compounds, compound use and pharmaceutical composition

专利摘要:
COMPOUNDS, USES OF COMPOUNDS, PHARMACEUTICAL COMPOSITIONS, OLIGONUCLEOTIDE, CONJUGATES AND METHODS. The invention provides new compounds and conjugates of these compounds useful for the distribution of biologically active substances. In addition to new design criteria for chemically stabilized siRNA particularly useful when covalently linked to a delivery polymer to achieve in vivo mRNA knockdown are disclosed in the present application.
公开号:BR112013016772B1
申请号:R112013016772-6
申请日:2011-12-22
公开日:2021-01-05
发明作者:Philipp Hadwiger；David B. Rozema；David L. Lewis；Eric A. Kitas；Guenther Ott；Hans Martin Mueller；Ingo Roehl；Kerstin Janh-Hofmann；Peter Mohr；Torsten Hoffmann
申请人:F. Hoffmann-La Roche Ag；
IPC主号:

专利说明:

[001] The present invention relates to new small molecule conjugates useful for the distribution of biologically active substances, such as nucleic acids, peptides and proteins. The distribution of nucleic acids and other substantially impermeable cell membrane compounds in a living cell is highly limited by the cell's complex membrane system.
[002] Another meaning that has been used to deliver a biologically active substance, such as nucleic acids in vivo, has been to bind the biologically active substance with either a small target molecule or a hydrophobic molecule such as a lipid or sterol. Although some distributions and activities have been observed with these conjugates, the dose of biologically active substance required with these methods has been prohibitively large, often resulting in undesirable effects of toxicity in vivo. Provided in the present application are small molecule compounds that can be conjugated to a biologically active substance and mediate the successful distribution of said biologically active substance in a cell. Surprisingly, it has been found that significantly reduced doses of the biologically active substance are now sufficient to successfully deliver when using the new compounds provided in the present application. In this way, the new compounds provide a powerful tool for the distribution of biologically active substances with considerably limited toxicity in vivo.
[003] In one embodiment, the present invention is directed to compounds of formula
where - 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; - 2 is hydrogen; - (CH2) kNC (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; - 3 is -NH-phenyl, which the phenyl group is further substituted with a substituent independently selected from - (CH2) -OH; or - (CH2) -OC (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; ep is an integer from 1 to 20.
[004] In another embodiment, the compounds of formula (I) may have the specific conformation, as shown in formula (Ia)
where all substituents R1, R2, R3 and Y as well as the variables k, m, n, and p have the meaning given above.
[005] In yet another embodiment, the present invention is directed to compounds of formula (I) or (Ia), where Y is - (CH2) 3-; and all the remaining substituent groups have the meaning given above.
[006] In yet another embodiment, the present invention is directed to compounds of formula (I) or (Ia), where Y is -C (O) -N- (CH2-CH2- O) p-CH2-CH2- ; and all substituent groups have the meaning given above.
[007] In yet another embodiment, compounds of formula (I) or (Ia), where 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, in which the phenyl group is further substituted - (CH2) -O-C (O) -O- (4-nitro-phenyl); n is 0; and R1 and k have the meaning given above.
[008] In yet another embodiment, compounds of formula (I) or (Ia) are provided, 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, in which the phenyl group is further substituted - (CH2) -O-C (O) -O- (4-nitro-phenyl); n is 0; and R1, k and p have the meaning given above.
[009] The term "C1-6 alkyl", as used in the present application, means a straight or branched hydrocarbon, saturated with 1 to 6 carbon atoms. Preferred C1-6 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, 2-butyl and the like.
[010] The term "halogen", as used in this application, means fluorine, chlorine, bromine or iodine with fluorine and chlorine being preferred.
[011] The compounds according to the present invention can generally be obtained using methods known to those skilled in the art of organic or medicinal chemistry. More particularly the compounds of formula (Ia), where Y is - (CH2) 3 and en = 0, can be obtained using compounds (A) depending on the starting material.

[012] The synthesis of (A) is described among other things in WO2001 / 070415.
[013] The compound of formula (A) is further reacted in the presence of a Huenig base and ethyl acetate (AcOEt), followed by the addition of dihydrofuran-2,5-dione in THF, to give the compounds of formula (B )

[014] The compounds of formula (B) are further reacted with an amine of formula (C),
to give the compounds of formula (Ia).
[015] The compounds of formula (I) or (Ia) are useful as binders in 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, for example, a primary amine group, in the biologically active substance with the activated carbonyl group in the -OC (O) -O- component of R3 as defined above in this application. Then, a conjugate comprising the compounds of formula (I) or (Ia) and a biologically active substance is provided in the present application.
[016] The term "biologically active substance" as used in this application refers to an inorganic or organic molecule, including a small molecule, peptide (for example, penetrating peptide cells), protein, carbohydrate (including monosaccharides, oligosaccharides and polysaccharides), nucleoprotein, mucoprotein, lipoprotein, polypeptide or synthetic protein, or a small molecule attached 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, which cause a biological effect when administered in vivo to an animal, which includes, but is not limited to birds and mammals, including humans. Preferably, said biologically active substance is a peptide or nucleic acid. Preferred nucleic acids used in the present application are siRNAs.
[017] The conjugate comprising the present compounds covalently linked to a biologically active substance has an improved ability to be absorbed by cells compared to said biologically active substance alone. Once the conjugate is released into the cell and transitions to the lysosome, the corresponding biologically active substance is released by enzymatic cleavage. This cleavage preferably occurs when a dipeptide 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 into the conjugate (see scheme 1). Most preferably, the conjugate contains the dipeptide motif and a spacer such as the p-aminobenzylcarbamate spacer (Bioconjugate Chem. 2002,13,855), which spontaneously breaks down once the C-terminal amide bond of the dipeptide motif and is cleaved as exemplified for the siRNAs in scheme 2. Then, conjugates comprising compounds of formula (I) or (Ia) are also cited as dipeptides that contain cholesterol conjugates. The enzymatic cleavage of the biologically active substance from the dipeptides containing cholesterol conjugates of that invention is catalyzed by innate proteases of the cell. An example of an innate protease capable of cleaving the dipeptide motif present in the compounds of formula (I) or (Ia) is Cathepsin B. Cathepsin B is a well-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 dipeptide motif described above is also cited as a dipeptide motif cleavable by Cathepsin.
[018] The present invention, therefore, also provides a method for delivering a biologically active substance, to cells in which said biologically active substance can subsequently be cleaved from the conjugate to develop a therapeutic activity. dipeptide motif with
Layout 1
[019] In a further embodiment of the present invention, a conjugate of the compounds of formula (I) or (Ia), covalently linked to a biologically active compound, preferably siRNA or a peptide component, is provided. Preferably, said peptide component is a peptide that exhibits properties that disturb membranes similar to penetrating peptides or amphiphilic peptides.
[020] Conjugates of formula (I) or (Ia) covalently linked to a biologically active substance are referred to in the present application as formula (II) or (IIa), respectively.
[021] Therefore, in a further embodiment, the present invention provides a compound of the formula.
R1 and k have the meaning given to formula (I) above; and the biologically active substance is a nucleic acid, a protein or a peptide.
[022] In a more specific embodiment, the present invention
where Ra is - (CH2) k-NH2; R1 and k have the meaning given to formula (I) above; and the biologically active substance is a nucleic acid, a protein or a peptide.
[023] In a preferred embodiment, the biologically active substance in formula (II) or (IIa) is a nucleic acid, most preferably an RNA si.
[024] In another preferred embodiment, the biologically active substance in formula (II) or (IIa) is a protein or a peptide.
[025] The compounds of formula (II) or (IIa) can have valuable properties in therapy. Therefore, in an additional embodiment, compounds of formula (II) or (1a) are provided for use as medicaments.
[026] Another embodiment of the invention is a pharmaceutical composition comprising the conjugates of the compounds of formula (I) or (Ia), covalently linked to a biologically active substance.
[027] In yet another embodiment of the invention, a pharmaceutical composition is provided which comprises the compounds of formula (IIa) together with pharmaceutically acceptable excipients.
[028] The embodiments below are exemplified for conjugates of the compounds of formula (I) or (Ia), covalently bound to siRNA, thus, in the compounds of formula (II) or (lIa) the active biological substance is siRNA. It is understood that these embodiments are also applicable to other biologically active substances, such as peptides and proteins.
[029] SiRNA covalent attachment to the compounds of formula (I) or (Ia) is obtained by reacting a suitable nucleophilic group, that is, 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 this group - C (O) - is achieved with a p-nitrophenoxy carbonate, as shown in scheme 2 below:

[030] The activated p-nitrophenyl carbonate can, for example, react with siRNA equipped with a hexylamino linker to generate a carbamate bond to produce the siRNA conjugate. Since siRNA is absorbed intracellularly and transfected into the lysosome, compounds of formula (II) or (lIa) in which the active biological substance is siRNA are cleaved by the protease activity releasing siRNA as also shown in scheme 2. The The cholesterol component of the conjugate of the compounds of formula (II) or (IIa) modifies the pharmacokinetic properties of siRNA in such a way that systemic administration allows silencing of the gene in vivo.

