compound Ex 29)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7840.4
7839.9
87.2


 


1
(Title compound Ex 1) (NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7931.1
7935.2
98.2


 


2
(Title compound Ex 2)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7992.2
7995.0
96.7


 


3
(Title compound Ex 3)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7911.1
7913.8
98.2


 


4
(Title compound Ex 4)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7955.1
7958.5
98.0


 


5
(Title compound Ex 5)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7920.5
7923.9
97.2


 


6
(Title compound Ex 6)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7936.1
7939.6
98.5


 


7
(Title compound Ex 7)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7904.2
7905.5
95.1


 


8
(Title compound Ex 8)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7904.2
7908.7
98.8


 


9
(Title compound Ex 9)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7904.2
7906.7
98.7


 


10
(Title compound Ex 10)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7918.2
7921.0
95.4


 


11
(Title compound Ex 11)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7902.0
7901.5
98.7


 


12
(Title compound Ex 12)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7934.54
7936.5
94.4


 


13
(Title compound Ex 13)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7916.09
7917.9
96.5


 


14
(Title compound Ex 14)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7886.07
7888.3
94.9


 


24
(Title compound Ex 24)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7781.8
7783.4
97.2


 


23
(Title compound Ex 23)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7815.8
7817.3
95.2


 


22
(Title compound Ex 22)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7781.8
7783.9
90.5


 


26
(Title compound Ex 26)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7905.9
7907.0
96.4


 


25
(Title compound Ex 25)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7871.9
7873.2
96.1


 


20
(Title compound Ex 20)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7840.7
7840.0
95.9


 


19
(Title compound Ex 19)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7857.8
7856.6
97.3


 


18
(Title compound Ex 18)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7741.6
7741.1
93.9


 


21
(Title compound Ex 21)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7798.6
7797.8
87.6


 


15
(Title compound Ex 15)(NHC6)GGAAUCuuAuAuuuGAUCcASA
284
7927.1
7926.8
97.2
















TABLE 2







Di-peptide containing cholesterol siRNA conjugates. The last entry


(SEQ ID NO pair 266/154) represents a siRNA conjugate lacking the


di-peptide motif. Key: lower case letters a, c, g, u, are 2′-O-Methyl


nucleotides; A phosphorothioate linkages is symbolized with a lower


case “s”. (NHC6) is the aminohexyl linker incorporated at the


5′-end of the sense strand.










SEQ ID

SEQ ID



No
Sense sequence (5′-3′)
No
Antisense sequence (5′-3′)





284
(Title compound Ex 16)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 31)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 33)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 32)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 17)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 30)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 27)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 28)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 29)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 1)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 2)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 3)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 4)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 5)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 6)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 7)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 8)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 9)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 10)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 11)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 12)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 13)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 14)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 24)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 23)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 22)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 26)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 25)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 20)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 19)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 18)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 21)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


284
(Title compound Ex 15)(NHC6)
154
uuGGAUcAAAuAuAAGAuUCcscsU



GGAAUCuuAuAuuuGAUCcAsA




 


285
(Chol)GGAAUCuuAuAuuuGAUCcAcA
154
uuGGAUcAAAuAuAAGAuUCcscsU









Example 35
In Vivo Experiments
Co-Administration of Di-Peptide Containing Cholesterol siRNA Conjugates and Delivery Polymer In Vivo
Six to eight week old mice (strain C57BL/6 or ICR,  ̃18-20 g each) were obtained from Harlan Sprague Dawley (Indianapolis Ind.). Mice were housed at least 2 days prior to injection. Feeding was performed ad libitum with Harlan Teklad Rodent Diet (Harlan, Madison Wis.).
Mice (n=3 per group) were injected with a mixture of 0.2 mL solution of delivery polymer and 0.2 ml di-peptide containing cholesterol siRNA conjugates. The injected dose was, unless otherwise stated, 15 mg/kg for the delivery polymer and 0.1 mg/kg with respect to the di-peptide containing cholesterol siRNA conjugates. Solutions were injected by infusion into the tail vein. 48 hours post injection serum ApoB levels were measured relative to isotonic glucose treated animals according to the procedure below.
Serum ApoB Levels Determination.
Mice were fasted for 4 h before serum collection by submandibular bleeding. Serum ApoB protein levels were determined by standard sandwich ELISA methods. Briefly, a polyclonal goat anti-mouse ApoB antibody and a rabbit anti-mouse ApoB antibody (Biodesign International) were used as capture and detection antibodies respectively. An HRP-conjugated goat anti-rabbit IgG antibody (Sigma) was applied afterwards to bind the ApoB/antibody complex. Absorbance of tetramethyl-benzidine (TMB, Sigma) colorimetric development was then measured by a Tecan Safire2 (Austria, Europe) microplate reader at 450 nm.
In FIG. 1 various di-peptide containing cholesterol siRNA conjugates were benchmarked against the same siRNA conjugated to cholesterol but lacking the cleavable motif elaborated earlier in this section. The effect of this siRNA conjugate (SEQ ID NO pair 266/154, “non-cleavable control”) on serum ApoB levels was set to 1 in order to evaluate the influence of the di-peptide containing conjugates relative to the non-cleavable control. Substituting the initially used Phe-Lys motif (siRNA with Title compound of Example 16) with the corresponding D-amino acids (siRNA with Title compound of Example 14) or just replacing the Lys with the unnatural enantiomer (siRNA with Title compound of Example 30) yielded ApoB reduction less pronounced or equivalent to the non-cleavable control siRNA. Replacing Lys by Gly (siRNA with Title compound of Example 23) or Phe by p-Methoxyphenylalanine (siRNA with Title compound of Example 13) reduced the potency compared to siRNA with Title compound of Example 16. Other di-peptide motifs containing siRNA conjugates were shown to be as efficacious as the original Phe-Lys containing conjugate.
FIG. 2 summarizes di-peptide containing cholesterol siRNA conjugates that were as efficacious or had improved efficacy compared to siRNA with Title compound of Example 16 consisting of the Phe-Lys motif. All these conjugates were significantly more active compared to the “non-cleavable” cholesterol siRNA conjugate SEQ ID NO pair 266/154. The best performing di-peptide containing cholesterol siRNA conjugates had a fluorine modified phenyl ring in the Phy-Lys motif (siRNA with Title compound of Example 8, siRNA with Title compound Example 9) or had the phenylalanine substituted with beta-phenylalanine (siRNA with Title compound of Example 11) or a derivative thereof (siRNA with Title compound of Example 10).
Since di-peptide containing cholesterol siRNA conjugates with di-peptide motifs consisting of D-amino acids are performing equal to the non-cleavable control conjugate it is conceivable that the other di-peptide sequences are indeed cleaved by a protease activity in vivo. However, given the broad acceptance of different amino acids and derivatives thereof it is likely that more than one enzyme is participating in the cleavage reaction as suggested in the literature (Bioconjugate Chem. 2002, 13, 855).
As shown in FIG. 3, the incorporation of a Cathepsin cleavable di-peptide motif (in this case Phe-Lys, siRNA with Title compound of Example 16) between the siRNA and the small molecule ligand cholesterol boosts the potency of the siRNA conjugate compared to the straight cholesterol siRNA conjugate (SEQ ID NO pair 266/154). Further spacing of the cholesterol ligand from the di-peptide motif by means of PEG based linkers diminishes the potency proportional to the length of the PEG linker.
In FIG. 4 the polymer dose was kept constant at 15 mg/kg. The siRNA dose was titrated and the effect on serum ApoB content was measured. The Di-peptide containing cholesterol siRNA conjugates containing the Phe-Lys (F-K) motif was significantly more potent compared to the control conjugate lacking the di-peptide sequence.
Example 36
2′-Modified Oligoribonucleotide Synthesis
Oligoribonucleotides were synthesized according to the phosphoramidite technology on solid phase. Depending on the scale either an ABI 394 synthesizer (Applied Biosystems) or an AKTA oligopilot 100 (GE Healthcare, Freiburg, Germany) was used. Syntheses were performed on a solid support made of controlled pore glass (CPG, 520 {acute over (Å)}, with a loading of 75 μmol/g, obtained from Prime Synthesis, Aston, Pa., USA). All 2′-modified RNA phosphoramidites as well as ancillary reagents were purchased from SAFC (Hamburg, Germany). Specifically, the following 2′-O-Methyl phosphoramidites 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. The 2′-Deoxy-2′-fluoro-phosphoramidites carried the same protecting groups as the 2′-O-methyl RNA amidites. All amidites were dissolved in anhydrous acetonitrile (100 mM) and molecular sieves (3 {acute over (Å)}) were added. To generate the 5′-phosphate the 2-[2-(4,4′-Dimethoxytrityloxy)ethylsulfonyl]ethyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite from Glen Research (Sterling, Va., USA) was used. In order to introduce the C-6 aminolinker at the 5′-end of the oligomers the 6-(Trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite from Thermo Fisher Scientific (Milwaukee, Wis., USA) was employed. The 5′-modifications were introduced without any modification of the synthesis cycle. 5-Ethyl thiotetrazole (ETT, 500 mM in acetonitrile) was used as activator solution. Coupling times were 6 minutes. In order to introduce phosphorothioate linkages a 50 mM solution of 3-((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from AM Chemicals, Oceanside, Calif., USA) in anhydrous Acetonitrile/pyridine (1:1 v/v) was employed.
Example 37
Cleavage and Deprotection of Support Bound Oligomer
After finalization of the solid phase synthesis, the dried solid support was transferred to a 15 mL tube and treated with concentrated aqueous ammonia (Aldrich) for 18 hours at 40° C. After centrifugation the supernatant was transferred to a new tube and the CPG was washed with aqueous ammonia. The combined solutions were evaporated and the solid residue was reconstituted in buffer A (see below).
Example 38
Purification of Oligoribonucleotides
Crude oligomers were purified by anionic exchange HPLC using a column packed with Source Q15 (GE Helthcare) and an AKTA Explorer system (GE Helthcare). Buffer A was 10 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4 (Fluka, Buchs, Switzerland) and contained 20% Acetonitrile and buffer B was the same as buffer A with the exception of 500 mM sodium perchlorate. A gradient of 22% B to 42% B within 32 column volumes (CV) was employed. UV traces at 280 nm were recorded Appropriate fractions were pooled and precipitated with 3M NaOAc, pH=5.2 and 70% Ethanol. Finally, the pellet was washed with 70% Ethanol.
Example 39
Annealing of Oligoribonucleotides to Generate siRNA
Complementary strands were mixed by combining equimolar RNA solutions. The mixture was lyophilized and reconstituted with an appropriate volume of annealing buffer (100 mM NaCl, 20 mM sodium phosphate, pH 6.8) to achieve the desired concentration. This solution was placed into a water bath at 95° C. which was cooled to rt within 3 h.
Example 40
In Vitro Activity of siRNAs Devoid of 2′-OH Residues
In order to investigate if siRNAs lacking any 2′-OH residues show potent in vitro knock down activity, we tested a panel of EGFP mRNA-targeted siRNAs with different 2′-modification chemistries (SEQ ID pairs 31/32 to 149/150, and see Table 3 for examples). The siRNAs were screened for sense and antisense activity with the Dual-Glo® Luciferase Assay System (Promega) using the psiCHECK2 vector (Promega) in COS7 cells (DSMZ, Braunschweig, Germany, cat. No. ACC-60). To address the silencing activity conferred by sense and antisense strand we cloned each corresponding 19mer target site sequence as separate psiCHECK2 construct (psiCHECK2-AT for antisense activity, psiCHECK2-ST for sense activity) into the multiple cloning region located 3′ to the translational stop codon of the synthetic Renilla luciferase. By using Lipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany, cat. No. 11668-019) COS7 cells were co-transfected with vector construct and 3 nM of the corresponding siRNA complementary to the cloned target site. Successful siRNA-mediated silencing was determined 24 hours after transfection via the activity of the renilla luciferase normalized to firefly luciferase levels to take transfection efficiency into account (see FIG. 5a for antisense activity and FIG. 5b for sense activity).
Table 3: Exemplary siRNA sequences and chemical modifications used for determination of in vitro knock down activity dependent on 2′-modifications. Reference duplices and selected examples of corresponding modification variants used in this study. Xf indicates a 2′-fluoro modification of the nucleotide X, small letters indicate a 2′-O-methyl modification, underlined letters indicate a DNA nucleotide, all other capital letters indicate ribonucleotides. The letter “p” indicates a 5′-phosphate.










RNA duplices
















unmod
5′- UGCCCAUCCUGGUCGAGCUTT -3′



(SEQ ID NO: 286)


 



3′- TTACGGGUAGGACCAGCUCGAp -5′



(SEQ ID NO: 287)


 


F/OMe
5′- UfgCfcCfaUfcCfuGfgUfcGfaGfcUfTsT -3′



(SEQ ID NO: 288)


 



3′- TsTaCfgGfgUfaGfgAfcCfaGfcUfcGfap -5′



(SEQ ID NO: 289)


 


F/DNA
5′- UfGCfCCfAUfCCfUGfGUfCGfAGfCUfTsT -3′



(SEQ ID NO: 290)


 



3′- TsTACfGGfGUfAGfGAfCCfAGfCUfCGfAp -5′



(SEQ ID NO: 291)


 


DNA/OMe
5′- UgCcCaUcCuGgUcGaGcUTsT -3′



(SEQ ID NO: 292)


 



3′- TsTaCgGgUaGgAcCaGcUcGap -5′



(SEQ ID NO: 293)









It was found that the 5 most potent modified siRNAs (≧60% knock-down) were designed in an alternating 2′-fluoro/2′-O-methyls (2′F/2′-OMe) pattern. While conferring antisense activity, this chemistry fully eliminated the activity of the corresponding sense strands, as shown by lack or minimal renilla luciferase activity for all tested 2′F/2′-OMe variants.
We concluded that such 2′F/2′-OMe pattern is promoting the siRNA's intended antisense strand activity while undesired off-target effects coming from the sense strand are fully suppressed. This design is specifically preferable for siRNAs, that come with the need for protection against 2′O-directed nucleolytic cleavage.
Example 41
Detection of DNAse II-Sensitive Sites by In Vitro Assay
An ion pairing (IP) reversed phase (RP) high performance liquid chromatography (HPLC) coupled to an electrospray ionization (ESI) mass spectrometry (MS) or an anion exchange (AEX)-HPLC based method was established to test the in vitro stability of selected single and double stranded RNAs.
Method description: For stability analysis a 10 μM solution of either single stranded or double stranded RNA was incubated at 37° C. in 5 mM sodium acetate buffer solution (pH 4.5) containing 0.8 or 8 units DNase II (from bovine spleen, Type V, Sigma Aldrich). The incubation reaction was stopped by adding a 100 mM triethyl ammonium acetate (TEAA) solution, shifting the pH to 7 and inactivating the DNase II enzyme. Analysis was done by either LC/MS combined with UV-detection or by AEX-HPLC with UV-detection. UV-detection traces at 260 nm were used for quantitative analysis, MS data served for cleavage site identification within the RNA sequence.