[031] In one embodiment, the compounds of formula (II) or (1a) in which the active biological substance is siRNA is co-administered with a delivery polymer. Distribution polymers provide a means of breaking down cell membranes and mediating endosomal release. In another embodiment, said delivery polymer and the siRNA conjugate of the invention are not covalently linked and are synthesized separately, and can be supplied in separate containers or in a single container. Delivery polymers for oligonucleotides such as siRNA are well known in the art. For example, Rozema et al., In US patent publication 20040162260 demonstrated a means for reversibly regulating the membrane breaking activity of an active membrane polyamine. Reversible regulation provided as a means of limiting the activity of endosomes of target cells thus limits toxicity. His method was dependent on the reaction of amines in the polyamine with 2-propionic-3-methylmaleic anhydride. This modification converted the polycation to a polyanion by converting primary amines to groups containing carboxyl, and reversibly inhibited the activity of the membrane polyamine. To allow for the codistribution of the nucleic acid with the delivery vehicle, the nucleic acid was covalently linked to the delivery polymer. In the provisional patent application US 61/307490 a new generation of distribution polymers is described. In this, active membrane polyamines are provided which comprise an amphiphatic terpolymer formed by random polymerization of amine-containing monomers, smaller hydrophobic monomers and larger hydrophobic monomers. This new generation of distribution polymers removed the need for the polynucleotide and the polymer to be associated, both by covalent bonding and by charge-charge interaction.
[032] Non-limiting examples of delivery polymers used for coadministration with the invented siRNA conjugates are membrane active polyamines and polyvinyl ether (PBAVE), Dynamic PolyConjugates (DPC; Rozema et al., 2007) and improved DPCs as disclosed in provisional US patent application 61/307490.
[033] In a further embodiment, a new siRNA chemical modification standard for in vivo distribution is provided. This new pattern of chemical modification of siRNA is especially useful with delivery vehicles that have relatively strong endosomal / lysosomal retention.
[034] SiRNA stabilization against degradation by nucleases in the endosome / lysosome located as DNAse II was found to greatly improve the target knock down. This stabilization can directly affect the amount of siRNA released into the cytoplasm, where the RNAi cellular machinery is located. Only the portion of siRNA available in the cytoplasm will trigger the RNAi effect.
[035] In addition to weak pharmacokinetic characteristics, siRNAs are susceptible to nucleases in the biological environment, when administered as such in the circulation without a protective delivery vehicle. Consequently, many siRNAs are rapidly degraded both extracellularly in tissue and in the bloodstream of or after intracellular absorption (endosome).
[036] A well-known nuclease located 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 4.5-5, reflecting current conditions in the acidified environment of the endosomal / lysosomal compartment. The following pathways of RNA degradation induced by DNase II have been identified in vitro and are disclosed in the present invention:
[037] A. RNA strips containing at least one 2'-OH nucleotide are rapidly degraded through a cyclic pentavalent phosphorus intermediate, taking 2'-3 'cyclic phosphates 5' cleavage product. The formation of the pentavalent intermediate can be inhibited by nucleotides devoid of a 2'-OH group, such as 2'-deoxy, 2'-O-methyl (2'-OMe) or 2'-deoxy-2'-fluoro (2 '-F).
[038] B. Additionally, the RNA is degraded in a 5 'exonucleolytic pathway independent of the 2' modification at the 5 'terminal nucleotides. This degradation pathway can be inhibited by 5 'terminal non-nucleotide components, such as, for example, cholesterol, aminoalkyl linker or a phosphotioate in the first internucleotide bond.
[039] C. A 5'-phosphate also protects and slows down the kinetics of exonucleolytic cleavage, but cannot fully block this pathway. This is most likely due to cleavage of 5'-phosphate by phosphatases or an inherent phosphatase activity of the DNase II enzyme preparation used in the stability assay.
[040] D. The best protection was achieved with oligonucleotides devoid of any 2'-OH nucleotide on the strip, starting with a 2-'OMe nucleotide at the 5 'termination connected by a phosphorothioate (PTO) link to the second nucleotide. Other non-2'-OH nucleotides also protect against exo 5 'degradation, but to a lesser extent compared to the 2-'OMe modification.
[041] Thus, the inventors of the present invention have found that siRNAs can be significantly stabilized using the following design, in which an oligonucleotide with an antisense tape is provided with the modification pattern: 5 '- (w) - ( Z1) - (Z2) - (Z3) na-3 'and a sense chain, with the 5'- (Z3) n s-3' modification pattern, where: W is, independently, a 5'-phosphate or phosphotioate 5 'or H, Z1 is independently a modified 2' nucleoside. Z2 is independently a 2 'deoxynucleoside or modified fluoro 2' nucleoside. Z3 is independently a 2 'modified nucleoside, na is 8-23 and ns is 8-25.
[042] In a preferred embodiment, an oligonucleotide with an antisense tape with the modification pattern is provided: 5 '- (w) - (Z1) - (Z2) - (Z3) at -3' and a sense tape with the pattern modification 5'- (Z3) ns -3 ', where Z1 is a modified fluoro 2' nucleoside or a 2 'deoxynucleoside, and all remaining substituents, as well as the variables na and ns have the meaning given above.
[043] In a preferred embodiment, an oligonucleotide with an antisense tape with the modification pattern is provided: 5 '- (w) - (Z1) - (Z2) - (Z3) at -3' and a sense tape with the pattern modification 5'- (Z3) ns -3 ', wherein Z3 is a modified O-methyl 2' nucleoside, a modified fluoro 2 'nucleoside or a 2' deoxynucleoside, and all remaining substituents, as well as the variables in and we have the meaning given above.
[044] In a preferred embodiment, an oligonucleotide with an antisense tape with the modification pattern is provided: 5 '- (w) - (Z1) - (Z2) - (Z3) at -3' and a sense tape with the pattern modification 5'- (Z3) ns -3 ', where Z1 is a modified 2' fluoro nucleoside or deoxynucleoside 2 'and Z3 is modified O-methyl 2' nucleoside, a modified fluoro 2 'nucleoside, a 2' deoxynucleoside , and all remaining substituents, as well as the variables na and ns have the meaning given above.
[045] The nucleosides in the nucleic acid sequence of the oligonucleotide with the new modification pattern can be linked via 5'-3 'phosphodiesters or 5'- 3' phosphorothioates as used in the present application, the "antisense" tape is the tape of siRNA that is complementary to the target mRNA and that will be the binding to the mRNA, once the siRNA is unwound.
[046] The sense tape of said siRNA that comprises the new modification pattern is complementary to the antisense tape.
[047] Said siRNA comprising the new modification pattern has proved to be particularly advantageous when covalently attached to a distribution polymer, as exemplified by Rozema et al. (Dynamic PolyConjugates (DPC; Rozema et al. 2007). The potency and duration of the effect can be significantly improved with the use of the siRNA modification strategy described in that invention.
[048] In another embodiment, said siRNA comprising the new modification pattern is especially useful when conjugated with small molecules that alter the pharmacokinetic properties of siRNA, such as cholesterol or the compounds of formula (I) and (Ia) provided in this application. In one embodiment, a conjugate of a small molecule and an oligonucleotide is provided, in which the oligonucleotide has the following modification pattern: the antisense tape with the modification pattern ::: 5 '- (w) - (Z1) - (Z2 ) - (Z3) na -3 'and a sense tape with the modification pattern 5'- (Z3) ns-, in which 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 (Ia), resulting in compounds of formula (II) or (1a).
[049] Preferably, said siRNA conjugates are co-administered with a delivery polymer. Such distribution polymers are described above.
[050] In one embodiment, said siRNA comprising the new pattern of modification is especially useful when conjugated to a ligand that is known to bind to a specific receptor that internalizes the conjugate in a cell. In particular, the asialoglycoprotein receptor (ASGPR), expressed in hepatocytes, is a receptor known to allow the release (endocytosis and lysosomal degradation) of deialized proteins from the circulation. N-acetyl-D-galactosamine has been shown to have high binding affinity for the receptor, especially when presented multivalent and when galactose residues are adequately spaced (J Biol Bhem, 2001, 276, 37577). In order to use this high-capacity receptor for receptor-mediated endocytosis of the biologically active substance, the synthetic ligand shown below has been prepared to be covalently linked to siRNAs that comprise the new modification pattern. Since this type of endocytosis leads to lysosomal degradation of the internalized material, siRNA must be prepared in such a way that it is stable in the lysosome, which is now clarified by the new modification pattern described above.
[051] The binder for the ASGPR is linked to the biologically active substance via an amide bond. The formation of the amide bond can be established with the help of NHS chemistry. The ligand used in the conjugation reaction is shown below (formula III). For interaction with ASGPR the O-acetate groups need to be removed as shown in (formula IV) by a siRNA.


[052] 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 tape with the 5 '- (w) - ( Z1) - (Z2) - (Z3) na -3 'and a sense chain with the modification pattern 5'- (Z3) ns -, where the substituents, as well as the variables na and ns have the meaning given above. Said conjugate is also referred to as GalNAc palmitoil conjugate. Preferably, said GalNAc palmitoil conjugate is co-administered with a delivery polymer. Such distribution polymers are described above.
[053] These patterns of modification of cleavable ligands have been found to be advantageous in comparison to small molecule ligands stably linked. Possible cleavable linkers are a dipeptide motif exemplified in scheme 1 or cleavable linker RNA comprising 2'-OH containing nucleotides. The cleavable linker RNA is especially useful in connection with siRNAs having the new modification pattern (fully modified 2 'siRNA) described above.
[054] In principle, a nuclease cleavage site can be introduced by 3 'or 5' protrusions containing at least one 2'-OH nucleotide on both the sense and antisense strips. The final active siRNA species is generated by processing intracellular nuclease. In addition, it is possible to use defined cleavage sites implemented by 2'-OH nucleotides in the region of paired bases. This can be done by using at least one 2'-OH nucleotide complementary to the opposite strand, or by introducing at least one incompatible 2'-OH nucleotide or a clamp / protrusion containing at least one 2'-OH nucleotide.
[055] Unlike other cleavable linker chemistries, the use of cleavage sites defined by the introduction of 2'-OH nucleotides leads to a more versatile conjugation approach. With the introduction of selective cleavage sites on one or both strands of siRNA, both at the 3 'and / or 5' end, and within the duplex structure, multiple conjugation is possible.
[056] Consequently, in one embodiment, a conjugate of a small molecule and an oligonucleotide is provided in which: a) the small molecule comprises a linker nucleotide comprising 1 to 10, preferably 1 to 5, more preferably 1 to 3 nucleotides 2 '-OH; b) the oligonucleotide has the following modification pattern: the antisense tape with the modification pattern 5 '- (w) - (Z1) - (Z2) - (Z3) at -3' and a sense tape with the modification pattern 5'- (Z3) ns -, where the substituents, as well as the variables na and ns have the meaning given above. c) the oligonucleotide is covalently linked to the small molecule-binding nucleotide.
[057] The linker nucleotide is cleaved by intracellular nucleases, such as DNAase II after internalization of the conjugate to the endosome, thus releasing the siRNA.
[058] Preferably, said conjugate is co-administered with a delivery polymer. Such distribution polymers are described above.
[059] In another embodiment of the invention, a compound of formula (V) is provided. Such a compound comprises a cholesterol component and a linker nucleotide comprising 1 to 10, preferably 1 to 5, more preferably 1 to 3 2'-OH nucleotides; this linker nucleotide is useful for covalently attaching an oligonucleotide as a siRNA to the compound of formula (V). Preferably, said oligonucleotide has the new pattern of modification described above. Thus, in another embodiment, a conjugate of a compound of formula (V) and an oligonucleotide is provided, wherein the oligonucleotide is covalently linked to the linker nucleotide of the compound of formula (V).

[060] The linker nucleotide is cleaved by intracellular nucleases, such as DNAase II after internalization of the conjugate of a compound of formula (V) and an oligonucleotide in the endosome, thus releasing the siRNA.
[061] Preferably, said conjugate of a compound of formula (V) and an oligonucleotide is co-administered with a delivery polymer. Such distribution polymers are described above.
[062] In another embodiment, said delivery polymer and the conjugate of a compound of formula (V) and an oligonucleotide of the invention are not covalently linked and synthesized separately, and can be supplied in separate containers or in a single container. DEFINITIONS
[063] The term "small molecule" as used in this application refers to organic or inorganic molecules both synthesized and found in nature, generally having a molecular weight of less than 10,000 grams per mole, optionally less than 5,000 grams per mole, and optionally less than 2,000 grams per mol.
[064] The term "peptide" as used in the present application refers to any polymer compound produced by formation of an amide bond 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 in the present application refers to polypeptides of specific sequence of more than about 50 residues.
[065] The term "dipeptide motif" as used in the present application refers to any motif comprising an amide bond formed by any 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.
[066] As used in the present application, the term "amino acid" refers to any molecule that contains both the amine and carboxyl functional groups. Thus, the term "amino acid" refers to both natural, unnatural and synthetic amino acids. Any natural amino acids used in the present invention are cited in the present application by their common abbreviations.
[067] The term "binder" as used in the present application refers to a component that is capable of covalently or otherwise chemically bonding to a biologically active substance. The term "binder" in the context of the invention is preferably a compound of formula (I) or (Ia) covalently linked to a biologically active substance.
[068] The term "biologically active substance" as used in this application refers to an inorganic or organic molecule, including a small molecule, peptide (for example, penetrating peptide cells), protein, carbohydrate (including monosaccharides, oligosaccharides and polysaccharides), nucleoprotein, mucoprotein, lipoprotein, polypeptide or synthetic protein, or a small molecule attached 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, which cause a biological effect when administered in vivo to an animal, which includes, but is not limited to birds and mammals, including humans . Preferably, said biologically active substance is a peptide or nucleic acid. Preferred nucleic acids used in the present application are siRNAs.
[069] The term "nucleic acid" as used in the present application means an oligomer or polymer composed of nucleotides, for example, deoxyribonucleotides or ribonucleotides or synthetically produced compounds (for example, PNA as described in US patent 5,948,902 and references cited therein) that can hybridize to naturally occurring nucleic acids in a specific sequence in a manner analogous to the two naturally occurring nucleic acids, for example, can participate in Watson-Crick base pairing interactions. Non-naturally occurring nucleic acids are oligomers or polymers that contain nucleobase sequences that do not occur in nature, or species that contain functional equivalents of naturally occurring nucleobases, sugars or inter-sugar bonds, such as peptide nucleic acids (PNA), threose nucleic acids (TNA), blocked nucleic acids (LNA) or glycerol nucleic acids (GNA). This term includes oligomers that contain naturally occurring nucleic acid nucleobases, adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U), as well as oligomers that contain base or nucleobase analogs. modified. Nucleic acids can be derived 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 various oligonucleotides that are being manufactured for use as investigative reagents, diagnostic agents or potentials and defined therapeutic agents. The term includes oligomers that comprise a single-stranded nucleic acid or a double-stranded nucleic acid.
[070] The term "2'-modified" as used in the present application refers to a β-D-ribonucleoside or β-D-ribonucleotide that comprises naturally occurring enucleobases that have the 2'-OH group replaced by H, F, O-CH3 or other substituents known in the art.
[071] The term "2'-OH nucleotide" as used in the present application refers to β-D-ribonucleotide which comprises naturally occurring nucleobases having a 2'-OH group.
[072] The term "5'-phosphate" as used in the present application refers to the formula -O-P (= O) (OH) OH. In another aspect, the phosphate is modified so that one of the groups O or OH is replaced by S and designated in the present application as "5'-phosphotioate".
[073] The term "phosphorothioate" as used in this application refers to an internucleotide bond in which one of the non-bridged oxygen atoms is replaced by sulfur.
[074] The term "delivery polymer" as used in the present application refers to polymers suitable for functional delivery of a biologically active substance. In the context of the present invention, the delivery polymer is both covalently linked and co-administered with the biologically conjugated substance to the compounds described in the present application and mediates endosomal leakage after internalization in the cell and absorption in the endosome. The term "polymer" in this context means any compound that is made up of two or more monomeric units covalently linked to each other, where the monomeric units can be the same or different, so that the polymer can be a homopolymer or a heteropolymer. Representative polymers include peptides, polysaccharides, nucleic acids and the like, where the polymers can be naturally occurring or synthetic. Non-limiting examples of distribution polymers are, for example, reviewed in the INTERNATIONAL JOURNAL OF PHARMACEUTICAL RESEARCH AND DEVELOPMENT, October - 2010 / Volume - 2 / Issue - 8 / Article N ° 2. Non-limiting examples of distribution polymers useful for distribution of nucleic acids are disclosed in patent applications EP 10165502.5 and 10191030.5, PCT publication document WO 2008/0022309 and provisional patent application US 61/307490 and references cited in the present application; which are all included for reference.
[075] As used in the present application, "pharmaceutical composition" includes the conjugates of the invention, a pharmaceutical carrier or diluent and any other medium or agent necessary for formulation.
[076] As used in the present application, "pharmaceutical carrier" includes any and all solvents, dispersion medium, coatings, antibacterial and antifungal agents, isotonic and delayed absorption agents and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (for example, by injection or infusion).
[077] A conjugate of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the person skilled in the art, the route and / or mode of administration will vary, depending on the desired results. To administer a conjugate of the invention by certain routes of administration, it may be necessary to coat the conjugate or co-administer the conjugate with a material that prevents its inactivation. For example, the conjugate can be administered to a subject in a suitable carrier or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile and post-sterile aqueous solutions or dispersions for the improvised preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art.
[078] The phrases “parenteral administration” and “parenterally administered” as used in this application mean modes of administration, except enteral and topical administration, usually by injection and include, without limitation, intravenous, intramuscular, intraarterial injection and infusion, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal.
[079] These carriers may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. The prevention of the presence of microorganisms can be ensured both by sterilization procedures, above 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 in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption, such as aluminum monoesterate and gelatin.
[080] Regardless of the route of administration selected, the conjugates of the present invention, which can be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention, are formulated in pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. on the subject.
[081] The current dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied in order to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a specific patient, composition and mode of administration, not being toxic to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors, including the activity of the specific compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the specific compound being employed, the duration of treatment , other drugs, compounds and / or materials used in combination with the specific compositions employed, the age, sex, weight, condition, general health and previous medical history of the patient being treated and similar factors well known in medical techniques.
[082] The pharmaceutical composition must be sterile and fluid as the composition can be dispensed by syringe. In addition to water, the carrier is preferably an isotonic buffered saline solution.
[083] Adequate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using 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. BRIEF DESCRIPTION OF THE FIGURES
[084] Figure 1 shows the co-administration of conjugated siRNAs comprising the compounds of formula (I) or (Ia) and a polymer for in vivo distribution.
[085] Figure 2 shows the co-administration of conjugated siRNAs comprising the compounds of formula (I) or (Ia) and a polymer of in vivo distribution.
[086] Figure 3 shows the co-administration of conjugated siRNAs comprising the compounds of formula (I) or (Ia) and a polymer for in vivo distribution.
[087] Figure 4 shows the co-administration of conjugated siRNAs comprising the compounds of formula (I) or (Ia) and a polymer for in vivo distribution.
[088] Figure 5a shows the antisense tape mediated by gene silencing with fully modified 2 'siRNAs. COS 7 cells were cotransfected with EGFP-directed to siRNAs at 3 nM and psiCHECK2-AT. The knockdown activity of siRNAs was assessed by measuring renilla activity against firefly luciferase from the reporter construct. SiRNAs were classified by knockdown activity of unmodified reference RNAs (2-19-2).
[089] Figure 5b shows the sense strand mediated by gene silencing with fully modified 2 'siRNAs. COS 7 cells were cotransfected with EGFP-directed to siRNAs at 3 nM and psiCHECK2-ST. The knockdown activity of siRNAs was assessed by measuring luciferase expression from the reporter construct. SiRNAs were classified by knockdown activity of unmodified reference RNAs (2-19-2).
[090] Figure 6a shows the reduction of FVII serum activity in non-human primates, through intravenous injection of several 2 'modified siRNAs covalently linked to a distribution polymer.
[091] Figure 6b shows the development of prothrombin time in non-human primates, by treatment with covalently modified 2 'siRNAs conjugated to a delivery polymer.
[092] The invention will be better understood by reference to the following examples. However, they should not be considered as limiting the scope of the invention. EXAMPLES EXAMPLE 1
[093] Step 1: 3 - [(3S, 8S, 9S, 10R, 13R, 14S, 17R) -17 - ((R) -1,5- Dimethylhexyl) -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