    A. IP-RP-HPLC was done employing a Waters XBridge C18 column (2.5×50 mm, 2.5 μm particle size) at 65° C. column temperature. Gradient elution was performed using 100 mM hexafluoroisopropanol (HFIP) and 16 mM triethylamine in 1% methanol as eluent A and composition A in 95% methanol as eluent B. A gradient from 1% B to 18% B in 30 minutes was employed.
    B. AEX-HPLC was performed on a Dionex DNA Pac200 column (4×250 mm) at 50° C. using a 20 mM phosphate buffer containing 10% ACN at pH=11. Eluent B contained 1 M NaBr in eluent A. A gradient from 25 to 62% B in 18 minutes was employed.









TABLE 4







Duplexes and the remaining intact strands evaluated for their


stability against DNase II. Key: lower case letters a, c, g,


u, are 2′-O-Methyl nucleotides; Upper case letters A, C, G,


U followed by “f” indicates a 2′-fluoro nucleotide. Lower


case “p” indicates a 5′-phosphate. (invdT) represents an


inverted deoxythimidine (3′-3′-linked). A phosphorothioate


linkages is symbolized with a lower case “s”. dT is deoxy-


thimidine. (NHC6) is the aminohexyl linker incorporated at


the 5′-end of the sense strand.














% Intact


% Intact


SEQ

strand
SEQ

strand


ID
Sense strand
after 6
ID
Antisense strand
after 6


NO
sequence (5′-3′)
hours
NO
sequence (5′-3′)
hours





157
GGAuGAAGuGGAGAuuAGud
  0
158
ACuAAUCUCcACUUcAUCCd
  0, 1



TsdT


TsdT



 


160
(NH2C6)GfgAfuGfaAfgUfgGfa
101
159
pasCfuAfaUfcUfcCfaCfuUfcAf
 97



GfaUfuAfgUf(invdT)


uCfc(invdT)



 


165
(NH2C6)GfcAfaAfgGfcGfuGfc
103
166
puGfaGfuUfgGfcAfcGfcCfuUfu
103



CfaAfcUfcAf(invdT)


Gfc(invdT)



 


167
(NH2C6)GcAAAGGcGuGccAA
 56
168
UGAGUUGGcACGCCUUUGC
 49



cucAdTsdT


dTsdT



 


169
(NH2C6)GGAUfCfAUfCfUfCf
 64
170
GUfAAGACfUfUfGAGAUfGA
 54



AAGUfCfUfUfACfdTsdT


UfCfCfdTsdT



 


153
GGAAUCuuAuAuuuGAUCcAs
  0, 1
154
uuGGAUcAAAuAuAAGAuUC
  0, 1



A


cscsU



 


173
(NH2C6)UfgAfcCfaCfaGfuCfg
102
174
pusUfuAfaUfcCfgAfcUfgUfgGf
102



GfaUfuAfaAf(invdT)


uCfa(invdT)



 


175
(NH2C6)uGAccAcAGucGGAu
  0, 4
176
puUuAAUCCGACUGUGGucA
  0, 3



uAAAdTsdT


dTsdT



 


175
(NH2C6)uGAccAcAGucGGAu
  6
177
UUuAAUCCGACUGUGGUcA
  3



uAAAdTsdT


dTsdT









CONCLUSIONS
A. RNA strands containing at least one 2′-OH nucleotide (e.g. both strands of SEQ ID NO pair 157/158) are rapidly degraded via a cyclic pentavalent intermediate, leading to 2′-3′ cyclic phosphates at the 5′-cleavage product. The formation of the pentavalent intermediate can be inhibited using nucleotides lacking a 2′-OH group, like e.g. 2′-deoxy, 2′-OMe or 2′-F.

B. Additionally, RNA is degraded via a 5′-exonucleolytic pathway, that is independent from the 2′-modification on the 5′-terminal nucleotides. This degradation pathway can be inhibited using 5′-terminal non-nucleotide moieties, like e.g. a C6-aminolinker (e.g. SEQ ID NO 160 in SEQ ID NO pair 160/159 or SEQ ID NO 165 in SEQ ID NO pair 165/166) or a phosphorothioate at the first internucleotide linkage (e.g. SEQ ID NO 160 in SEQ ID NO pair 160/159).

C. A 5′-phosphate group slows down the exonucleolytic cleavage kinetics, but can not fully block the degradation starting at this end (e.g. SEQ ID NO 160 in SEQ ID NO pair 160/159). This is most probably due to the cleavage of the 5′-phosphate by either phosphatases or by an inherent phosphatase activity of the DNase II enzyme.

D. The best protection for RNA strands was achieved with oligonucleotides containing no 2′-OH nucleotide within the strand, starting with a 2′-OMe nucleotide at the 5′-end connected by a phosphorothioate linkage to the second nucleotide (e.g. SEQ ID NO 173 in SEQ ID NO pair 173/174). Other terminal non-2′-OH nucleotides also protect against the 5′-exo degradation, but to a lower extent compared to the 2′-OMe modification (refer to Table 9)

Example 42
In Vivo Knock Down Activity of siRNAs Devoid of 2′-OH Residues
In vivo experiments were conducted with mice injected with Factor VII (FVII)-targeting siRNAs (SEQ ID NO pairs 179/166 and 180/168, see Table 5) co-administered with DPC-GalNac.







TABLE 5 a







Sequences of siRNAs for in vivo experiment. Key: lower


case letters a, c, g, u, are 2′-O-Methyl nucleotides;


Upper case letters A, C, G, U followed by “f” indicates


a 2′-fluoro nucleotide. Lower case “p” indicates a 5′-


phosphate. (invdT) represents an inverted deoxythimidine


(3′-3′-linked). A phosphorothioate linkages is symbolized


with a lower case “s”. dT is deoxythimidine. (NHC6) is


the aminohexyl linker incorporated at the 5′-end of the


sense strand. GalNAc refers to the structure in formula


(IV).









SEQ ID
SEQ ID



NO pair
NOs
Sequence 5′->3′





179/166
179
GalNAc-(NHC6)- GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)


 



166
puGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)


 


180/168
180
GalNAc-(NHC6)-GcAAAGGcGuGccAAcucAdTsdT


 



168
UGAGUUGGcACGCCUUUGCdTsdT









A FVII siRNA with an alternating 2′-OMe/2′-F pattern on sense and antisense strand was generated with a 5′-terminal 2′-OMe nucleotide on the antisense and a 5′-terminal 2′-F strand on the sense strand. Both strands are protected by an inv(dT) at the 3′-terminal overhang. The antisense strand was bearing a 5′-phosphate group to maintain activity of the siRNA. The sense strand was conjugated to a GalNAc-palmitoyl construct at its 5′ end for targeting to hepatocytes by the asialyloglycoprotein-receptor. siRNA (2.5 mg/kg) was co-administered with GalNAc-targeted PBAVE (15 mg/kg) in mice.
FVII mRNA measurements were done from liver homogenates using QuantiGene 1.0 branched DNA (bDNA) Assay Kit (Panomics, Fremont, Calif., USA, Cat-No: QG0004).
At necropsy 1-2 g liver tissue was snap frozen in liquid nitrogen. Frozen tissue was powderized with mortar and pistil on dry ice. 15-25 mg of tissue was transferred to a chilled 1.5 mL reaction tube, 1 mL 1:3 Lysis Mixture prediluted in MilliQ water and 3.3 μL Proteinase K (50 μg/μL) was added and tissue was lysed by several seconds ultrasound sonication at 30-50% power (HD2070, Bandelin, Berlin, Germany). Lysates were stored at −80° C. until analysis. For mRNA analysis lysate was thawed and digested with Proteinase K for 15 min at 1000 rpm in a thermomixer at 65° C. (Thermomixer comfort, Eppendorf, Hamburg, Germany). FVII and GAPDH mRNA levels were determined using QuantiGene 1.0 bDNA Assay Kit reagents according to the manufacturer's recommendations. FVII mRNA expression was analyzed using 20 μL lysate and a mouse FVII probe set. GAPDH mRNA expression was analysed using 40 μL lysate and rattus norwegicus probe sets shown to be cross-react with mice (sequences of probesets see above). As assay readout the chemiluminescence signal at end of the assay was measured in a Victor 2 Light luminescence counter (Perkin Elmer, Wiesbaden, Germany) as relative light units (RLU). The signal for FVII mRNA was divided by signal for GAPDH mRNA from the same lysate Values are reported as FVII mRNA expression normalized to GAPDH.
Results demonstrate a 79% FVII mRNA knock down at 48 hours post dosing after administration of SEQ ID NO pair 179/166. In contrast, the 2′-OH nucleotide bearing siRNA SEQ ID NO pair 180/168 showed no significant knock down (<25%), as shown in Table 5.







TABLE 5 b







Results of in vivo knockdown studies









Time
SEQ ID NO pair 179/166
SEQ ID NO pair 180/168


[hour]
Remaining mRNA [%]
Remaining mRNA [%]












1
84
92


6
83
88


24
53
100


48
21
76









Example 43
Tissue Distribution of siRNAs Devoid of 2′-OH Residues
The siRNA concentration in the liver tissue samples was determined using a proprietary oligonucleotide detection method as described in WO2010043512. Briefly, the siRNA quantification is based on the hybridization of a fluorescently (Atto-425) labeled PNA-probe (Atto425-OO-GCAAAGGCGTGCCAACT (SEQ ID NO: 270), obtained from Panagene Inc, Korea) complementary to the antisense strand of the siRNA duplex, followed by AEX-HPLC based separation. Quantification was done by fluorescence detection against an external calibration curve that was generated from a dilution series of the two FVII siRNA used in the in vivo experiment (see example 42). For plasma samples between 0.2 to 2 μL and for tissue  ̃1 mg aliquots were injected onto the HPLC system.
Liver tissue analysis of the stabilized siRNA lacking 2′-OH nucleotide showed high concentrations of intact antisense strand in the liver in the ug/g range, but  ̃95% was present in the 5′-dephosphorylated inactive form (see table IR.04). The resulting RNA with a terminal 2′-OMe nucleotide is not prone for rephosphorylation in the cytoplasm by the phosphokinase hClp1 (see below). In contrast, the antisense strand of the 2′-OH containing siRNA was completely degraded in the tissue within the first 6 hours post dosing.







TABLE 6







Liver tissue analysis of the stabilized


siRNA containing no 2′-OH nucleotide










SEQ ID NO pair 181/186
SEQ ID NO pair 181/185


Time
in Liver [ng/g]
in Liver [ng/g]











[hour]
−5′-Phosphat
+5′-Phosphat
−5′-Phosphat
+5′-Phosphat














1
873
171
9
BDL


6
1351
106
 BDL*
BDL


24
1043
65
BDL
BDL


48
1062
66
BDL
BDL





*BDL = below detection limit






Example 44
In Vitro Knock Down Activity of siRNAs with Optimized 5′-Ends
An additional in vitro screen for FVII siRNAs was conducted in order to identify siRNAs that may be intracellularly (re-)phosphorylated at the antisense's 5′-end to result in the RNAi-competent species All siRNAs from this screen are shown in Table 7. The alternating 2′-OMe/2′-F modification pattern was identical to the 1st generation design (without any 2′-OH residues) with exception of various modifications at the first two nucleotides at the 5′-end of the antisense strand. The two 5′-terminal nucleotides of the antisense strand were generated as 2′-F or 2′-deoxy modified nucleotides in various combinations with and without an additional 5′-phosphate or 5′-phosphothioate. All siRNAs were screened in dose response (24 nM to 0.00037 nM in 4fold dilutions) for knock down activity after transfection of primary mouse hepatocytes (30000 cells per well; 96 well plate formate) using Lipofectamine 2000 according to the manufacturer's instructions. Two siRNAs were comparable active to the parental duplex (SEQ ID NO pair 182/168); comparable active siRNAs: SEQ ID NO pairs 181/186 and 181/185) in terms of IC50 values, one with a 5′-terminal 2′-F and a phosphate group and one with two 5′-terminal 2′-deoxy nucleotides and a 5′-phosphorothioate (see Table 7 for IC50 values). Both of them are  ̃5-6-fold more active compared to the siRNA (SEQ ID NO pair 181/166) used in the first animal experiment with the terminal 2′-OMe nucleotide.