[094] The amine titer was prepared from its nitrile precursor according to a literature protocol [Lollo et al., WO2001 / 070415].
[095] 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

[096] In a 2 L rounded-bottom flask, 3- ((3S, 8S, 9S, 10R, 13R, 14S, 17R) -10,13-dimethyl-17 - ((R) -6-methylheptan-2 -il) - 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta [a] phenantren-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 room temperature overnight => white suspension. All volatiles have been removed i. v., the residue dissolved in CH2Cl2, the organic layer washed with NH4Cl and brine, dried with Na2SO4, and evaporated to dryness. The crude product was dissolved in CH3CN / H2O and lyophilized to produce 29.8 g of the compound's title as a soft powder.
[097] MS (ISP): (M-H) 542.5.
[098] 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

[099] In a 10 mL round-bottom flask, the above preparation 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] phenantren-3- yloxy) propylamino) -4-oxobutanoic acid (106 mg, 184 μmol, Eq: 1.00), (S) -2 - ((S) -2-amino-3- (4-nitrophenyl) propanamide) -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 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 room temperature overnight. TLC indicated the consumption of starting material. All volatiles have been removed i. V. and the crude product purified by flash chromatography SiO2 / MeOH 7% / NEt3 0.1% in CH2Cl2 to yield 128 mg of the title of the compound as a light yellow solid.
[0100] MS: expected mass: 1240,7552, mass found: 1240,7518.
[0101] Step 4:

[0102] In a 10 ml round bottom flask, the above preparation 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] phenantren-3- yloxy) propyl) -N4 - ((S) -1 - ((S) -1- (4- (hydroxymethyl) phenylamine) -6 - ((4-methoxyphenyl) diphenylmethylamine) -1-oxohexan-2-ylamine) - 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 proceeded overnight. The mixture was poured over crushed ice, extracted 2x with AcOEt, washed with H2O, dried with Na2SO4 and evaporated to dryness. After trituration with approximately 10 ml of diethyl ether, 99 mg of the product title was obtained as an off-white solid.
[0103] MS: expected mass: 1405.7614, found mass: 1405.7518.
[0104] The dipeptide building block required 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

[0105] 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) were 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.
[0106] In a second 50 ml round-bottom 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 mixture reaction was stirred for 20 '. The solution of the first flask containing the corresponding monosilylamine silyl ester was added and the reaction was stirred for another 3 hours. The mixture was poured over crushed ice / NH4Cl, extracted 2x with AcOEt, washed with H2O and brine, dried with Na2SO4 and evaporated to dryness. Flash chromatography of SiO2 / MeOH 10% / NEt3 0.1% in CH2Cl2 produced 1.38 g of the compound's title as brownish foam.
[0107] MS (ISP): (M + H) 833.5, (M + Na) 855.4.
[0108] 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

[0109] In a 250 mL pear-shaped bottle, the one synthesized above (S) -2 - ((S) -2 - ((((9H-fluoren-9-yl) methoxy) carbonylamino) -3- (4 - nitrophenyl) propanamide) -6 - ((4-methoxyphenyl) diphenylmethylamino) 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 proceeded overnight. The mixture was poured over crushed ice / NH4Cl (pH approximately 7), extracted 2x with AcOEt, washed with H2O, dried with Na2SO4 and evaporated to dryness. The crude product was triturated with diethyl ether (1 x 50 ml); the resulting solid was filtered and dried to yield 1.214 g of the compound's title as a light brown solid.
[0110] MS (ISP): (M + H) 938.7.
[0111] 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

[0112] In a 50 ml round bottom flask, the above preparation [(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 room temperature for 3 h, when the MS indicated the disappearance of the starting material. All volatiles were evaporated i. V .; resulting from SiO2 / NEt3 flash chromatography 0.1% in CH2Cl2 => MeOH10% / NEt3 0.1% in CH2Cl2, followed by a second flash chromatography of SiO2 / MeOH5% / NEt3 0.1% in CH2Cl2 produced 502 mg of compound title as light brown foam.
[0113] MS: expected mass: 715.337, mass found: 715.3362. EXAMPLE 2
[0114] O-Benzyl-N- [4 - ({3 - [(3beta) -colest-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 with use 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 a coupling partner. The first was prepared from (S) -2 - ((((9H-fluoren-9-yl) methoxy) carbonylamino) - 3- (4- (benzyloxy) phenyl) propanoic acid as described above in steps a] - c ].
[0115] MS: expected mass: 1466.8182, found mass: 1466.8136. EXAMPLE 3
[0116] N- [4 - ({3 - [(3Beta) -colest-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 with use 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 a coupling partner. The first was prepared from (S) -2 - ((((9H-fluoren-9-yl) methoxy) carbonylamino) - 3- (4-cyanophenyl) propanoic acid as described above in steps a] - c].
[0117] MS: expected mass: 1385.7716, mass found: 1385.7696. EXAMPLE 4
[0118] 3,4-Dichloro-N- [4 - ({3 - [(3beta) -colest-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 with use 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 first was prepared from (S) -2 - ((((9H-fluoren-9-yl) methoxy) carbonylamino) - 3- (3,4-dichlorophenyl) propanoic acid as described above in steps a] - c] .
[0119] MS: expected mass: 1428.6984, mass found: 1428.695. EXAMPLE 5
[0120] 4-Chloro-N- [4 - ({3 - [(3beta) -colest-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 with use 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) diphenylmethylamino) hexanamide as coupling partner. The first was prepared from (S) -2 - ((((9H-fluoren-9-yl) methoxy) carbonylamino) -3- (4-chlorophenyl) propanoic acid as described above in steps a] - c].
[0121] MS: expected mass: 1394.7373, mass found: 1394.7342. EXAMPLE 6
[0122] 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] phenantren-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 a coupling partner. The first was prepared from (S) -2 - ((((9H-fluoren-9-yl) methoxy) carbonylamino) -3- (naphthalen-1-yl) propanoic acid as described above in steps a] - c] .
[0123] MS: expected mass: 1410.792, found mass: 1410.7918. EXAMPLE 7
[0124] N- [4 - ({3 - [(3Beta) -colest-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 first was prepared from (S) -2 - ((((9H-fluoren-9-yl) methoxy) carbonylamino) - 3- (4-fluorophenyl) propanoic acid as described above in steps a] - c].
[0125] MS: expected mass: 1378.7669, mass found: 1378.7609. EXAMPLE 8
[0126] N- [4 - ({3 - [(3Beta) -colest-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 a coupling partner. The first was prepared from (S) -2 - ((((9H-fluoren-9-yl) methoxy) carbonylamino) - 3- (2-fluorophenyl) propanoic acid as described above in steps a] - c].
[0127] MS: expected mass: 1378.7669, mass found: 1378.7689. EXAMPLE 9
[0128] N- [4 - ({3 - [(3Beta) -colest-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 with use 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 first was prepared from (S) -2 - ((((9H-fluoren-9-yl) methoxy) carbonylamino) - 3- (3-fluorophenyl) propanoic acid as described above in steps a] - c].
[0129] MS: expected mass: 1378.7669, found mass: 1378.7659. EXAMPLE 10
[0130] 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