TABLE 7







IC 50 values











SEQ

SEQ




ID

ID

IC50


NO
Sense strand sequence (5′-3′)
NO
Sense strand sequence (5′-3′)
(nM)





181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
185
pUfsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)
0.17


 


182
GcAAAGGcGuGccAAcucAdTsdT
168
UGAGUUGGcACGCCUUUGCdTsdT
0.228


 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
186
psdTdGaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)
0.228


 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
187
psdTsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)
0.554


 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
188
pdTsdGaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)
0.631


 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
189
pdTsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)
0.702


 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
190
pusGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)
0.749


 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
166
puGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)
1.002


 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
191
psUfGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)
1.185


 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
192
UfsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)
2.257


 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
193
psUfsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)
2.428


 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
194
psdTsdGaGfuUfgGfcAfcGfcCfuUfuGfc(invdT
3.208


 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
195
usGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)
3.974


 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
196
uGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)
4.235


 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
197
dTsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)
4.235


 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
198
psdTGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)
4.704


 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
199
dTsdGaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)
5.341


 


183
(Chol)GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invd
190
pusGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)
9.771









Example 45
In Vitro 5′-Phosphorylation of siRNAs with Optimized 5′-Termini
All siRNAs without a 5′-phosphate or 5′-phosphorothioate listed in Table 7 were assessed for phosphorylation by hClp1 in a HeLa S100 cell extract.
5′-phosphorylation s was analyzed from S100 HeLa extracts as described by Weitzer and Martinez (S. Weitzer and J. Martinez. hClp1: a novel kinase revitalizes RNA metabolism. Cell Cycle 6 (17):2133-2137, 2007). Directly after incubation of 1 μM siRNAs in the S100 HeLa extract containing 5 mM ATP, the solution was analyzed by either IP-RP-HPLC or AEX-HPLC under denaturing conditions by injection of 5 μL sample solution:

    A. IP-RP-HPLC was done employing a Waters XBridge C18 column (2.5×50 mm, 2.5 μm particle size) at 65° C. column temperature. Gradient elution was performed using 100 mM hexafluoroisopropanol (HFIP) and 16 mM triethylamine in 1% methanol as eluent A and composition A in 95% methanol as eluent B. A gradient from 1% B to 18% B in 30 minutes was employed.
    B. AEX-HPLC was performed on a Dionex DNA Pac200 column (4×250 mm) at 50° C. using a 20 mM phosphate buffer containing 10% ACN at pH=11. Eluent B contained 1 M NaBr in eluent A. A gradient from 25 to 62% B in 18 minutes was employed.


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

%(5′-phosphorylation)=100*PA[5′-phosphorylated strand]/(PA[5′-phosphorylated strand]+PA[parent strand])

In Table 8 is shown, that the antisense strand of an siRNA cannot be 5′-phosphorylated, when a 2′-OMe nucleotide is located at the 5′-terminus (SEQ ID NO pair 181/196 and SEQ ID NO pair 181/195). In contrast the antisense strand is susceptible to 5′-phosphorylation, when a 2′-F, 2′-deoxy or 2′-OH nucleotide is 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). The two siRNAs, that were comparably active in the in vitro assay as the parental SEQ ID NO pair 182/168 (SEQ ID NO pair 181/186 and 181/185), are susceptible to 5′-phosphorylation once the synthetically introduced 5′-phosphate/5′-PTO group is cleaved in vivo, eg. by phosphatases.







TABLE 8







Percentage of 5′-phosphorylated strand after 4 hours incubation in S100


HeLa cell extract. Key: lower case letters a, c, g, u, are 2′-O-Methyl


nucleotides; Upper case letters A, C, G, U followed by “f” indicates a


2′-fluoro nucleotide.(invdT) represents an inverted deoxythimidine


(3′-3′-linked). A phosphorothioate linkages is symbolized with a lower


case “s”. dT is deoxythimidine.












SEQ

sense
SEQ

antisense


ID
Sense strand sequence
5′P
ID
Antisense strand sequence 
5′P


NO
(5′-3′)
[%]
NO
(5′-3′)
[%]





181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf
52
196
uGfaGfuUfgGfcAfcGfcCfuUfuGfc
  0



(invdT)


(invdT)



 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf
53
195
usGfaGfuUfgGfcAfcGfcCfuUfuGfc
  0



(invdT)


(invdT)



 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf
44
192
UfsGfaGfuUfgGfcAfcGfcCfuUfuGfc
 22



(invdT)


(invdT)



 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf
42
197
dTsGfaGfuUfgGfcAfcGfcCfuUfuGfc
 22



(invdT)


(invdT)



 


181
GfcAfaAfgGfcGfuGfcCfaAfcUfcAf
47
199
dTsdGaGfuUfgGfcAfcGfcCfuUfuGf
 13



(invdT)


c(invdT)



 


182
GcAAAGGcGuGccAAcucAdTsdT
31
168
UGAGUUGGcACGCCUUUGCdTsd
 42






T



 


184
GfcAfaAfgGfcGfuGfcCfaAfcUfcA
22
168
UGAGUUGGcACGCCUUUGCdTsd
100






T









Example 46
In Vitro DNAse II-Stability of siRNAs with Optimized 5′ Ends
All antisense strands were screened for DNAse II stability as described in example 41. The two antisense strands present in the siRNAs that were comparable active to the parental duplex (SEQ ID NO 186 and SEQ ID NO pair 185 one with a 5′-terminal 2′-F and a phosphate group and one with two 5′-terminal 2′-deoxy nucleotides and a 5′-phosphorthioate are stable towards DNAse II cleavage II (>70% intact strand after 20 hr incubation).







TABLE 9







In vitro stability of siRNAs towards DNase II after


20 hours incubation










Sense
Antisense

%


SEQ ID
SEQ ID

intact


NO
NO
Sequence (5′-3′)
strand





181
192
UfsGfaGfuUfgGfcAfcGfc70.CfuUfuGfc(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 Activity of siRNAs with Optimized 5′ Ends
In order to evaluate if the in vitro improvement by optimized 5′-ends transfers to the in vivo situation, we conducted further mouse experiments with GalNAc-palmitoyl conjugates of selected siRNAs (see Table 10). SiRNAs were administered as under identical conditions as described for the first mouse experiment (example 42, this patent application).
For measurement of FVII levels, plasma samples from mice were prepared by collecting blood (9 volumes) by submandibular bleeding into microcentrifuge tubes containing 0.109 mol/L sodium citrate anticoagulant (1 volume) following standard procedures. FVII activity in plasma was measured with a chromogenic method using a BIOPHEN VII kit (Hyphen BioMed/Aniara, Mason, Ohio) following manufacturer's recommendations. Absorbance of colorimetric development was measured using a Tecan Safire2 microplate reader at 405 nm.
The siRNAs under investigation showed improved in vivo activity, fully correlating with the in vitro screening results. FVII activity in serum was reduced by more than 80% for both siRNAs 48 hours post dosing, compared to 49% using the first generation DNase II stable siRNA design (see Table 10). This result clearly underscores the importance of a 5′-terminal nucleotide on the antisense strand that can be effectively phosphorylated, in case phosphatases in vivo cleave the synthetically generated 5′-phosphate or 5′-phosphothioate group. In case of a 5′-terminal 2′-OMe nucleotide as used in the first design or described in the literature as a more potent siRNA design based on in vitro comparison with canonical siRNAs. (Allerson et al. J. Med Chem. 2005, 48, 901-904), the cleavage of the synthetic phosphate in vivo would lead to a strong reduction in potency of the corresponding siRNA.







TABLE 10







In vivo knockdown activity of siRNAs with optimized 5′ends. Key:


lower case letters a, c, g, u, are 2′-O-Methyl nucleotides; Upper


case letters A, C, G, U followed by “f” indicates a 2′-fluoro


nucleotide. Lower case “p” indicates a 5′-phosphate. (invdT)


represents an inverted deoxythimidine (3′-3′-linked). A phos-


phorothioate linkages is symbolized with a lower case “s”.(NHC6)


is the aminohexyl linker incorporated at the 5′-end of the sense


strand. GalNAc refers to the structure in formula (IV).















%


SEQ

SEQ

remaining


ID
Sense strand sequence
ID
Antisense strand sequence
FVII in


NO
(5′-3′)
NO
(5′-3′)
serum





179
GalNAc-(NH2C6)-
166
puGfaGfuUfgGfcAfcGfcCfuUfuGfc
27



GfcAfaAfgGfcGfuGfcCfaAfcUfc

(invdT)




Af(invdT)





 


179
GalNAc-(NH2C6)-
190
pusGfaGfuUfgGfcAfcGfcCfuUfuGfc
51



GfcAfaAfgGfcGfuGfcCfaAfcUfc

(invdT)




Af(invdT)





 


179
GalNAc-(NH2C6)-
185
pUfsGfaGfuUfgGfcAfcGfcCfuUfuGfc
17



GfcAfaAfgGfcGfuGfcCfaAfcUfc

(invdT)




Af(invdT)





 


179
GalNAc-(NH2C6)-
263
psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc
13



GfcAfaAfgGfcGfuGfcCfaAfcUfc

(invdT)




Af(invdT)









Example 48
In Vitro Knock Down Activity of siRNAs with Optimized 3′ Ends
To further increase activity of the DNase II stable siRNAs an SAR study of the 3′-overhang was performed. Various combinations of invdT, dTinvdT or dTsdT on either the sense or the antisense strand 3′-overhang were applied to Aha1- and EGFP-targeting siRNAs (see Tables 11 and 12, respectively) and were pairwise compared for composition of both 3′ends in most potent siRNAs. All siRNAs were screened in dose response (24 nM to 0.00037 nM in 4-fold dilutions) for knock down activity after transfection of primary mouse hepatocytes (30000 cells/well; 96 well plate format) using Lipofectamine2000 according to the manufacturer's instructions.







TABLE 11







In vitro knock down activity of EGFP-targeting siRNAs with different 3′-ends.











SEQ

SEQ




ID
Sense strand sequence
ID
Antisense strand sequence
IC50


NO
(5′-3′)
NO
(5′-3′)
(nM





 45
GCUGGAGUUCGUGACCGCCdTdT
 46
GGCGGUCACGAACUCCAGCdTdT
1.0490


 


212
GcuGGAGuucGuGAccGccdTsdT
225
GGCGGUcACGAACUCcAGCdTsdT
#N/A


 


201
gcUfgGfaGfuUfcGfuGfaCfcGfcCf(invdT)
221
dGsGfcGfgUfcAfcGfaAfcUfcCfaGfc(invdT)
0.4377


 


201
gcUfgGfaGfuUfcGfuGfaCfcGfcCf(invdT)
214
dGsGfcGfgUfcAfcGfaAfcUfcCfaGfcdTsdT
0.1479


 


211
gcUfgGfaGfuUfcGfuGfaCfcGfcCfdT(invdT)
223
dGsGfcGfgUfcAfcGfaAfcUfcCfaGfcdT(invdT)
0.5833


 


203
gcUfgGfaGfuUfcGfuGfaCfcGfcCfdTsdT
214
dGsGfcGfgUfcAfcGfaAfcUfcCfaGfcdTsdT
0.2166


 


204
GfcUfgGfaGfuUfcGfuGfaCfcGfcCf(invdT)
224
pGfsGfcGfgUfcAfcGfaAfcUfcCfaGfc(invdT)
0.9100


 


204
GfcUfgGfaGfuUfcGfuGfaCfcGfcCf(invdT)
215
pGfsGfcGfgUfcAfcGfaAfcUfcCfaGfcdTsdT
0.2241


 


207
GfcUfgGfaGfuUfcGfuGfaCfcGfcCfdT(invdT)
218
pGfsGfcGfgUfcAfcGfaAfcUfcCfaGfcdT(invdT)
0.3474


 


206
GfcUfgGfaGfuUfcGfuGfaCfcGfcCfdTsdT
215
pGfsGfcGfgUfcAfcGfaAfcUfcCfaGfcdTsdT
0.2392


 


205
GfscUfgGfaGfuUfcGfuGfaCfcGfcCf(invdT)
220
GfsGfcGfgUfcAfcGfaAfcUfcCfaGfc(invdT)
0.4251


 


205
GfscUfgGfaGfuUfcGfuGfaCfcGfcCf(invdT)
216
GfsGfcGfgUfcAfcGfaAfcUfcCfaGfcdTsdT
0.2349


 


210
GfscUfgGfaGfuUfcGfuGfaCfcGfcCfdT(invdT)
222
GfsGfcGfgUfcAfcGfaAfcUfcCfaGfcdT(invdT)
0.5230


 


209
GfscUfgGfaGfuUfcGfuGfaCfcGfcCfdTsdT
216
GfsGfcGfgUfcAfcGfaAfcUfcCfaGfcdTsdT
0.4937


 


200
gscUfgGfaGfuUfcGfuGfaCfcGfcCf(invdT)
217
pdGsGfcGfgUfcAfcGfaAfcUfcCfaGfc(invdT)
0.2643


 


200
gscUfgGfaGfuUfcGfuGfaCfcGfcCf(invdT)
213
pdGsGfcGfgUfcAfcGfaAfcUfcCfaGfcdTsdT
0.0936


 


208
gscUfgGfaGfuUfcGfuGfaCfcGfcCfdT(invdT)
219
pdGsGfcGfgUfcAfcGfaAfcUfcCfaGfcdT(invdT)
0.3776


 


202
gscUfgGfaGfuUfcGfuGfaCfcGfcCfdTsdT
213
pdGsGfcGfgUfcAfcGfaAfcUfcCfaGfcdTsdT
0.1569
















TABLE 12







In vitro knock down activity of Aha1-targeting siRNAs with different 3′-ends.











SEQ

SEQ




ID
Sense strand sequence
ID
Antisense strand sequence
IC50


NO
(5′-3′)
NO
(5′-3′)
(nM





157
GGAuGAAGuGGAGAuuAGudTsdT
158
ACuAAUCUCcACUUcAUCCdTsdT
0.094


 


234
GfgAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT)
246
AfsCfuAfaUfcUfcCfaCfuUfcAfuCfc(invdT)
0.081


 


234
GfgAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT)
240
AfsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT
0.036


 


233
GfgAfuGfaAfgUfgGfaGfaUfuAfgUfdT(invdT)
239
AfsCfuAfaUfcUfcCfaCfuUfcAfuCfcdT(invdT)
0.034


 


236
GfgAfuGfaAfgUfgGfaGfaUfuAfgUfdTsdT
240
AfsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT
0.040


 


231
GfsgAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT)
241
pAfsCfuAfaUfcUfcCfaCfuUfcAfuCfc(invdT)
0.037


 


231
GfsgAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT)
267
pAfsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT
0.030


 


229
GfsgAfuGfaAfgUfgGfaGfaUfuAfgUfdT(invdT)
268
pAfsCfuAfaUfcUfcCfaCfuUfcAfuCfcdT(invdT)
0.024


 


228
GfsfAfuGfaAfgUfgGfaGfaUfuAfgUfdTsdT
267
pAfsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT
0.021


 


232
ggAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT)
245
dAsCfuAfaUfcUfcCfaCfuUfcAfuCfc(invdT)
0.060


 


232
ggAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT)
238
dAsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT
0.030


 


237
ggAfuGfaAfgUfgGfaGfaUfuAfgUfdT(invdT)
244
dAsCfuAfaUfcUfcCfaCfuUfcAfuCfcdT(invdT) 
0.045


 


230
ggAfuGfaAfgUfgGfaGfaUfuAfgUfdTsdT
238
dAsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT
0.025


 


227
gsgAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT)
243
pdAsCfuAfaUfcUfcCfaCfuUfcAfuCfc(invdT)
0.045


 


227
gsgAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT)
266
pdAsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT
0.015


 


235
gsgAfuGfaAfgUfgGfaGfaUfuAfgUfdT(invdT) 
242
pdAsCfuAfaUfcUfcCfaCfuUfcAfuCfcdT(invdT)
0.039


 


226
gsgAfuGfaAfgUfgGfaGfaUfuAfgUfdTsdT
266
pdAsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT
0.014









It was found, that siRNAs with 2 nucleotide dTsdT-overhangs on the antisense strand performed always better than those with a single invdT overhang at the antisense's 3′-end (while sense strands were the same). Further beneficial was the combination with a sense strand modified with a single invdT-overhang as 3′ overhang.
Example 49
In Vivo Knock Down Activity of siRNAs in Non-Human Primates
Preparation of DPCs and Dosing
DPCs were prepared by covalently attaching polymer “149 RAFT” to the indicated siRNA targeting coagulation Factor VII (siF7) at 4:1 wt:wt ratio (polymer:siRNA) through a disulfide linkage and then modifying the polymer-siRNA conjugate with a 2:1 wt:wt mixture of CDM-PEG:CDM-NAG at a 7× wt:wt ratio (CDM:polymer). Cynomolgous monkeys were dosed with 1 mg/kg DPC (polymer weight) and 0.25 mg/kg of the indicated siRNA. One animal received DPC containing siF7 SEQ ID NO pair 151/152, two animals received DPC containing siF7 SEQ ID NO pair 253/254), #1 and #2), and two animals received DPC containing SEQ ID NO pair 251/255, #1 and #2). F7 values were normalized to the average of the two pre-dose values. Animals receiving DPCs containing SEQ ID NO pair 253/254 or SEQ ID NO pair 251/255 had greater levels of F7 knockdown and longer PT than the animal 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 7 mg/kg) and dexmedetomidine (up to 0.03 mg/kg) and moved to a procedure room. In the procedure room, animals were placed on a water jacketed heating pad and the injection site was shaved and prepped with an antiseptic. An intravenous catheter (20 to 22 gauge) was inserted into a systemic vein (cephalic or small saphenous) and the DPC solution was infused (2 ml/kg) slowly over 1 to 2 minutes. A pulse oximeter was used to monitor the heart rate and oxygen saturation during and immediately following the injection procedure. Each injection procedure took about 20 minutes to perform. After injection the catheter was removed and gentle
pressure was applied to the venipuncture site. Animals were taken back to their cages and given an IM injection of the reversal drug atipamezole (antisedan) (0.10 to 0.15 mg/kg). Animals were monitored until they regained normal activity.
Blood Collection Procedure
Blood samples (1-5 ml) were obtained for the measurement of gene inhibition (F7 activity, coagulation time), blood chemistries, and markers of liver damage (CBC, chemistry panel, ALT, cytokines, complement). For these blood collection procedures, animals were given an IM injection containing a combination of ketamine (up to 7 mg/kg) and dexmedetomidine (up to 0.03 mg/kg). Once sedated, animals were moved on to a portable procedure table and a 22 gauge needle and syringe were used to collect blood from the femoral vein. Immediately after the blood collection, pressure was applied to the venipuncture site and the blood was divided into the appropriate sample tubes for each blood test. Animals were then given an IM injection of the reversal drug atipamezole (antisedan) (0.10 to 0.15 mg/kg) and returned to their cage. No more than 20% of total blood volume was drawn in any 30-day period (estimated blood volume=60 ml/kg). Each blood collection procedure took about 10 minutes to perform.
Factor VII (F7) Activity Measurements
Blood samples from non-human primates were prepared by filling serum separator tubes with whole blood and allowing the blood to clot at room temperature for at least 20 minutes. After clotting, blood tubes were centrifuged for 3 minutes at 9000 rpm, aliquoted into eppendorf tubes, and stored at −20° C. until assayed. F7 activity in serum was measured with a chromogenic method using a BIOPHEN VII kit (Hyphen BioMed/Aniara, Mason, Ohio) following manufacturer's recommendations. Absorbance of colorimetric development was measured using a Tecan Safire2 microplate reader at 405 nm.
Coagulation Tests (Protime, Partial Protime and Fibrinogen)
Blood samples from non-human primates were prepared by completely filling sodium citrate tubes (BD Vacutainer) with whole blood and gently mixing to prevent clot formation. Tubes were transported to a clinical testing lab within one hour and coagulation assays were performed within 4 hours from the time of collection.







TABLE 13







FVII SiRNA used for NHP experiment: Key: lower case letters


a, c, g, u, are 2′-O-Methyl nucleotides; Upper case letters


A, C, G, U followed by “f” indicates a 2′-fluoro nucleotide.


Lower case “p” indicates a 5′-phosphate. (invdT) represents


an inverted deoxythimidine (3′-3′-linked). A phosphorothioate


linkages is symbolized with a lower case “s”. dT is


deoxythimidine.










SEQ

SEQ



ID
Sense strand sequence
ID
Antisense strand sequence


NO
(5′-3′)
NO
(5′-3′)





251
(NH2C6)GfuUfgGfuGfaAfuGfgAfgCfuCf
252
pCfsUfgAfgCfuCfcAfuUfcAfcCfaAfc



aGf(invdT)

(invdT)


 


253
(NH2C6)GfgUfcCfuGfuUfgUfuGfgUfgAf
254
pAfsUfuCfaCfcAfaCfaAfcAfgGfaCfcd



aUf(invdT)

TsdT


 


251
(NH2C6)GfuUfgGfuGfaAfuGfgAfgCfuCf
255
pCfsUfgAfgCfuCfcAfuUfcAfcCfaAfcd



aGf(invdT)

TsdT









Changing from an 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 otherwise constant modification pattern lead to a more pronounced serum FVII reduction and a significantly prolonged duration of this effect in non-human primates (see FIG. 6a). This observation is supported by an expected biologic consequence, namely a more pronounced effect on the prothrombin time corresponding to the extent of Factor 7 reduction (see FIG. 6b).
Example 50
In Vivo Knock Down Activity of siRNAs with Cleavable RNA Linkers
In Table 14 the in vivo efficacy based on FVII protein inhibition in serum was compared using cholesterol or the GalNAc-palmitoyl siRNA conjugate in the same sequence context in mice. The in vivo experiment was conducted as described in example 42. FVII inhibition was strongly decreased for the cholesterol conjugated siRNAs containing no 2′-OH nucleotide compared to the GalNAc-palmitoyl conjugated counterparts (SEQ ID NO pair 179/166 vs. 179/190, SEQ ID NO pair 257/264 vs. SEQ ID NO pair 179/262, SEQ ID NO pair 257/263 vs. SEQ ID NO pair 179/163 and SEQ ID NO pair 257/166 vs. (SEQ ID NO pair 179/166). In contrast for a 2′-OH containing siRNA the cholesterol conjugate lead to higher FVII inhibition compared to the GalNAc-palmitoyl derivative (SEQ ID NO pair 180/168 vs. SEQ ID NO pair 258/168).
The small molecule ligands GalNAc-palmitoyl and cholesterol used in the described in vivo experiment are connected to the siRNA via a non-cleavable linker to the 5′-end of the sense strand. In case the sense strand exhibit 2′-OH nucleotides the ligand is still cleavable by nucleases (e.g. DNase II in the endosomal or lysosomal compartment). The cleavage reaction releases the free siRNA that is then released into the cytoplasm by the endosomal perturbing activity of the delivery polymer.
For siRNAs lacking a 2′-OH nucleotide in the sense strand, the ligands are stably connected to the duplex, as no enzymatic (nuclease/protease/esterase etc.) or chemical mechanism triggers the cleavage of the ligand. Therefore, fully stable cholesterol conjugated siRNA can be trapped in cell membranes due to the membrane interaction of the lipophilic cholesterol ligand. Even high concentrations of the siRNA in the tissue is not sufficient for effective release of the siRNA into in the cytoplasm. In contrast, the less lipophilic GalNAc-palmitoyl conjugated siRNA can be released into the cytoplasm, due to a less pronounced interaction with cell membranes. For this reason a stable, non-cleavable GalNAc-palmitoyl siRNA conjugate is more efficacious compared to a cholesterol conjugated to the same siRNA.
Developing cleavable linker constructs would help to circumvent the issue of membrane trapping for stably conjugated cholesterol siRNA. Using disulfide linker chemistry is described as an attractive possibility to introduce a defined cleavage site but cleavage is most probably restricted to the reducing environment of specific organelles within the cell (PNAS, 2006, 103, 13872). As cleavage is expected to be slow in the endosomal/lysosomal compartment most of the cholesterol-disulfide conjugated siRNA can still be trapped in membranes as described for the non-cleavable cholesterol conjugates.











TABLE 14







SEQ

% FVII



SEQ ID
ID
activity
activity


Conjugate
NO pair
NO
Sequence 5′->3′
in serum







GalNA 
179/166
179
GalNAc-NH2C6-GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
27




166
puGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)



 



179/190
179
GalNAc-NH2C6-GfcAfaAfgGfcGfuGFcCfaAfcUfcAf(invdT)
51




190
pusGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)



 



179/262
179
GalNAc-NH2C6-GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
17




262
pUfsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)



 



179/263
179
GalNAc-NH2C6-GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
13




263
psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)



 



180/168
180
GalNAc-NH2C6-GcAAAGGcGuGccAAcucAdTsdT
86




168
UGAGUUGGcACGCCUUUGCdTsdT



 


Cholesterol
257/166
257
Cholesterol-NH2C6-GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
48




166
puGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)



 



257/190
257
Cholesterol-NH2C6-GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
93




190
pusGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)



 



257/264
257
Cholesterol-NH2C6-GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
63




264
pUfsGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)



 



257/263
257
Cholesterol-NH2C6-GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT)
41




263
psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc(invdT)



 



258/168
258
Cholesterol-NH2C6-GcAAAGGcGuGccAAcucAdTsdT
50




168
UGAGUUGGcACGCCUUUGCdTsdT









In addition to the well-established disulfide cleavable linker chemistry another possibility is the generation of defined cleavage sites by using 2′-OH nucleotides at certain positions. Introduction of 2′-OH nucleotides at selective positions is a new approach to achieve cleavage of the conjugates from RNA strands. The 2′-OH nucleotides can either be implemented by adding single stranded overhangs with at least one 2′-OH-nucleotide at the 3′- or 5′-end of the RNA strand or by using 2′-OH nucleotides within the duplex region of an siRNA. The enzymatic activity of nucleases present in the endosome/lysosome cleaves selectively at this positions. In a first design the cholesterol was connected to the sense strand via a single stranded overhang containing 3 2′-OH nucleotides (AUC) at the 5′-terminus.
Cholesterol conjugated siRNAs comparing various cleavable linker chemistries are shown in Table 15. All siRNAs have the identical sequence context, just the linker chemistry was altered. Cholesterol was connected to the sense strand via single stranded overhang comprised of a three 2′-OH nucleotides (AUC) to the 5′-terminus. When co-administered with a delivery polymer this siRNA (SEQ ID NO pair 260/263) lead to 77% FVII down modulation in serum in mice, compared to only 60% when using the identical siRNA with a stably attached cholesterol (SEQ ID NO pair 257/263). The same siRNA with a cholesterol conjugated via a linker according to formula Ia to the 5′-terminus of the sense strand (SEQ ID NO pair 261/263) lead to 93% FVII activity reduction in serum. All results were achieved by co-administration of 15 mg/kg of a delivery polymer with 2.5 mg/kg of the cholesterol conjugated siRNA in mice.