[0131] In a 10 mL round bottom flask, the above preparation 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] phenantren-3- yloxy) propylamine) -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 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 room temperature overnight. TLC indicated the consumption of starting material. All volatiles have been removed i. V. and the crude product purified by flash chromatography SiO2 / MeOH 5% / NEt3 0.1% in CH2Cl2 to yield 197 mg of the compound title as an off-white solid.
[0132] MS: expected mass: 1227.7763, mass found: 1227.7714.
[0133] 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] phenantren- 3-yloxy) propylamino) -4-oxobutanamido) -4- (4-fluorophenyl) butanamido) -6 - ((4-methoxyphenyl) diphenylmethylamino) hexanamido) -benzyl 4-nitrophenyl carbonate

[0134] In a 10 mL round-bottom flask, the above preparation 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] phenantren-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 CH2 Cl2 (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 proceeded at room temperature overnight. The mixture was poured over crushed ice / NH4Cl (pH approximately 6), extracted 2x with AcOEt, washed with H2O and brine, dried with Na2SO4 and evaporated to dryness. After trituration with AcOEt / heptane, 123 mg of the title of the compound was obtained as a light yellow solid.
[0135] MS: expected mass: 1392.7825, found mass: 1392.7819.
[0136] The dipeptide building block required for step 1 was prepared as follows:
[0137] Step a: (S) -2 - [(S) -3- (9H-Fluoren-9-ylmethoxycarbonylamino) - 4- (4-fluoro-phenyl) -butyrylamino] -6 - {[(4-methoxy -phenyl) -diphenyl-methyl] -amino} -hexanoic acid

[0138] In a 25 mL round-bottomed flask, (S) -2-amino-6 - ((4-methoxyphenyl) diphenylmethyl-amino) hexanoic acid (Bioconjugate Chem. 2002, 13, 855-869, 1,040 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 room temperature for 20 min.
[0139] In a second 50 ml round-bottomed vial, (S) -3 - ((((9H-fluoren-9-yl) methoxy) carbonyl-amino) -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 mixture reaction was stirred for 15 min. The solution of the first flask containing the corresponding monosilylamine silyl ester was added and the reaction was stirred for another 3 hours at room temperature. The mixture was poured over crushed ice / NH4Cl, extracted 2x with AcOEt, washed with H2O and brine, dried with Na2SO4 and evaporated to dryness. Flash chromatography of SiO2 / MeOH 5% / NEt3 0.1% in CH2Cl2 produced 2.10 g of the title of the compound as yellow foam. MS (ISP): (M + H) 820.6.
[0140] 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

[0141] In a 250 mL pear-shaped bottle, the one synthesized above {(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 CH2 Cl2 (12.5 mL). Huenig's base (662 mg, 871 μL, 5.21 mmol, Eq: 2.00) was added and the reaction proceeded overnight. The mixture was poured over crushed ice / NH4Cl (pH approximately 7), extracted 2x with AcOEt, washed with H2O and brine, dried with Na2SO4 and evaporated to dryness. The crude product was triturated with diethyl ether (1 x 50 ml); the resulting solid was filtered and dried to yield 0.796 g of the compound title as a light brown solid. MS (ISP): (M + H) 925.6.
[0142] 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

[0143] In a 50 ml round bottom flask, the above preparation {(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 μmol, 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 room temperature overnight. The mixture was poured over crushed ice / NH4Cl (pH approximately 7), extracted 2x with AcOEt, washed with H2O and brine, dried with Na2SO4 and evaporated to dryness. Flash chromatography of SiO2 / 10% MeOH / 0.1% NEt3 in CH2Cl2 produced 500 mg of the compound title as an off-white solid.
[0144] MS: expected mass: 702.3581, found mass: 702.3578. EXAMPLE 11
[0145] 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] phenantren-3-yloxy) propylamino) -4-oxobutanamido) -4-phenylbutanamido) -6 - ((4-methoxyphenyl) diphenylmethylamino) hexanamido) benzyl 4-nitrophenyl carbonate
was prepared in analogy to Example 10, but with use 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 a coupling partner. The first was prepared from (S) -3 - ((((9H-fluoren-9-yl) methoxy) carbonylamino) -4-phenylbutanoic acid as described above in steps a] - c].
[0146] MS: expected mass: 1374.792, found mass: 1374.7877. EXAMPLE 12
[0147] 4 - ({N ~ 2 ~ - [(3S) -4- (4-chlorophenyl) -3 - {[4 - ({3 - [(3beta) -colest-5- en-3-yloxy]] propyl} amino) -4-oxobutanoyl] amino} butanoyl] -N ~ 6 ~ - [(4-methoxyphenyl) (diphenyl) methyl] -L-lysyl} amino) benzyl 4-nitrophenyl carbonate
NO2 was prepared in analogy to Example 10, but with use 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 a coupling partner. The first was prepared from (S) -3 - ((((9H-fluoren-9-yl) methoxy) carbonylamino) -4- (4-chlorophenyl) -butanoic acid as described above in steps a] - c].
[0148] MS (ISP): (M + H) 1409.9. EXAMPLE 13
[0149] N- [4 - ({3 - [(3Beta) -colest-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 with use 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) diphenylmethylamino) hexanamide as a coupling partner. The first was prepared from (S) -2 - ((((9H-fluoren-9-yl) methoxy) carbonylamino) -3- (4-methoxyphenyl) propanoic acid as described above in steps a] - c] of the example 1.
[0150] MS (ISP): (M + H) 1391.9. EXAMPLE 14
[0151] N- [4 - ({3 - [(3Beta) -colest-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 with use in step 3 (R) -2 - ((R) -2-amino-3-phenyl-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) diphenylmethylamino) 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].
[0152] MS: expected mass: 1360.7763, mass found: 1360.7774. EXAMPLE 15
[0153] 4 - ({N ~ 2 ~ - [(3S) -3 - {[4 - ({3 - [(3Beta) -colest-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 carbonate
was prepared in analogy to example 10, but with use 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 a coupling partner. The first was prepared from (S) -3 - ((((9H-fluoren-9-yl) methoxy) carbonylamino) - 4- (4-cyanophenyl) butanoic acid as described above in steps a] - c].
[0154] MS: expected mass: 1399.7872, mass found: 1399.7857. EXAMPLE 16
[0155] N- [4 - ({3 - [(3beta) -colest-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

[0156] Step 1: (S) -2 - ((S) -2-Amino-3-phenyl-propionylamino) -6 - {[(4-methoxy-phenyl) -diphenyl-methyl] -amino} -hexanoic acid (4-hydroxymethyl-phenyl) -amide

[0157] The (S) -2 - ((S) -2-amino-3-phenyl-propionylamino) -6 - {[(4-methoxy-phenyl) -diphenyl-methyl] -amino} -acid building block hexanoic (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
[0158] 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) phenylamine) -6 - (((4-methoxyphenyl) diphenylmethylamino) -1-oxohexan-2-ylamino) -1-oxo-3-phenylpropan-2- il) succinamide

[0159] 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-dimethylhexyl) -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) TLC was added at t = 1 h showed that 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 of the sat. (1 X), 0.05M potassium hydrogen phthalate solution (2 X), water (1 X) and brine (1 X). The organic extract was dried with MgSO4 and concentrated under reduced pressure. The crude material was purified by chromatography to obtain the title product (682 mg, 513, μmol) as a light brown solid. MS (ISP): (M + H) 1196.8
[0160] Step 3: Hunig's base (465 mg, 629 ul, 3.6 mmol, eq: 6) was added to a solution of the previous alcohol (718 mg, 600 umol, 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 of the compound (800 mg, 529 μmol) as a light brown solid. MS (ISP): (M + H) 1361.9 EXAMPLE 17
[0161] Step 1: (S) -2 - [(S) -2- (9H-Fluoren-9-ylmethoxycarbonylamino) - 3-phenyl-propionylamino] -hexanoic acid

[0162] Commercially available as 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 0oC 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 proceeded at room temperature for 14 h. The mixture was poured over crushed ice / citric acid (pH approximately 3), extracted 2x with ethyl acetate, washed with H2O and brine, dried with Na2SO4 and evaporated to dryness. Flash chromatography of SiO2 / AcOEt produced 0.870 mg of the title of the compound as a white solid. MS (ISP): (M + H) 501.2.
[0163] Step 2: {(S) -1 - [(S) -1- (4-Hydroxymethyl-phenylcarbamoyl) - pentylcarbamoyl] -2-phenyl-ethyl} -carbamic acid 9H-fluoren-9-ylmethyl ester

[0164] In a pear-shaped bottle, the one synthesized above (S) - 2 - [(S) -2- (9H-fluoren-9-ylmethoxy-carbonylamino) -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 an Air flask. The mixture was poured over crushed ice / NH4Cl, extracted 2x with AcOEt, washed with H2O, dried with Na2SO4 and the volume reduced to 300 ml. The precipitate was filtered and dried to give 5.25 g of the title of the compound as a light brown solid. MS (ISP): (M + H) 606.3.
[0165] Step 3: (S) -2 - ((S) -2-Amino-3-phenyl-propionylamino) -hexanoic acid (4-hydroxymethyl-phenyl) -amide

[0166] In a round-bottomed flask, the above preparation {(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 room temperature overnight. All volatiles were evaporated i. V .; resulting from 10% SiO2 / CH2Cl2 / MeOH chromatography, followed by crystallization from AcOEt, produced 2.116 g of the compound title as light brown crystals. MS (ISP): (M + H) 384.2.
[0167] 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 with the same in analogy to example 16 step 2 MS (ISP): (M + H) 909.7 (M + Na) 931.8.
[0168] Step 5: N- [4 - ({3 - [(3beta) -colest-5-en-3-yloxy] propyl} amino) - 4-oxobutanoyl] -L-phenylalanyl-N- [4- ( {[(4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] -L- norleucinamide
was prepared with the same in analogy to example 16 step 3 MS expected mass: 1073.6453, mass found 1073.642 EXAMPLE 18
[0169] N- [4 - ({3 - [(3beta) -colest-5-en-3-yloxy] propyl} amino) -4-oxobutanoyl] -L-alanyl-N- [4 - ({[( 4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] glycinamide

[0170] Step 1: Addition of FMOC-4-Aminobenzyl alcohol to the 2-chlorotritil resin
2-Chlorotritil chloride resin (Novabiochem 01-64-0114, 100 to 200 mesh), 1% DVB (18 g, 21.6 mmol, Eq: 1.00) was expanded 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 stirred overnight. The resin was drained and covered with a 10% solution of Hünig's Base in methanol (300 mL). The resin was washed with DMF and DCM and dried overnight with HV to obtain 21.7 g of resin. The load determination resulted in 0.41 mmoL / g.
[0171] 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

[0172] The resin from step 1 (1 g, 410 μmol, Eq: 1.00) was pre-washed with DMF (2X) and treated with piperidine / DMF = 1/4 (10 mL) for 5 and 10 minutes. The resin was washed alternately with DMF and IPA (3 X 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 stirred with the resin for one hour. The resin was washed alternately with DMF and isopropyl alcohol (3X).
[0173] The following Fmoc cleavages and subsequent Fmoc-Ala-OH couplings (511 mg, 1.64 mmol, Eq: 4) and N- {3- [(3S, 8S, 9S, 10R, 13R, 14S, 17R) -17 - ((R) -1,5-Dimethylhexyl) -10,13-dimethyl- 2,3,4,7,8,9,10,11,12,13,14,15,16 , 17-tetradecahydro-1H-cyclopenta [a] phenanthrene-3-yloxy] -propyl} -succinamic acid (example 1, step 2) (892 mg, 1.64 mmol, Eq: 4) were consequently performed. The dry peptide resin was stirred for about 2 X 30 min in 1% TFA / DCM (2 X 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 (2x). After purification by flash chromatography, the product (84 mg, 97.3 μmol) was obtained as a white solid. Expected mass MS: 776.5452, mass found 776.5455
[0174] Step 3: The alcohol prepared above N1- (3- ((3S, 8S, 9S, 10R, 13R, 14S, 17R) -10,13-dimethyl-17 - ((R) -6-methylheptan-2 -il) - 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta [a] phenantren- 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 left to react overnight. The solvent was removed in vacuo. The resulting solid was triturated with diethyl ether. The solid was collected by filtration and washed with diethyl ether. The product was vacuum dried to obtain the title of the compound (84 mg, 80.2 μmol) as a light brown solid.
[0175] MS expected mass: 941.5514, mass found 941.5518 EXAMPLE 19
[0176] N- [4 - ({3 - [(3beta) -colest-5-en-3-yloxy] propyl} amino) -4-oxobutanoyl] -L-leucyl-N- [4 - ({[( 4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] -L- methioninamide

[0177] Step 1: Addition of FMOC-4-aminobenzyl alcohol to the 2-chlorotritil resin
was prepared in analogy to example 18, step 1
[0178] 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.
[0179] The product (208 mg, 210 μmol) was obtained as a light yellow solid. MS (ISP): (M + H) 893.6183
[0180] Step 3: was prepared in analogy to example 18, step 3. After purification on silica gel, the title of the compound (161 mg, 137 μmol) was obtained as a light brown solid.
[0181] MS expected mass: 1057.6174, mass found 1057.6184 EXAMPLE 20
[0182] N- [4 - ({3 - [(3beta) -colest-5-en-3-yloxy] propyl} amino) -4-oxobutanoyl] -L-leucyl-N ~ 1 ~ - [4- ( {[(4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] -L- aspartamide

[0183] STEP 1: Addition of FMOC-4-aminobenzyl alcohol to the 2-chlorotritil resin
was carried out in analogy to example 18, step 1
[0184] 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] phenantren-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.
[0185] The product (87 mg, 89.4 μmol) was obtained as a light yellow solid.
[0186] MS expected mass: 875.6136, mass found 875.6133
[0187] Step 3: The title of the compound was prepared in analogy to example 18, step 3. After purification on silica gel (87 mg, 89.4 μmol) the title of the compound was obtained as a light brown solid.
[0188] MS expected mass: 1040.6198, mass found 1040.6188 EXAMPLE 21
[0189] N- [4 - ({3 - [(3beta) -colest-5-en-3-yloxy] propyl} amino) -4-oxobutanoyl] -L-alanyl-N ~ 1 ~ - [4- ( {[(4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] -L- aspartamide

[0190] Step 1: Addition of FMOC-4-aminobenzyl alcohol to the 2-chlorotritil resin
was carried out in analogy to example 18, step 1
[0191] 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] phenantren-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.
[0192] The product (140 mg, 159 μmol) was obtained as a light yellow solid. MS (ISP): (M + H) 834.8 (M + Na) 856.7
[0193] Step 3: The title of the compound was prepared in analogy to example 18, step 3. After purification on silica gel (169 mg, 152 μmol) it was obtained as a light brown solid.
[0194] MS expected mass: 998.5729, mass found 998.5739 EXAMPLE 22
[0195] N ~ 2 ~ - [4 - ({3 - [(3beta) -colest-5-en-3-yloxy] propyl} amino) -4-oxobutanoyl] -L-asparaginyl-N- [4- ( {[(4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] glycinamide

[0196] Step 1: Addition of FMOC-4-Aminobenzyl alcohol to the 2-chlorotritil resin
was carried out in analogy to example 18, step 1
[0197] 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] phenantren-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.
[0198] The product (140 mg, 162 μmol) was obtained as a white solid.
[0199] MS expected mass: 819.551, mass found 819.5503
[0200] Step 3: The title of the compound was obtained in analogy to example 18, step 3 (176 mg, 161 μmol) as a light brown solid.
[0201] MS expected mass: 984.5572, mass found 984.5489 EXAMPLE 23
[0202] N- [4 - ({3 - [(3beta) -colest-5-en-3-yloxy] propyl} amino) -4-oxobutanoyl] -L-phenylalanyl-N- [4 - ({[( 4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] glycinamide

[0203] Step 1: Addition of FMOC-4-aminobenzyl alcohol to the 2-chlorotritil resin
was performed in analogy to example 18, step 1
[0204] 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.
[0205] The product (259 mg, 288 μmol) was obtained as a white solid.
[0206] MS expected mass: 852.5765, mass found 852.5754
[0207] Step 3: The title of the compound was obtained in analogy to example 18, step 3. (280 mg, 247 μmol) as a light brown solid.
[0208] MS expected mass: 1017.5827, mass found 1017.5775 EXAMPLE 24
[0209] N- [4 - ({3 - [(3beta) -colest-5-en-3-yloxy] propyl} amino) -4-oxobutanoyl] -L-leucyl-N- [4 - ({[( 4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] glycinamide

[0210] Step 1: Addition of FMOC-4-aminobenzyl alcohol to the 2-chlorotritil resin
was carried out in analogy to example 18, step 1
[0211] 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.
[0212] The product (240 mg, 278 μmol) was obtained as a light yellow solid.
[0213] MS expected mass: 818.5921, mass found 818.5921
[0214] Step 3: The title of the compound was prepared in analogy to example 18, step 3. After purification on silica gel, (194 mg, 177 μmol) was obtained as a light yellow solid.
[0215] MS expected mass: 983.5983, mass found 983.6004 EXAMPLE 25
[0216] N- [4 - ({3 - [(3beta) -colest-5-en-3-yloxy] propyl} amino) -4-oxobutanoyl] -L-leucyl-N- [4 - ({[( 4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] -L- phenylalaninamide

[0217] Step 1: Addition of FMOC-4-aminobenzyl alcohol to the 2-chlorotritil resin
was carried out in analogy to example 18, step 1
[0218] 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.
[0219] The product (153 mg, 151 μmol) was obtained as a light yellow solid.
[0220] MS expected mass: 908.6391, mass found 908.637
[0221] Step 3: The title of the compound was prepared in analogy to example 18, step 3. After purification on silica gel, (117 mg, 98 μmol) was obtained as a white solid.
[0222] MS expected mass: 1073.6453, mass found 1073.646 EXAMPLE 26
[0223] N- [4 - ({3 - [(3beta) -colest-5-en-3-yloxy] propyl} amino) -4-oxobutanoyl] -L-phenylalanyl-N- [4 - ({[( 4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] -L- phenylalaninamide

[0224] Step 1: Addition of FMOC-4-aminobenzyl alcohol to the 2-chlorotritil resin
was performed in analogy to example 18, step 1
[0225] 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 the amino acid.
[0226] The product (240 mg, 204 μmol) was obtained as a light yellow solid.
[0227] MS expected mass: 942.6234, mass found 942.6218
[0228] Step 3: The title of the compound was prepared analogously to example 18, step 3. After purification on silica gel, (190 mg, 154 μmol) was obtained as a white solid.
[0229] MS expected mass: 1107.6296, mass found 1107.6287 EXAMPLE 27
[0230] N- [4 - ({3 - [(3beta) -colest-5-en-3-yloxy] propyl} amino) -4-oxobutanoyl] -L-leucyl-N- [4 - ({[( 4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] -L-leucinamide