TABLE 15







In vivo comparison of various linker chemistries for


cholesterol conjugated siRNAs











SEQ

SEQ

% FVII


ID
Sense strand sequence
ID
Antisense strand sequence
activity


NO
(5′-3′)
NO
(5′-3′)
in serum





257
Chol-(NH2C6)-
263
psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc
40



GfcAfaAfgGfcGfuGfcCfaAfcUfcAf

(invdT)




(invdT)





 


259
Chol-C6SSC6-
263
psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc
22



GfcAfaAfgGfcGfuGfcCfaAfcUfcAf

(invdT)




(invdT)





 


260
Chol-AUC-
263
psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc
23



GfcAfaAfgGfcGfuGfcCfaAfcUfcAf

(invdT)




(invdT)





 


261
Chol-Cathepsin-
263
psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc
7



GfcAfaAfgGfcGfuGfcCfaAfcUfcAf

(invdT)




(invdT)









These results indicate, that the use of a cleavable linker improves the in vivo potency of siRNAs containing no 2′-OH nucleotide. The cleavable linker can either comprised of 2′-OH containing nucleotides, a di-peptide cleavage motive or a disulfide linker chemistry. All cleavable linker constructs improve the in vivo potency in a co-administration setup of a cholesterol conjugated siRNAs with a slow endosomal release delivery polymer.
Example 51
In Vitro Serum Stability of siRNAs with Cleavable Linkers
The stability of the cleavable linker was evaluated in an in vitro stability assay. The cholesterol conjugated sense strands were incubated in 90% mouse serum at 37° C. for various time points. The incubation reaction was stopped by addition of proteinase K in a sodium dodecyl sulfate (SDS) containing buffer—The treatment degrades all proteins and enzymes without interfering with the RNA strand integrity. 25 μL of this solution was directly injected onto a AEX-HPLC system connected to a UV detector at 260 nm. AEX-HPLC was performed on a Dionex DNA Pac200 column (4×250 mm) at 75° C. using a 20 mM Tris buffer containing 50% ACN at pH=8. 800 mM NaBr in eluent B serves as eluent salt. A gradient from 25 to 62% B in 18 minutes was employed.
The cholesterol containing single stranded RNA elutes from the HPLC column as a broad peak at 260 nm After cleavage of the cholesterol sharp symmetric peaks is observed at lower retention time. Cleavage rate of cholesterol was determined by the following equitation (PA=Peak Area):

%(free RNA)=100*PA[free RNA]/(PA[free RNA]+PA[cholesterol conjugated RNA])

In vitro it was shown, that the 3 nt nucleotide (AUC)-overhang is quantitatively cleaved in less than 1 hour in 90% mouse serum. The cleavage occurs 3′ to the two pyrimidine nucleotides in the overhang, leading to two distinct cleavage metabolites (peak areas of metabolites were summarized for data evaluation). In contrast, the di-peptide containing linker according to formula Ia, the disulfide and the stably linked cholesterol are fully stable in mouse serum.
Example 52
Tissue Distribution of siRNAs with Cleavable Linkers
The siRNA concentration in the liver tissue samples was determined using a proprietary oligonucleotide detection method as described in WO2010043512. Briefly, the siRNA quantification is based on the hybridization of a fluorescently (Atto-425) labeled PNA-probe (Atto425-OO-TGAGTTGGCACGCCTTT (SEQ ID NO: 269) obtained from Panagene Inc, Korea) complementary to the sense strand of the siRNA duplex, followed by AEX-HPLC based separation. Quantification was done by fluorescence detection against an external calibration curve that was generated from a dilution series of the two FVII siRNA used in the in vivo experiment (see example 42). For plasma samples between 0.2 to 2 μL and for tissue  ̃1 mg aliquots were injected onto the HPLC system.
In Table 16 results from liver tissue analysis are shown. When analyzing the siRNA content it was found, that the sense strand that is present in liver tissue, is quantitatively cleaved from cholesterol when using either the di-peptide linker motive or the 3 nt 5′-overhang with the unmodified linker sequence AUC. In contrast, only 15% of the disulfide linked siRNA that is present in the liver is cleaved from cholesterol within the first 48 hours post dosing and nothing of the stably attached cholesterol is cleaved from the siRNA.
When comparing the absolute amounts of cholesterol-free siRNA in liver tissue similar amounts were found for the disulfide linker and for the RNA AUC-linker, nicely correlating with equal FVII serum activity 48 hours post dosing The lower FVII activity achieved with the di-peptide linked cholesterol siRNA fully correlates with the higher absolute amount of the cleaved cholesterol-free siRNA.
The total amount of cholesterol siRNA conjugate equipped with an (AUC)-linker on the sense strand delivered into the liver is  ̃6-fold lower as compared to the stably or the disulfide attached cholesterol and  ̃3-fold lower compared to the di-peptide conjugated cholesterol siRNA. The 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 circulation as shown in the in vitro incubation with mouse serum. When the cholesterol ligand is cleaved from the siRNA already in circulation the resulting siRNA is prone to renal clearance and is rapidly excreted into urine without delivery into tissue.












TABLE 16









Total
% sense






siRNA
cleaved


SEQ

SEQ

in
ligand


ID
Sense strand sequence
ID
Antisense strand sequence
Liver
in


NO
(5′-3′)
NO
(5′-3′)
[ng/g]
liver




















257
Chol-(NH2C6)-
263
psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc
5837
0



GfcAfaAfgGfcGfuGfcCfaAfeUf

(invdT)





cAf(invdT)






 


259
Chol-C6SSC6-
263
psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc
4357
14.8



GfcAfaAfgGfcGfuGfcCfaAfcUf

(invdT)





cAf(invdT)






 


260
Chol-AUC-
263
psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc
 912
96.1



GfcAfaAfgGfcGfuGfcCfaAfcUf

(invdT)





cAf(invdT)






 


261
Chol-Cathepsin-
263
psdTdGfaGfuUfgGfcAfcGfcCfuUfuGfc
2760
99.8



GfcAfaAfgGfcGfuGfcCfaAfcUf

(invdT)





cAf(invdT)









In the following tables siRNAs used in the examples are summarized:








TABLE 17







Core sequences










SEQ

SEQ



ID
Sense strand
ID
Antisense strand


NO
sequence (5′-3′)
NO
sequence (5′-3′)





  1
ACAUGAAGCAGCACGACUU
  2
AAGUCGUGCUGCUUCAUGU


 


  3
GCCCGACAACCACUACCUG
  4
CAGGUAGUGGUUGUCGGGC


 


  5
CGAGAAGCGCGAUCACAUG
  6
CAUGUGAUCGCGCUUCUCG


 


  7
AUAUCAUGGCCGACAAGCA
  8
UGCUUGUCGGCCAUGAUAU


 


  9
ACAAGCUGGAGUACAACUA
 10
UAGUUGUACUCCAGCUUGU


 


 11
GCAGCUCGCCGACCACUAC
 12
GUAGUGGUCGGCGAGCUGC


 


 13
CGUCCAGGAGCGCACCAUC
 14
GAUGGUGCGCUCCUGGACG


 


 15
GCUGGAGUUCGUGACCGCC
 16
GGCGGUCACGAACUCCAGC


 


 17
CCACCCUGACCUACGGCGU
 18
ACGCCGUAGGUCAGGGUGG


 


 19
CGACUUCAAGGAGGACGGC
 20
GCCGUCCUCCUUGAAGUCG


 


 21
UUCAAGAUCCGCCACAACA
 22
UGUUGUGGCGGAUCUUGAA


 


 23
GGCAACUACAAGACCCGCG
 24
CGCGGGUCUUGUAGUUGCC


 


 25
CCGGCAAGCUGCCCGUGCC
 26
GGCACGGGCAGCUUGCCGG


 


 27
UGCCCAUCCUGGUCGAGCU
 28
AGCUCGACCAGGAUGGGCA


 


 29
CAAGUUCAGCGUGUCCGGC
 30
GCCGGACACGCUGAACUUG


 


151
GGAAUCUUAUAUUUGAUCCAA
152
UUGGAUCAAAUAUAAGAUUCCCU


 


155
GGAUGAAGUGGAGAUUAGU
156
ACUAAUCUCCACUUCAUCC


 


161
GCAAAGGCGUGCCAACUCA
162
UGAGUUGGCACGCCUUUGC


 


161
GCAAAGGCGUGCCAACUCA
178
TGAGUUGGCACGCCUUUGC


 


161
GCAAAGGCGUGCCAACUCA
256
TGAGUUGGCACGCCUUUGC


 


163
GGAUCAUCUCAAGUCUUAC
164
GUAAGACUUGAGAUGAUCC


 


171
UGACCACAGUCGGAUUAAA
172
UUUAAUCCGACUGUGGUCA


 


247
GUUGGUGAAUGGAGCUCAG
248
CUGAGCUCCAUUCACCAAC


 


249
GGUCCUGUUGUUGGUGAAU
250
AUUCACCAACAACAGGACC
















TABLE 18







Mapping of core sequences and modified sequence








Core sequences













Sense

Antisense
Modified sequences














SEQ
strand
SEQ
strand
SEQ

SEQ



ID
sequence 
ID
sequence
ID
Sense strand
ID
Antisense strand


NO
(5′-3′)
NO
(5′-3′)
NO
sequence (5′-3′)
NO
sequence (5′-3′)





  1
ACAUGAAGC
  2
AAGUCGUGC
 31
ACAUGAAGCAG
 32
AAGUCGUGCUGCU



AGCACGACU

UGCUUCAUG

CACGACUUdTdT

UCAUGUdTdT



U

U






 


  1
ACAUGAAGC
  2
AAGUCGUGC
 61
AfcAfuGfaAfgCfa
 62
paAfgUfcGfuGfcUfg



AGCACGACU

UGCUUCAUG

GfcAfcGfaCfuUfd

CfuUfcAfuGfudTsdT



U

U

TsdT




 


  1
ACAUGAAGC
  2
AAGUCGUGC
 91
dAcdAudGadAgdC
 92
padAgdTcdGudGcdTg



AGCACGACU

UGCUUCAUG

adGcdAcdGadCud

dCudTcdAudGudTsd



U

U

TdTsdT

T


 


  1
ACAUGAAGC
  2
AAGUCGUGC
121
AfdCAfdTGfdAAf
122
pdAAfdGUfdCGfdTG



AGCACGACU

UGCUUCAUG

dGCfdAGfdCAfdC

fdCUfdGCfdTUfdCAf



U

U

GfdACfdTUfdTsdT

dTGfdTdTsdT


 


  3
GCCCGACAA
  4
CAGGUAGUG
 33
GCCCGACAACC
 34
CAGGUAGUGGUU



CCACUACCU

GUUGUCGGG

ACUACCUGdTdT

GUCGGGCdTdT



G

C






 


  3
GCCCGACAA
  4
CAGGUAGUG
 63
GfcCfcGfaCfaAfcC
 64
pcAfgGfuAfgUfgGfu



CCACUACCU

GUUGUCGGG

faCfuAfcCfuGfdTs

UfgUfcGfgGfcdTsdT



G

C

dT




 


  3
GCCCGACAA
  4
CAGGUAGUG
 93
dGcdCcdGadCadA
 94
pcdAgdGudAgdTgdG



CCACUACCU

GUUGUCGGG

cdCadCudAcdCud

udTgdTcdGgdGcdTsd



G

C

GdTsdT

T


 


  3
GCCCGACAA
  4
CAGGUAGUG
123
GfdCCfdCGfdACf
124
pdCAfdGGfdTAfdGU



CCACUACCU

GUUGUCGGG

dAAfdCCfdACfdT

fdGGfdTUfdGUfdCG



G

C

AfdCCfdTGfdTsdT

fdGGfdCdTsdT


 


  5
CGAGAAGCG
  6
CAUGUGAUC
 35
CGAGAAGCGCG
 36
CAUGUGAUCGCGC



CGAUCACAU

GCGCUUCUC

AUCACAUGdTdT

UUCUCGdTdT



G

G






 


  5
CGAGAAGCG
  6
CAUGUGAUC
 65
CfgAfgAfaGfcGfc
 66
pcAfuGfuGfaUfcGfc



CGAUCACAU

GCGCUUCUC

GfaUfcAfcAfuGfd

GfcUfuCfuCfgdTsdT



G

G

TsdT




 


  5
CGAGAAGCG
  6
CAUGUGAUC
 95
dCgdAgdAadGcdG
 96
pcdAudGudGadTcdG



CGAUCACAU

GCGCUUCUC

cdGadTcdAcdAud

cdGcdTudCudCgdTsd



G

G

GdTsdT

T


 


  5
CGAGAAGCG
  6
CAUGUGAUC
125
CfdGAfdGAfdAGf
126
pdCAfdTGfdTGfdAU



CGAUCACAU

GCGCUUCUC

dCGfdCGfdAUfdC

fdCGfdCGfdCUfdTCf



G

G

AfdCAfdTGfdTsdT

dTCfdGdTsdT


 


  7
AUAUCAUGG
  8
UGCUUGUCG
 37
AUAUCAUGGCC
 38
UGCUUGUCGGCCA



CCGACAAGC

GCCAUGAUA

GACAAGCAdTdT

UGAUAUdTdT



A

U






 


  7
AUAUCAUGG
  8
UGCUUGUCG
 67
AfuAfuCfaUfgGfc
 68
puGfcUfuGfuCfgGfc



CCGACAAGC

GCCAUGAUA

CfgAfcAfaGfcAfd

CfaUfgAfuAfudTsdT



A

U

TsdT




 


  7
AUAUCAUGG
  8
UGCUUGUCG
 97
dAudAudCadTgdG
 98
pudGcdTudGudCgdG



CCGACAAGC

GCCAUGAUA

cdCgdAcdAadGcd

cdCadTgdAudAudTsd



A

U

AdTsdT

T


 


  7
AUAUCAUGG
  8
UGCUUGUCG
127
AfdTAfdTCfdAUf
128
pdTGfdCUfdTGfdTCf



CCGACAAGC

GCCAUGAUA

dGGfdCCfdGAfdC

dGGfdCCfdAUfdGAf



A

U

AfdAGfdCAfdTsd

dTAfdTdTsdT







T




 


  9
ACAAGCUGG
 10
UAGUUGUAC
 39
ACAAGCUGGAG
 40
UAGUUGUACUCCA



AGUACAACU

UCCAGCUUG

UACAACUAdTdT

GCUUGUdTdT



A

U






 


  9
ACAAGCUGG
 10
UAGUUGUAC
 69
AfcAfaGfcUfgGfa
 70
puAfgUfuGfuAfcUfc



AGUACAACU

UCCAGCUUG

GfuAfcAfaCfuAfd

CfaGfcUfuGfudTsdT



A

U

TsdT




 


  9
ACAAGCUGG
 10
UAGUUGUAC
 99
dAcdAadGcdTgdG
100
pudAgdTudGudAcdT



AGUACAACU

UCCAGCUUG

adGudAcdAadCud

cdCadGcdTudGudTsd



A

U

AdTsdT

T


 