[0231] Step 1: Addition of FMOC-4-aminobenzyl alcohol to the 2-chlorotritil resin
was performed analogously to example 18, step 1
[0232] 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 the amino acid.
[0233] The product (254 mg, 284 μmol) was obtained as a white solid
[0234] MS expected mass: 874.6547, mass found 874.6527
[0235] Step 3: The title of the compound was prepared in analogy to example 18, step 3. After purification on silica gel it was obtained as a white solid (178 mg, 168 μmol).
[0236] MS expected mass: 1039.6609, mass found 1039.6588 EXAMPLE 28
[0237] N- [4 - ({3 - [(3beta) -colest-5-en-3-yloxy] propyl} amino) -4-oxobutanoyl] -L-alanyl-N- [4 - ({[( 4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] -L-alaninamide

[0238] Step 1: {(S) -1 - [(S) -1- (4-Hydroxymethyl-phenylcarbamoyl) - ethylcarbamoyl] -ethyl} -carbamic acid 9H-fluoren-9-ylmethyl ester

[0239] 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 overnight at room temperature. The mixture was diluted with 2-propanol / 10% ethyl acetate (100 ml) and the solution was washed with 5% KHSO4 / 10% K2SO4 (2X), water (1X) and brine (1X), dried with MgSO4 and evaporated vacuum. 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 a light brown solid. MS (ISP): (M + H) 488.3
[0240] Step 2: (S) -2-Amino-N - [(S) -1- (4-hydroxymethyl-phenylcarbamoyl) -ethyl] -propionamide

[0241] The compound was prepared in analogy to the example, 1 step c to obtain the product (245 mg, 877 μmol) as a light yellow solid. MS (ISP): (M + H) 266.3, (M + Na) 288.2 (2M + H) 531.3
[0242] 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

[0243] The compound was prepared in analogy to example 16, step 2 (165 mg, 198 μmol) as a light brown solid. Expected mass MS: 790.5608, mass found 790.5587
[0244] Step 4: The title of the compound was prepared in analogy to example 18, step 3. After purification on silica gel it was obtained as a white solid (99 mg, 98.4 μmol).
[0245] MS expected mass: 955.567, mass found 955.5651 EXAMPLE 29
[0246] N- [4 - ({3 - [(3beta) -colest-5-en-3-yloxy] propyl} amino) -4-oxobutanoyl] -L-isoleucyl-N ~ 1 ~ - [4- ( {[(4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] -L- aspartamide

[0247] Step 1: (S) -2 - [(2S, 3S) -2- (9H-Fluoren-9-ylmethoxycarbonylamino) -3-methyl-pentanoylamino] -succinamic acid

[0248] The 2-chlorotrityl chloride resin (5 g, 7.5 mmol, Eq: 1.00) was expanded in DCM and then treated with a Fmoc-Asn (Trt) -OH solution (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 covered with a 10% Huenig Base solution 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 vacuo to obtain the product (2.85 g, 5.79 mmol) as a white solid.
[0249] MS expected mass: 467.2056, mass found 467.2056
[0250] Step 2: {(1S, 2S) -1- [2-Carbamoyl-1 - ((S) -4-hydroxymethyl-phenylcarbamoyl) -ethylcarbamoyl] -2-methyl-butyl} -carbamic acid 9H-fluoren 9-ylmethyl ester

[0251] The compound was prepared in analogy to example 28, step 1 (620 mg, 336 μmol) as a light yellow solid.
[0252] Step 3: (S) -2 - ((2S, 3S) -2-Amino-3-methyl-pentanoylamino) - N * 1 * - (4-hydroxymethyl-phenyl) -succinamide

[0253] The compound was prepared in analogy to example 1, step c (100 mg, 228 μmol) as a light yellow solid.
[0254] 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] phenantren- 3-yloxy) propylamino) -4-oxobutanamido) -3-methylpentanamido) -N1- (4- (hydroxymethyl) phenyl) succinamide

[0255] The compound was prepared in analogy to example 16, step 2 (89 mg, 91.4 μmol) as a light yellow solid.
[0256] Step 5: The compound from the previous step was reacted with the title of the compound analogously to example 18, step 3. After purification on silica gel, (42 mg, 36.3 μmol) was obtained as a light brown solid.
[0257] MS expected mass: 1040.6198, mass found 1040.6177 EXAMPLE 30
[0258] N- [4 - ({3 - [(3beta) -colest-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

[0259] The compound was prepared in analogy to example 16, step 1 starting with Fmoc-D-Lys (Boc) -OH, (158 mg, 116 μmol) as a light brown solid.
[0260] MS (ISP): (M + H) 1362.8 (M + Na) 1383.8 EXAMPLE 31
[0261] N- {15 - [(3beta) -colest-5-en-3-yloxy] -4,15-dioxo-8,11-dioxa- 5,14-diazapentadecan-1-oil} -L-phenylalanyl -N ~ 6 ~ - [(4-methoxyphenyl) (diphenyl) methyl] -N- [4 - ({[(4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] -L-lysinamide

[0262] The title of the compound was prepared analogous to example 16 with the use of a cholesterol-oligo-PEG derivative in step 2 of the synthesis. MS (ISP): (M + H) 1479.8
[0263] Cholesterol-PEG required 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] phenantren- 3-yloxycarbonylamino ] -ethoxy} -ethoxy) -ethyl] -succinamic acid for step 2 was prepared as follows:
[0264] 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] phenantren- 3-yl ester

[0265] A solution of cholesteryl chloroformate (1 g, 2.23 mmol) in 25 mL of dichloromethane was added dropwise, with stirring, to a solution of 2.2 '- (ethylenedioxy) bis- (ethylamine) (495 mg, 3.34 mmol) in 75 ml of dichloromethane. The reaction was stirred overnight at room temperature. The reaction was diluted with dichloromethane and extracted with water. The organic extract was dried with 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
[0266] Step b: N- [2- (2- {2 - [(3S, 8S, 9S, 10R, 13R, 14S, 17R) -17 - ((R) - 1,5-Dimethylhexyl) - 10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta [a] phenanthren-3-yloxycarbonylamino] -etoxy } -ethoxy) -ethyl] - succinamic acid

[0267] The amine from step a (480 mg, 0.856 mmol) and triethylamine (0.13 mL, 0.94 mmol) were dissolved in 5 mL of dichloromethane. After adding succinic anhydride (90 mg, 0.9 mmol), the solution was stirred overnight at room temperature. The TLC check also showed 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 mixture of 5% KHSO4 / 10% K2SO4. The organic extract was dried with 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
[0268] N- {30 - [(3beta) -colest-5-en-3-yloxy] -4,30-dioxo- 8,11,14,17,20,23,26-heptaoxa-5,29- diasatriacontan-1-oil} -L-phenylalanyl-N ~ 6 ~ - [(4-methoxyphenyl) (diphenyl) methyl] -N- [4 - ({[(4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] - L- lysinamide

[0269] The title of the compound was prepared analogous to example 16 with the use of a cholesterol-PEG derivative in step 2 of the synthesis. MS (ISP): (M + H) 1699.9
[0270] The required intermediate cholesterol-PEG 1 - [(3beta) - colest-5-en-3-yloxy] -1,27-dioxo-5,8,11,14,17,20,23-heptaoxa-2 , 26- diasatriacontan-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] phenantren-3-yl} oxy) -25-oxo-3,6,9,12,15,18,21- heptaoxa-24-asapentacos-1-yl] carbamate Cholesteryl chloroform (476 mg, 1.06 mmol) and triethylamine (155 µL, 1.113 mmol) were dissolved in 5 mL of dichloromethane. Then, a solution of alpha-amino-omega-boc-amino-octa (ethylene glycol) (497 mg, 1.06 mmol) dissolved in 1 ml of dichloromethane was added. The solution was stirred overnight at room temperature and diluted with dichloromethane and extracted with an aqueous mixture of 5% KHSO4 / 10% K2SO4. 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
[0271] Step b: 1 - [(3beta) -colest-5-en-3-yloxy] -1,27-dioxo- 5,8,11,14,17,20,23-heptaoxa-2,26- diasatriacontan-30-oic acid

[0272] The previous Boc derivative (450 mg, 0.511 mmol) was dissolved in 4M HCl 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 of dichloromethane and treated with triethylamine (32 µL, 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 5% KHSO4 / 10% K2SO4 buffer. The organic extract was dried with anhydrous MgSO4, filtered and evaporated to obtain 390 mg of the desired product.
[0273] MS (ISP): (M + H) 881.7 EXAMPLE 33
[0274] N- {66 - [(3beta) -colest-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- diasahexahexacontan-1-oil} -L-phenylalanyl-N ~ 6 ~ - [(4-methoxyphenyl) (diphenyl) methyl ] -N- [4 - ({[(4-nitrophenoxy) carbonyl] oxy} methyl) phenyl] -L-lysinamide

[0275] The title of the compound was prepared analogous to example 16 with the use of a cholesterol-PEG derivative in step 2 of the synthesis. MS (ISP): (M + H) 2228.1
[0276] The required intermediate cholesterol-PEG 1 - [(3beta) -colest-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-diasahexahexacontan-66-oic acid for step 2 was prepared as follows:
[0277] Step a: (3beta) -colest-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