  9
ACAAGCUGG
 10
UAGUUGUAC
129
AfdCAfdAGfdCUf
130
pdTAfdGUfdTGfdTA



AGUACAACU

UCCAGCUUG

dGGfdAGfdTAfdC

fdCUfdCCfdAGfdCUf



A

U

AfdACfdTAfdTsdT

dTGfdTdTsdT


 


 11
GCAGCUCGC
 12
GUAGUGGUC
 41
GCAGCUCGCCG
 42
GUAGUGGUCGGC



CGACCACUA

GGCGAGCUG

ACCACUACdTdT

GAGCUGCdTdT



C

C






 


 11
GCAGCUCGC
 12
GUAGUGGUC
 71
GfcAfgCfuCfgCfc
 27
pgUfaGfuGfgUfcGfg



CGACCACUA

GGCGAGCUG

GfaCfcAfcUfaCfdT

CfgAfgCfuGfcdTsdT



C

C

sdT




 


 11
GCAGCUCGC
 12
GUAGUGGUC
101
dGcdAgdCudCgdC
102
pgdTadGudGgdTcdG



CGACCACUA

GGCGAGCUG

cdGadCcdAcdTad

gdCgdAgdCudGcdTsd



C

C

CdTsdT

T


 


 11
GCAGCUCGC
 12
GUAGUGGUC
131
GfdCAfdGCfdTCf
132
pdGUfdAGfdTGfdGU



CGACCACUA

GGCGAGCUG

dGCfdCGfdACfdC

fdCGfdGCfdGAfdGC



C

C

AfdCUfdACfdTsd

fdTGfdCdTsdT







T




 


 13
CGUCCAGGA
 14
GAUGGUGCG
 43
CGUCCAGGAGC
 44
GAUGGUGCGCUCC



GCGCACCAU

CUCCUGGAC

GCACCAUCdTdT

UGGACGdTdT



C

G






 


 13
CGUCCAGGA
 14
GAUGGUGCG
 73
CfgUfcCfaGfgAfg
 74
pgAfuGfgUfgCfgCfu



GCGCACCAU

CUCCUGGAC

CfgCfaCfcAfuCfdT

CfcUfgGfaCfgdTsdT



C

G

sdT




 


 13
CGUCCAGGA
 14
GAUGGUGCG
103
dCgdTcdCadGgdA
104
pgdAudGgdTgdCgdC



GCGCACCAU

CUCCUGGAC

gdCgdCadCcdAud

udCcdTgdGadCgdTsd



C

G

CdTsdT

T


 


 13
CGUCCAGGA
 14
GAUGGUGCG
133
CfdGUfdCCfdAGf
134
pdGAfdTGfdGUfdGC



GCGCACCAU

CUCCUGGAC

dGAfdGCfdGCfdA

fdGCfdTCfdCUfdGGf



C

G

CfdCAfdTCfdTsdT

dACfdGdTsdT


 


 15
GCUGGAGUU
 16
GGCGGUCAC
 45
GCUGGAGUUCG
 46
GGCGGUCACGAAC



CGUGACCGC

GAACUCCAG

UGACCGCCdTdT

UCCAGCdTdT



C

C






 


 15
GCUGGAGUU
 16
GGCGGUCAC
 75
GfcUfgGfaGfuUfc
 76
pgGfcGfgUfcAfcGfa



CGUGACCGC

GAACUCCAG

GfuGfaCfcGfcCfd

AfcUfcCfaGfcdTsdT



C

C

TsdT




 


 15
GCUGGAGUU
 16
GGCGGUCAC
105
dGcdTgdGadGudT
106
pgdGcdGgdTcdAcdG



CGUGACCGC

GAACUCCAG

cdGudGadCcdGcd

adAcdTcdCadGcdTsd



C

C

CdTsdT

T


 


 15
GCUGGAGUU
 16
GGCGGUCAC
135
GfdCUfdGGfdAGf
136
pdGGfdCGfdGUfdCA



CGUGACCGC

GAACUCCAG

dTUfdCGfdTGfdA

fdCGfdAAfdCUfdCCf



C

C

CfdCGfdCCfdTsdT

dAGfdCdTsdT


 


 15
GCUGGAGUU
 16
GGCGGUCAC
200
gscUfgGfaGfuUfc
213
pdGsGfcGfgUfcAfcGf



CGUGACCGC

GAACUCCAG

GfuGfaCfcGfcCf

aAfcUfcCfaGfcdTsdT



C

C

(invdT)




 


 15
GCUGGAGUU
 16
GGCGGUCAC
201
gcUfgGfaGfuUfcG
214
dGsGfcGfgUfcAfcGfa



CGUGACCGC

GAACUCCAG

fuGfaCfcGfcCf

AfcUfcCfaGfcdTsdT



C

C

(invdT)




 


 15
GCUGGAGUU
 16
GGCGGUCAC
202
gscUfgGfaGfuUfc
213
pdGsGfcGfgUfcAfcGf



CGUGACCGC

GAACUCCAG

GfuGfaCfcGfcCfd

aAfcUfcCfaGfcdTsdT



C

C

TsdT




 


 15
GCUGGAGUU
 16
GGCGGUCAC
203
gcUfgGfaGfuUfcG
214
dGsGfcGfgUfcAfcGfa



CGUGACCGC

GAACUCCAG

fuGfaCfcGfcCfdTs

AfcUfcCfaGfcdTsdT



C

C

dT




 


 15
GCUGGAGUU
 16
GGCGGUCAC
204
GfcUfgGfaGfuUfc
215
pGfsGfcGfgUfcAfcGf



CGUGACCGC

GAACUCCAG

GfuGfaCfcGfcCf

aAfcUfcCfaGfcdTsdT



C

C

(invdT)




 


 15
GCUGGAGUU
 16
GGCGGUCAC
205
GfscUfgGfaGfuUfc
216
GfsGfcGfgUfcAfcGfa



CGUGACCGC

GAACUCCAG

GfuGfaCfcGfcCf

AfcUfcCfaGfcdTsdT



C

C

(invdT)




 


 15
GCUGGAGUU
 16
GGCGGUCAC
206
GfcUfgGfaGfuUfc
215
pGfsGfcGfgUfcAfcGf



CGUGACCGC

GAACUCCAG

GfuGfaCfcGfcCfd

aAfcUfcCfaGfcdTsdT



C

C

TsdT




 


 15
GCUGGAGUU
 16
GGCGGUCAC
200
gscUfgGfaGfuUfc
217
pdGsGfcGfgUfcAfcGf



CGUGACCGC

GAACUCCAG

GfuGfaCfcGfcCf

aAfcUfcCfaGfc(invdT)



C

C

(invdT)




 


 15
GCUGGAGUU
 16
GGCGGUCAC
207
GfcUfgGfaGfuUfc
218
pGfsGfcGfgUfcAfcGf



CGUGACCGC

GAACUCCAG

GfuGfaCfcGfcCfd

aAfcUfcCfaGfcdT



C

C

T(invdT)

(invdT)


 


 15
GCUGGAGUU
 16
GGCGGUCAC
208
gscUfgGfaGfuUfc
219
pdGsGfcGfgUfcAfcGf



CGUGACCGC

GAACUCCAG

GfuGfaCfcGfcCfd

aAfcUfcCfaGfcdT



C

C

T(invdT)

(invdT)


 


 15
GCUGGAGUU
 16
GGCGGUCAC
205
GfscUfgGfaGfuUfc
220
GfsGfcGfgUfcAfcGfa



CGUGACCGC

GAACUCCAG

GfuGfaCfcGfcCf

AfcUfcCfaGfc(invdT)



C

C

(invdT)




 


 15
GCUGGAGUU
 16
GGCGGUCAC
201
gcUfgGfaGfuUfcG
221
dGsGfcGfgUfcAfcGfa



CGUGACCGC

GAACUCCAG

fuGfaCfcGfcCf

AfcUfcCfaGfc(invdT)



C

C

(invdT)




 


 15
GCUGGAGUU
 16
GGCGGUCAC
209
GfscUfgGfaGfuUfc
216
GfsGfcGfgUfcAfcGfa



CGUGACCGC

GAACUCCAG

GfuGfaCfcGfcCfd

AfcUfcCfaGfcdTsdT



C

C

TsdT




 


 15
GCUGGAGUU
 16
GGCGGUCAC
210
GfscUfgGfaGfuUfc
222
GfsGfcGfgUfcAfcGfa



CGUGACCGC

GAACUCCAG

GfuGfaCfcGfcCfd

AfcUfcCfaGfcdT



C

C

T(invdT)

(invdT)


 


 15
GCUGGAGUU
 16
GGCGGUCAC
211
gcUfgGfaGfuUfcG
223
dGsGfcGfgUfcAfcGfa



CGUGACCGC

GAACUCCAG

fuGfaCfcGfcCfdT

AfcUfcCfaGfcdT



C

C

(invdT)

(invdT)


 


 15
GCUGGAGUU
 16
GGCGGUCAC
204
GfcUfgGfaGfuUfc
224
pGfsGfcGfgUfcAfcGf



CGUGACCGC

GAACUCCAG

GfuGfaCfcGfcCf

aAfcUfcCfaGfc



C

C

(invdT)

(invdT)


 


 15
GCUGGAGUU
 16
GGCGGUCAC
 45
GCUGGAGUUCG
 46
GGCGGUCACGAAC



CGUGACCGC

GAACUCCAG

UGACCGCCdTdT

UCCAGCdTdT



C

C






 


 15
GCUGGAGUU
 16
GGCGGUCAC
212
GcuGGAGuucGuG
225
GGCGGUcACGAAC



CGUGACCGC

GAACUCCAG

AccGccdTsdT

UCcAGCdTsdT



C

C






 


 17
CCACCCUGA
 18
ACGCCGUAG
 47
CCACCCUGACC
 48
ACGCCGUAGGUCA



CCUACGGCG

GUCAGGGUG

UACGGCGUdTdT

GGGUGGdTdT



U

G






 


 17
CCACCCUGA
 18
ACGCCGUAG
 77
CfcAfcCfcUfgAfc
 78
paCfgCfcGfuAfgGfuC



CCUACGGCG

GUCAGGGUG

CfuAfcGfgCfgUfd

faGfgGfuGfgdTsdT



U

G

TsdT




 


 17
CCACCCUGA
 18
ACGCCGUAG
107
dCcdAcdCcdTgdA
108
padCgdCcdGudAgdG



CCUACGGCG

GUCAGGGUG

cdCudAcdGgdCgd

udCadGgdGudGgdTs



U

G

TdTsdT

dT


 


 17
CCACCCUGA
 18
ACGCCGUAG
137
CfdCAfdCCfdCUf
138
pdACfdGCfdCGfdTA



CCUACGGCG

GUCAGGGUG

dGAfdCCfdTAfdC

fdGGfdTCfdAGfdGG



U

G

GfdGCfdGUfdTsd

fdTGfdGdTsdT







T




 


 19
CGACUUCAA
 20
GCCGUCCUCC
 49
CGACUUCAAGG
 50
GCCGUCCUCCUUG



GGAGGACGG

UUGAAGUCG

AGGACGGCdTdT

AAGUCGdTdT



C








 


 19
CGACUUCAA
 20
GCCGUCCUCC
 79
CfgAfcUfuCfaAfg
 80
pgCfcGfuCfcUfcCfuU



GGAGGACGG

UUGAAGUCG

GfaGfgAfcGfgCfd

fgAfaGfuCfgdTsdT



C



TsdT




 


 19
CGACUUCAA
 20
GCCGUCCUCC
109
dCgdAcdTudCadA
110
pgdCcdGudCcdTcdCu



GGAGGACGG

UUGAAGUCG

gdGadGgdAcdGgd

dTgdAadGudCgdTsd



C



CdTsdT

T


 


 19
CGACUUCAA
 20
GCCGUCCUCC
139
CfdGAfdCUfdTCf
140
pdGCfdCGfdTCfdCU



GGAGGACGG

UUGAAGUCG

dAAfdGGfdAGfdG

fdCCfdTUfdGAfdAGf



C



AfdCGfdGCfdTsd

dTCfdGdTsdT







T




 


 21
UUCAAGAUC
 22
UGUUGUGGC
 51
UUCAAGAUCCG
 52
UGUUGUGGCGGA



CGCCACAAC

GGAUCUUGA

CCACAACAdTdT

UCUUGAAdTdT



A

A






 


 21
UUCAAGAUC
 22
UGUUGUGGC
 81
UfuCfaAfgAfuCfc
 82
puGfuUfgUfgGfcGfg



CGCCACAAC

GGAUCUUGA

GfcCfaCfaAfcAfdT

AfuCfuUfgAfadTsdT



A

A

sdT




 


 21
UUCAAGAUC
 22
UGUUGUGGC
111
dTudCadAgdAudC
112
pudGudTgdTgdGcdG



CGCCACAAC

GGAUCUUGA

cdGcdCadCadAcd

gdAudCudTgdAadTsd



A

A

AdTsdT

T


 


 21
UUCAAGAUC
 22
UGUUGUGGC
141
UfdTCfdAAfdGAf
142
pdTGfdTUfdGUfdGG



CGCCACAAC

GGAUCUUGA

dTCfdCGfdCCfdA

fdCGfdGAfdTCfdTUf



A

A

CfdAAfdCAfdTsd

dGAfdAdTsdT







T




 


 23
GGCAACUAC
 24
CGCGGGUCU
 53
GGCAACUACAA
 54
CGCGGGUCUUGUA



AAGACCCGC

UGUAGUUGC

GACCCGCGdTdT

GUUGCCdTdT



G

C






 


 23
GGCAACUAC
 24
CGCGGGUCU
 83
GfgCfaAfcUfaCfa
 84
pcGfcGfgGfuCfuUfg



AAGACCCGC

UGUAGUUGC

AfgAfcCfcGfcGfd

UfaGfuUfgCfcdTsdT



G

C

TsdT




 


 23
GGCAACUAC
 24
CGCGGGUCU
113
dGgdCadAcdTadC
114
pcdGcdGgdGudCudT



AAGACCCGC

UGUAGUUGC

adAgdAcdCcdGcd

gdTadGudTgdCcdTsd



G

C

GdTsdT

T


 


 23
GGCAACUAC
 24
CGCGGGUCU
143
GfdGCfdAAfdCUf
144
pdCGfdCGfdGGfdTC



AAGACCCGC

UGUAGUUGC

dACfdAAfdGAfdC

fdTUfdGUfdAGfdTUf



G

C

CfdCGfdCGfdTsdT

dGCfdCdTsdT


 


 25
CCGGCAAGC
 26
GGCACGGGC
 55
CCGGCAAGCUG
 56
GGCACGGGCAGCU



UGCCCGUGC

AGCUUGCCG

CCCGUGCCdTdT

UGCCGGdTdT



C

G






 


 25
CCGGCAAGC
 26
GGCACGGGC
 85
CfcGfgCfaAfgCfu
 86
pgGfcAfcGfgGfcAfg



UGCCCGUGC

AGCUUGCCG

GfcCfcGfuGfcCfd

CfuUfgCfcGfgdTsdT



C

G

TsdT




 


 25
CCGGCAAGC
 26
GGCACGGGC
115
dCcdGgdCadAgdC
116
pgdGcdAcdGgdGcdA



UGCCCGUGC

AGCUUGCCG

udGcdCcdGudGcd

gdCudTgdCcdGgdTsd



C

G

CdTsdT

T


 


 25
CCGGCAAGC
 26
GGCACGGGC
145
CfdCGfdGCfdAAf
146
pdGGfdCAfdCGfdGG



UGCCCGUGC

AGCUUGCCG

dGCfdTGfdCCfdC

fdCAfdGCfdTUfdGCf



C

G

GfdTGfdCCfdTsdT

dCGfdGdTsdT


 


 27
UGCCCAUCC
 28
AGCUCGACC
 57
UGCCCAUCCUG
 58
AGCUCGACCAGGA



UGGUCGAGC

AGGAUGGGC

GUCGAGCUdTdT

UGGGCAdTdT



U

A






 


 27
UGCCCAUCC
 28
AGCUCGACC
 87
UfgCfcCfaUfcCfu
 88
paGfcUfcGfaCfcAfgG



UGGUCGAGC

AGGAUGGGC

GfgUfcGfaGfcUfd

faUfgGfgCfadTsdT



U

A

TsdT




 


 27
UGCCCAUCC
 28
AGCUCGACC
117
dTgdCcdCadTcdCu
118
padGcdTcdGadCcdAg



UGGUCGAGC

AGGAUGGGC

dGgdTcdGadGcdT

dGadTgdGgdCadTsdT



U

A

dTsdT




 


 27
UGCCCAUCC
 28
AGCUCGACC
147
UfdGCfdCCfdAUf
148
pdAGfdCUfdCGfdAC



UGGUCGAGC

AGGAUGGGC

dCCfdTGfdGUfdC

fdCAfdGGfdAUfdGG



U

A

GfdAGfdCUfdTsd

fdGCfdAdTsdT







T




 


 29
CAAGUUCAG
 30
GCCGGACAC
 59
CAAGUUCAGCG
 60
GCCGGACACGCUG



CGUGUCCGG

GCUGAACUU

UGUCCGGCdTdT

AACUUGdTdT



C

G






 


 29
CAAGUUCAG
 30
GCCGGACAC
 89
CfaAfgUfuCfaGfc
 90
pgCfcGfgAfcAfcGfcU



CGUGUCCGG

GCUGAACUU

GfuGfuCfcGfgCfd

fgAfaCfuUfgdTsdT



C

G

TsdT




 


 29
CAAGUUCAG
 30
GCCGGACAC
119
dCadAgdTudCadG
120
pgdCcdGgdAcdAcdG



CGUGUCCGG

GCUGAACUU

cdGudGudCcdGgd

cdTgdAadCudTgdTsd



C

G

CdTsdT

T


 


 29
CAAGUUCAG
 30
GCCGGACAC
149
CfdAAfdGUfdTCf
150
pdGCfdCGfdGAfdCA



CGUGUCCGG

GCUGAACUU

dAGfdCGfdTGfdT

fdCGfdCUfdGAfdAC



C

G

CfdCGfdGCfdTsdT

fdTUfdGdTsdT


 


151
GGAAUCUUA
152
UUGGAUCAA
153
GGAAUCuuAuAu
154
uuGGAUcAAAuAuA



UAUUUGAUC

AUAUAAGAU

uuGAUCcAsA

AGAuUCcscsU



CAA

UCCCU






 


151
GGAAUCUUA
152
UUGGAUCAA
265
(Chol)GGAAUCuu
154
uuGGAUcAAAuAuA



UAUUUGAUC

AUAUAAGAU

AuAuuuGAUCcAs

AGAuUCcscsU



CAA

UCCCU

A




 


155
GGAUGAAGU
156
ACUAAUCUC
157
GGAuGAAGuGG
158
ACuAAUCUCcACU



GGAGAUUAG

CACUUCAUC

AGAuuAGudTsdT

UcAUCCdTsdT



U

C






 


155
GGAUGAAGU
156
ACUAAUCUC
160
(NH2C6)GfgAfuGf
159
pasCfuAfaUfcUfcCfa



GGAGAUUAG

CACUUCAUC

aAfgUfgGfaGfaUf

CfuUfcAfuCfc(invdT)



U

C

uAfgUf(invdT)




 


155
GGAUGAAGU
156
ACUAAUCUC
226
gsgAfuGfaAfgUfg
266
pdAsCfuAfaUfcUfcCf



GGAGAUUAG

CACUUCAUC

GfaGfaUfuAfgUfd

aCfuUfcAfuCfcdTsdT



U

C

TsdT




 


155
GGAUGAAGU
156
ACUAAUCUC
227
gsgAfuGfaAfgUfg
266
pdAsCfuAfaUfcUfcCf



GGAGAUUAG

CACUUCAUC

GfaGfaUfuAfgUf

aCfuUfcAfuCfcdTsdT



U

C

(invdT)




 


155
GGAUGAAGU
156
ACUAAUCUC
228
GfsgAfuGfaAfgUf
267
pAfsCfuAfaUfcUfcCf



GGAGAUUAG

CACUUCAUC

gGfaGfaUfuAfgUf

aCfuUfcAfuCfcdTsdT



U

C

dTsdT




 


155
GGAUGAAGU
156
ACUAAUCUC
229
GfsgAfuGfaAfgUf
268
pAfsCfuAfaUfcUfcCf



GGAGAUUAG

CACUUCAUC

gGfaGfaUfuAfgUf

aCfuUfcAfuCfcdT



U

C

dT(invdT)

(invdT)


 


155
GGAUGAAGU
156
ACUAAUCUC
230
ggAfuGfaAfgUfgG
238
dAsCfuAfaUfcUfcCfa



GGAGAUUAG

CACUUCAUC

faGfaUfuAfgUfdTs

CfuUfcAfuCfcdTsdT



U

C

dT




 


155
GGAUGAAGU
156
ACUAAUCUC
231
GfsgAfuGfaAfgUf
267
pAfsCfuAfaUfcUfcCf



GGAGAUUAG

CACUUCAUC

gGfaGfaUfuAfgUf

aCfuUfcAfuCfcdTsdT



U

C

(invdT)




 


155
GGAUGAAGU
156
ACUAAUCUC
232
ggAfuGfaAfgUfgG
238
dAsCfuAfaUfcUfcCfa



GGAGAUUAG

CACUUCAUC

faGfaUfuAfgUf

CfuUfcAfuCfcdTsdT



U

C

(invdT)




 


155
GGAUGAAGU
156
ACUAAUCUC
233
GfgAfuGfaAfgUfg
239
AfsCfuAfaUfcUfcCfa



GGAGAUUAG

CACUUCAUC

GfaGfaUfuAfgUfd

CfuUfcAfuCfcdT



U

C

T(invdT)

(invdT)


 


155
GGAUGAAGU
156
ACUAAUCUC
234
GfgAfuGfaAfgUfg
240
AfsCfuAfaUfcUfcCfa



GGAGAUUAG

CACUUCAUC

GfaGfaUfuAfgUf

CfuUfcAfuCfcdTsdT



U

C

(invdT)




 


155
GGAUGAAGU
156
ACUAAUCUC
231
GfsgAfuGfaAfgUf
241
pAfsCfuAfaUfcUfcCf



GGAGAUUAG

CACUUCAUC

gGfaGfaUfuAfgUf

aCfuUfcAfuCfc



U

C

(invdT)

(invdT)


 


155
GGAUGAAGU
156
ACUAAUCUC
234
GfgAfuGfaAfgUfg
159
pasCfuAfaUfcUfcCfa



GGAGAUUAG

CACUUCAUC

GfaGfaUfuAfgUf

CfuUfcAfuCfc(invdT)



U

C

(invdT)




 


155
GGAUGAAGU
156
ACUAAUCUC
235
gsgAfuGfaAfgUfg
242
pdAsCfuAfaUfcUfcCf



GGAGAUUAG

CACUUCAUC

GfaGfaUfuAfgUfd

aCfuUfcAfuCfcdT



U

C

T(invdT)

(invdT)


 


155
GGAUGAAGU
156
ACUAAUCUC
236
GfgAfuGfaAfgUfg
240
AfsCfuAfaUfcUfcCfa



GGAGAUUAG

CACUUCAUC

GfaGfaUfuAfgUfd

CfuUfcAfuCfcdTsdT



U

C

TsdT




 


155
GGAUGAAGU
156
ACUAAUCUC
227
gsgAfuGfaAfgUfg
243
pdAsCfuAfaUfcUfcCf



GGAGAUUAG

CACUUCAUC

GfaGfaUfuAfgUf

aCfuUfcAfuCfc



U

C

(invdT)

(invdT)


 


155
GGAUGAAGU
156
ACUAAUCUC
237
ggAfuGfaAfgUfgG
244
dAsCfuAfaUfcUfcCfa



GGAGAUUAG

CACUUCAUC

faGfaUfuAfgUfdT

CfuUfcAfuCfcdT



U

C

(invdT)

(invdT)


 


155
GGAUGAAGU
156
ACUAAUCUC
232
ggAfuGfaAfgUfgG
245
dAsCfuAfaUfcUfcCfa



GGAGAUUAG

CACUUCAUC

faGfaUfuAfgUf

CfuUfcAfuCfc(invdT)



U

C

(invdT)




 


155
GGAUGAAGU
156
ACUAAUCUC
234
GfgAfuGfaAfgUfg
246
AfsCfuAfaUfcUfcCfa



GGAGAUUAG

CACUUCAUC

GfaGfaUfuAfgUf

CfuUfcAfuCfc(invdT)



U

C

(invdT)




 


155
GGAUGAAGU
156
ACUAAUCUC
157
GGAuGAAGuGG
158
ACuAAUCUCcACU



GGAGAUUAG

CACUUCAUC

AGAuuAGudTsdT

UcAUCCdTsdT



U

C






 


161
GCAAAGGCG
162
UGAGUUGGC
165
(NH2C6)GfcAfaAf
166
puGfaGfuUfgGfcAfc



UGCCAACUC

ACGCCUUUG

gGfcGfuGfcCfaAfc

GfcCfuUfuGfc(invdT)



A

C

UfcAf(invdT)




 


161
GCAAAGGCG
162
UGAGUUGGC
167
(NH2C6)GcAAAG
168
UGAGUUGGcACGC



UGCCAACUC

ACGCCUUUG

GcGuGccAAcucAd

CUUUGCdTsdT



A

C

TsdT




 


161
GCAAAGGCG
162
UGAGUUGGC
179
GalNAc-(NH2C6)-
166
puGfaGfuUfgGfcAfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

GfcCfuUfuGfc(invdT)



A

C

GfcCfaAfcUfcAf









(invdT)




 


161
GCAAAGGCG
162
UGAGUUGGC
180
GalNAc-(NH2C6)-
168
UGAGUUGGcACGC



UGCCAACUC

ACGCCUUUG

GcAAAGGcGuGcc

CUUUGCdTsdT



A

C

AAcucAdTsdT




 


161
GCAAAGGCG
162
UGAGUUGGC
181
GfcAfaAfgGfcGfu
185
pUfsGfaGfuUfgGfcAf



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

cGfcCfuUfuGfc



A

C

(invdT)

(invdT)


 


161
GCAAAGGCG
178
TGAGUUGGC
181
GfcAfaAfgGfcGfu
186
psdTdGaGfuUfgGfcA



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

fcGfcCfuUfuGfc



A

C

(invdT)

(invdT)


 


161
GCAAAGGCG
178
TGAGUUGGC
181
GfcAfaAfgGfcGfu
187
psdTsGfaGfuUfgGfcA



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

fcGfcCfuUfuGfc



A

C

(invdT)

(invdT)


 


161
GCAAAGGCG
178
TGAGUUGGC
181
GfcAfaAfgGfcGfu
188
pdTsdGaGfuUfgGfcA



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

fcGfcCfuUfuGfc



A

C

(invdT)

(invdT)


 


161
GCAAAGGCG
178
TGAGUUGGC
181
GfcAfaAfgGfcGfu
189
pdTsGfaGfuUfgGfcAf



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

cGfcCfuUfuGfc



A

C

(invdT)

(invdT)


 


161
GCAAAGGCG
162
UGAGUUGGC
181
GfcAfaAfgGfcGfu
190
pusGfaGfuUfgGfcAfc



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

GfcCfuUfuGfc(invdT)



A

C

(invdT)




 


161
GCAAAGGCG
162
UGAGUUGGC
181
GfcAfaAfgGfcGfu
166
puGfaGfuUfgGfcAfc



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

GfcCfuUfuGfc(invdT)



A

C

(invdT)




 


161
GCAAAGGCG
162
UGAGUUGGC
181
GfcAfaAfgGfcGfu
191
psUfGfaGfuUfgGfcAf



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

cGfcCfuUfuGfc



A

C

(invdT)

(invdT)


 


161
GCAAAGGCG
162
UGAGUUGGC
181
GfcAfaAfgGfcGfu
192
UfsGfaGfuUfgGfcAfc



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

GfcCfuUfuGfc(invdT)



A

C

(invdT)




 


161
GCAAAGGCG
162
UGAGUUGGC
181
GfcAfaAfgGfcGfu
193
psUfsGfaGfuUfgGfcA



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

fcGfcCfuUfuGfc



A

C

(invdT)

(invdT)


 


161
GCAAAGGCG
178
TGAGUUGGC
181
GfcAfaAfgGfcGfu
194
psdTsdGaGfuUfgGfc



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

AfcGfcCfuUfuGfc



A

C

(invdT)

(invdT)


 


161
GCAAAGGCG
162
UGAGUUGGC
181
GfcAfaAfgGfcGfu
195
usGfaGfuUfgGfcAfcG



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

fcCfuUfuGfc(invdT)



A

C

(invdT)




 


161
GCAAAGGCG
162
UGAGUUGGC
181
GfcAfaAfgGfcGfu
196
uGfaGfuUfgGfcAfcGf



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

cCfuUfuGfc(invdT)



A

C

(invdT)




 


161
GCAAAGGCG
178
TGAGUUGGC
181
GfcAfaAfgGfcGfu
197
dTsGfaGfuUfgGfcAfc



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

GfcCfuUfuGfc(invdT)



A

C

(invdT)




 


161
GCAAAGGCG
178
TGAGUUGGC
181
GfcAfaAfgGfcGfu
198
psdTGfaGfuUfgGfcAf



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

cGfcCfuUfuGfc



A

C

(invdT)

(invdT)


 


161
GCAAAGGCG
178
TGAGUUGGC
181
GfcAfaAfgGfcGfu
199
dTsdGaGfuUfgGfcAf



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

cGfcCfuUfuGfc



A

C

(invdT)

(invdT)


 


161
GCAAAGGCG
162
UGAGUUGGC
183
(Chol)GfcAfaAfgG
190
pusGfaGfuUfgGfcAfc



UGCCAACUC

ACGCCUUUG

fcGfuGfcCfaAfcUf

GfcCfuUfuGfc(invdT)



A

C

cAf(invdT)




 


161
GCAAAGGCG
162
UGAGUUGGC
181
GfcAfaAfgGfcGfu
196
uGfaGfuUfgGfcAfcGf



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

cCfuUfuGfc(invdT)



A

C

(invdT)




 


161
GCAAAGGCG
162
UGAGUUGGC
181
GfcAfaAfgGfcGfu
195
usGfaGfuUfgGfcAfcG



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

fcCfuUfuGfc(invdT)



A

C

(invdT)




 


161
GCAAAGGCG
162
UGAGUUGGC
181
GfcAfaAfgGfcGfu
192
UfsGfaGfuUfgGfcAfc



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

GfcCfuUfuGfc(invdT)



A

C

(invdT)




 


161
GCAAAGGCG
178
TGAGUUGGC
181
GfcAfaAfgGfcGfu
197
dTsGfaGfuUfgGfcAfc



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

GfcCfuUfuGfc(invdT)



A

C

(invdT)




 


161
GCAAAGGCG
178
TGAGUUGGC
181
GfcAfaAfgGfcGfu
199
dTsdGaGfuUfgGfcAf



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcAf

cGfcCfuUfuGfc



A

C

(invdT)

(invdT)


 


161
GCAAAGGCG
162
UGAGUUGGC
182
GcAAAGGcGuGcc
168
UGAGUUGGcACGC



UGCCAACUC

ACGCCUUUG

AAcucAdTsdT

CUUUGCdTsdT



A

C






 


161
GCAAAGGCG
162
UGAGUUGGC
184
GfcAfaAfgGfcGfu
168
UGAGUUGGcACGC



UGCCAACUC

ACGCCUUUG

GfcCfaAfcUfcA

CUUUGCdTsdT



A

C






 


161
GCAAAGGCG
162
UGAGUUGGC
179
GalNAc-(NH2C6)-
166
puGfaGfuUfgGfcAfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

GfcCfuUfuGfc(invdT)



A

C

GfcCfaAfcUfcAf









(invdT)




 


161
GCAAAGGCG
162
UGAGUUGGC
179
GalNAc-(NH2C6)-
190
pusGfaGfuUfgGfcAfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

GfcCfuUfuGfc(invdT)



A

C

GfcCfaAfcUfcAf









(invdT)




 


161
GCAAAGGCG
162
UGAGUUGGC
179
GalNAc-(NH2C6)-
185
pUfsGfaGfuUfgGfcAf



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

cGfcCfuUfuGfc



A

C

GfcCfaAfcUfcAf

(invdT)







(invdT)




 


161
GCAAAGGCG
178
TGAGUUGGC
179
GalNAc-(NH2C6)-
263
psdTdGfaGfuUfgGfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

AfcGfcCfuUfuGfc



A

C

GfcCfaAfcUfcAf

(invdT)







(invdT)




 


161
GCAAAGGCG
162
UGAGUUGGC
179
GalNAc-(NH2C6)-
166
puGfaGfuUfgGfcAfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

GfcCfuUfuGfc(invdT)



A

C

GfcCfaAfcUfcAf









(invdT)




 


161
GCAAAGGCG
162
UGAGUUGGC
179
GalNAc-(NH2C6)-
190
pusGfaGfuUfgGfcAfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

GfcCfuUfuGfc(invdT)



A

C

GfcCfaAfcUfcAf









(invdT)




 


161
GCAAAGGCG
162
UGAGUUGGC
179
GalNAc-(NH2C6)-
262
pUfsGfaGfuUfgGfcAf



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

cGfcCfuUfuGfc



A

C

GfcCfaAfcUfcAf

(invdT)







(invdT)




 


161
GCAAAGGCG
256
TGAGUUGGC
179
GalNAc-(NH2C6)-
263
psdTdGfaGfuUfgGfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu





A

C

GfcCfaAfcUfcAf

AfcGfcCfuUfuGfc







(invdT)

(invdT)


 


161
GCAAAGGCG
162
UGAGUUGGC
257
Chol-(NH2C6)-
166
puGfaGfuUfgGfcAfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

GfcCfuUfuGfc(invdT)



A

C

GfcCfaAfcUfcAf









(invdT)




 


161
GCAAAGGCG
162
UGAGUUGGC
257
Chol-(NH2C6)-
190
pusGfaGfuUfgGfcAfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

GfcCfuUfuGfc(invdT)



A

C

GfcCfaAfcUfcAf









(invdT)




 


161
GCAAAGGCG
162
UGAGUUGGC
257
Chol-(NH2C6)-
264
pUfsGfaGfuUfgGfcAf



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

cGfcCfuUfuGfc



A

C

GfcCfaAfcUfcAf

(invdT)







(invdT)




 


161
GCAAAGGCG
256
TGAGUUGGC
257
Chol-(NH2C6)-
263
psdTdGfaGfuUfgGfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

AfcGfcCfuUfuGfc



A

C

GfcCfaAfcUfcAf

(invdT)







(invdT)




 


161
GCAAAGGCG
162
UGAGUUGGC
258
Chol-(NH2C6)-
168
UGAGUUGGcACGC



UGCCAACUC

ACGCCUUUG

GcAAAGGcGuGcc

CUUUGCdTsdT



A

C

AAcucAdTsdT




 


161
GCAAAGGCG
256
TGAGUUGGC
257
Chol-(NH2C6)-
263
psdTdGfaGfuUfgGfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

AfcGfcCfuUfuGfc



A

C

GfcCfaAfcUfcAf

(invdT)







(invdT)




 


161
GCAAAGGCG
256
TGAGUUGGC
259
Chol-C6SSC6-
263
psdTdGfaGfuUfgGfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

AfcGfcCfuUfuGfc



A

C

GfcCfaAfcUfcAf

(invdT)







(invdT)




 


161
GCAAAGGCG
256
TGAGUUGGC
260
Chol-AUC-
263
psdTdGfaGfuUfgGfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

AfcGfcCfuUfuGfc



A

C

GfcCfaAfcUfcAf

(invdT)







(invdT)




 


161
GCAAAGGCG
256
TGAGUUGGC
261
Chol-Cathepsin-
263
psdTdGfaGfuUfgGfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

AfcGfcCfuUfuGfc



A

C

GfcCfaAfcUfcAf

(invdT)







(invdT)




 


161
GCAAAGGCG
256
TGAGUUGGC
257
Chol-(NH2C6)-
263
psdTdGfaGfuUfgGfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

AfcGfcCfuUfuGfc



A

C

GfcCfaAfcUfcAf

(invdT)







(invdT)




 


161
GCAAAGGCG
256
TGAGUUGGC
259
Chol-C6SSC6-
263
psdTdGfaGfuUfgGfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

AfcGfcCfuUfuGfc



A

C

GfcCfaAfcUfcAf

(invdT)







(invdT)




 


161
GCAAAGGCG
256
TGAGUUGGC
260
Chol-AUC-
263
psdTdGfaGfuUfgGfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

AfcGfcCfuUfuGfc



A

C

GfcCfaAfcUfcAf

(invdT)







(invdT)




 


161
GCAAAGGCG
256
TGAGUUGGC
261
Chol-Cathepsin-
263
psdTdGfaGfuUfgGfc



UGCCAACUC

ACGCCUUUG

GfcAfaAfgGfcGfu

AfcGfcCfuUfuGfc



A

C

GfcCfaAfcUfcAf

(invdT)







(invdT)




 


163
GGAUCAUCU
164
GUAAGACUU
169
(NH2C6)GGAUfCf
170
GUfAAGACfUfUfGA



CAAGUCUUA

GAGAUGAUC

AUfCfUfCfAAGUf

GAUfGAUfCfCfdTsd



C

C

CfUfUfACfdTsdT

T


 


171
UGACCACAG
172
UUUAAUCCG
173
(NH2C6)UfgAfcCf
174
pusUfuAfaUfcCfgAfc



UCGGAUUAA

ACUGUGGUC

aCfaGfuCfgGfaUfu

UfgUfgGfuCfa(invdT)



A

A

AfaAf(invdT)




 


171
UGACCACAG
172
UUUAAUCCG
175
(NH2C6)uGAccAc
176
puUuAAUCCGACU



UCGGAUUAA

ACUGUGGUC

AGucGGAuuAAA

GUGGucAdTsdT



A

A

dTsdT




 


171
UGACCACAG
172
UUUAAUCCG
175
(NH2C6)uGAccAc
177
UUuAAUCCGACUG



UCGGAUUAA

ACUGUGGUC

AGucGGAuuAAA

UGGUcAdTsdT



A

A

dTsdT




 


247
GUUGGUGAA
248
CUGAGCUCC
251
(NH2C6)GfuUfgGf
252
pCfsUfgAfgCfuCfcAf



UGGAGCUCA

AUUCACCAA

uGfaAfuGfgAfgCf

uUfcAfcCfaAfc



G

C

uCfaGf(invdT)

(invdT)


 


247
GUUGGUGAA
248
CUGAGCUCC
251
(NH2C6)GfuUfgGf
255
pCfsUfgAfgCfuCfcAf



UGGAGCUCA

AUUCACCAA

uGfaAfuGfgAfgCf

uUfcAfcCfaAfcdTsdT



G

C

uCfaGf(invdT)




 


249
GGUCCUGUU
250
AUUCACCAA
253
(NH2C6)GfgUfcCf
254
pAfsUfuCfaCfcAfaCf



GUUGGUGAA

CAACAGGAC

uGfuUfgUfuGfgUf

aAfcAfgGfaCfcdTsdT



U

C

gAfaUf(invdT)

## Claims

1. A compound of formula (I):




wherein
Y is a linker group selected from —(CH2)3— or —C(O)—NH—(CH2—CH2—O)p—CH2—CH2—;
R1 is —(C1-6) alkyl;
—(CH2)-naphthyl; or
—(CH2)m-phenyl, which phenyl is unsubstituted or up to four times substituted with a substituent independently selected from
—NO2,
—CN,
Halogen,
—O—(CH2)-phenyl,
—O—(C1-6) alkyl, or
—C(O)—NH2;


R2 is hydrogen;
—(CH2)k—NH—C(Ph)3, which phenyl rings are unsubstituted or independently substituted with —O—(C1-4)alkyl;
—(CH2)k—C(O)—NH2;
—(CH2)k-phenyl;
—(C1-6) alkyl, which is unsubstituted or once substituted with —S—CH3;

R3 is —NH-phenyl, which phenyl group is further substituted with a substituent independently selected from
—(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;
n is 0 or 1; and
p is an integer from 1 to 20.

2. The compound of claim 1, having the conformation as shown in formula (Ia):

3. The compound of claim 1 wherein Y is —(CH2)3—.

4. The compound of claim 3, wherein:
Y is —(CH2)3—;
R2 is —(CH2)k—NH—C(Ph)3, which phenyl rings are unsubstituted or independently substituted with —O—(C1-4)alkyl; and
R3 is —NH-phenyl, which phenyl group is further substituted with —(CH2)—O—C(O)—O-(4-nitro-phenyl);
n is 0; and
R1 and k are as defined in claim 1.

5. The compound of claim 1, wherein Y is —C(O)—NH—(CH2—CH2—O)p—CH2—CH2—.

6. The compound of claim 5, wherein:
Y is —C(O)—NH—(CH2—CH2—O)p—CH2—CH2—;
R2 is —(CH2)k—NH—C(Ph)3, which phenyl rings are unsubstituted or independently substituted with —O—(C1-4)alkyl; and
R3 is —NH-phenyl, which phenyl group is further substituted with —(CH2)—O—C(O)—O-(4-nitro-phenyl);
n is 0; and

R1, k and p have the meanings given above.