[0278] Alpha, omega-bis-amino-20 (ethylene glycol) (538 mg, 0.6 mmol) and triethylamine (92 µL, 0.66 mmol) were dissolved in 15 mL of dry dichloromethane. A solution of cholesteryl chloroformate (270 mg, 0.6 mmol) in 2 ml of dry dichloromethane was added by dropping 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
[0279] Step b: 1 - [(3beta) -colest-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- diasahexahexacontan-66-oic acid

[0280] The amine from step a (329 mg, 0.251 mmol), succinic anhydride (26.4 mg, 0.264 mmol) and triethylamine (40 µL, 0.286 mmol) were dissolved in 5 ml of dry dichloromethane. After adding more triethylamine (40 µL, 0.286 mmol), the solution (pH> 8) was stirred overnight at room temperature. The reaction was diluted with dichloromethane and washed twice with an aqueous mixture of 5% KHSO4 / 10% K2SO4. The organic extract was dried with anhydrous MgSO4, filtered and evaporated to obtain the product (260 mg, 0.175 mmol) as a colorless waxy solid.
[0281] MS (ISP): (M + NH4) 1408.9 EXAMPLE 34 GENERAL PROCEDURE FOR THE PREPARATION OF MATERIAL RNA CONJUGATES
[0282] Dimethyl sulfoxide solution (DMSO), N, N-Diisopropylethylamine (DIPEA) and sodium acetate (3 M, pH 5.2) were purchased from Sigma Aldrich Chemie GmbH (Traufkirchen, Germany).
[0283] Triethylammonium acetate (TEAA) (2.0 M, pH 7.0) and Acetonitrile (ACN, HPLC grade) for RP-HPLC were purchased from Biosolve (Valkenswaard, Netherlands).
[0284] Ethanol (EtOH, p.a.) was purchased from Merck (Darmstadt, Germany). Purified water from an Optilab HF system (Membra Pure, Germany) was used.
[0285] The 3 mL Resource RPC column (10 x 0.64 cm; 15 μm particle size) was purchased from GE Healthcare (Freiburg, Germany).
[0286] HPLC purification was performed using an ÃKTA Explorer 100 system (GE Healthcare). AMINO MODIFIED RNA SYNTHESIS
[0287] The RNA equipped with a hexylamino ligand at the 5 'end of the sense strand was produced by standard solid phase phosphoramidide chemistry on a 1215 mmol scale using an ÃKTA Oligopilot 100 (GE Healthcare, Freiburg, Germany) and pore glass controlled as a solid support (First Synthesis, Aston, PA, USA). RNA containing 2'-O-methyl nucleotides were generated using the corresponding phosphoramidites, 2'-O-methyl phosphoramidites and TFA-hexylaminolinker amidite (Sigma-Aldrich, SAFC, Hamburg, Germany). Cleavage and deprotection, as well as purification was performed by methods known in the art (Wincott F., et al., NAR 1995, 23,14, 2677-84).
[0288] The amino modified RNA was characterized by anion exchange HPLC (purity: 96.1%) and the identity was confirmed by ESI-MS ([M + H] 1+ calculated: 6937.4; [M + H ] 1 + measure: 6939.0.
[0289] Sequence: 5 ’- (NH2C6) GGAAUCuuAuAuuuGAUCcAsA-3 '; u, c: 2'-O-methyl nucleotides of corresponding RNA nucleotides, s: phosfortioate. GENERAL EXPERIMENTAL CONJUGATION PROCEDURE
[0290] The title of the compounds of examples 1 to 33 were coupled via the amino modified RNA according to the following procedure: RNA equipped with a C-6 aminolinker at the 5 'end (16.5 mg, 1 equivalent) is dissolved in 500 μL of DMSO and 150 μL of water. The p-Nitrophenylcarbonate derivative (10 equivalents) dissolved in 1 ml of DMSO is added, followed by 8 μL of DIPEA. The reaction mixture is stirred at 35 ° C in the dark and monitored using RP-HPLC (3 ml Resource RPC, buffer: A: 0.1M TEAA in water, B: 0.1M TEAA in 95% ACN , gradient: B 3% to B 100% at 20 CV). Once the reaction was carried out to complete the RNA conjugate, it was precipitated with sodium acetate (3 M) in EtOH at -20 ° C. For examples, an MMT protecting group is missing from the dipeptide motif whose corresponding conjugates are purified using the conditions described above. The pure fractions are pooled and the material is precipitated using sodium acetate / EtOH to give the desired RNA conjugate.
[0291] RNA conjugates that contain an MMT protecting group in the dipeptide sequence are transformed according to the procedure given below. GENERAL PROCEDURE FOR CLAIMING MMT
[0292] The crude RNA conjugate pellet is dissolved in 500 μL of water and 1.5 mL of sodium acetate buffer (3 M, pH 5.2 or 0.1 M, pH 4.0). The solution is stirred for 2 days at 30 ° C. The reaction mixture is monitored using RP-HPLC (3 ml Resource RPC, buffer: A: 0.1M TEAA in water, B: 0.1M TEAA in 95% ACN, gradient: B 3% to B 100% on 20 CV). After complete cleavage of the MMT protecting group, the RNA conjugate is directly purified using the conditions mentioned above. The pure fractions are grouped and the desired conjugate is precipitated using sodium acetate / EtOH.
[0293] As a control, an RNA conjugate without the dipeptide motif was synthesized. For that purpose, cholesterol was linked to the 5 'end through a ligand described in the literature (Nature Biotech, 2007, 25, 1149). This conjugate is referred to as "not cleavable".
[0294] All RNA conjugates were analyzed by RP HPLC for purity and identity and confirmed by ESI MS (negative mode) Briefly, RP-HPLC was performed on a Dionex Ultimate system (Dionex, Idstein, Germany) equipped with XBridge C18 column (2.5 x 50 mm, 2.5 μm particle size, Waters, Eschborn, Germany), with column temperature of 65 ° C. Gradient elution was performed using hexafluoroisopropanol (HFIP) 100 mM 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 spectrometry analysis, a ThermoFinnigan LCQ DecaXP ESI-MS system with micro-spray source and ion trap detector was coupled online to the HPLC system.
[0295] Examples of specific compounds of the formula (IIa) are described in Table 1. The resulting compounds are cited as “cholesterol-containing siRNA conjugates containing dipeptides”, where cholesterol-containing siRNA conjugates containing specific dipeptides are further cited as “examples of title of compound X- (NHC6) - (siRNA sequence) ”and“ siRNA with title of the compound of Example X ”. SIRNA PREPARATION Antisense sequence: 5'-uuGGAUcAAAuAuAAGAuUCcscsU-3 'u, c: 2'-O-methyl nucleotides of corresponding RNA nucleotides, s: phosfortioate
[0296] SiRNA cholesterol conjugates containing dipeptides directed against apolipoprotein B mRNA were generated by mixing an equimolar solution of complementary strips in ring buffer (20 mM sodium phosphate, pH 6.8, 100 mM sodium chloride) , heated in a water bath at 80 to 85 ° C for 3 minutes and cooled to room temperature over a period of 3 to 4 hours. Duplex formation was confirmed by native gel electrophoresis.
[0297] All dipeptide preparations containing siRNA cholesterol conjugates are listed in table 2. TABLE 1
[0298] Dipeptides containing siRNA cholesterol conjugates (5 'and 3') and analytical data. Important: the lower case letters a, c, g, u are 2'-O-Methyl nucleotides; Phosphorothioate A bonds are symbolized with a lowercase letter "s". (NHC6) is the aminohexyl linker incorporated at the 5 'end of the sense tape.

TABLE 2
[0299] Dipeptide containing siRNA cholesterol conjugates. The last item (SEQ ID NO 266/154 pair) represents a siRNA conjugate without the dipeptide motif. Important: the lower case letters a, c, g, u are 2'-O-Methyl nucleotides; Phosphorothioate A bonds are symbolized with a lowercase letter "s". (NHC6) is the aminohexyl linker incorporated at the 5 'end of the sense tape.


EXAMPLE 35 IN VIVO EXPERIMENTS DIPEPTIDE COADMINISTRATION CONTAINING SYRNA CHOLESTEROL CONJUGATES AND IN VIVO POLYMER DISTRIBUTION
[0300] Mice aged six to eight weeks (strain C57BL / 6 or ICR, approximately 18 to 20 g each) were obtained from Harlan Sprague Dawley (Indianapolis IN). The mice were accommodated at least 2 days before the injection. Feeding was done at will with the Rodent Diet of Harlan Teklad (Harlan, Madison WI).
[0301] The mice (n = 3 per group) were injected with a mixture of 0.2 ml of polymer distribution solution and 0.2 ml of dipeptide containing siRNA cholesterol conjugates. The injected dose was, except where otherwise specified, 15 mg / kg for the delivery polymer and 0.1 mg / kg for siRNA cholesterol conjugates containing dipeptide. The solutions were injected by infusion into the tail vein. 48 hours after injection, serum ApoB levels were measured in relation to animals treated with isotonic glucose according to the procedure below. DETERMINATION OF SERUM APOB LEVELS
[0302] Mice were fasting for 4 h before collection of serum due to submandibular bleeding. Serum levels of ApoB protein were determined by standard sandwich ELISA methods. Briefly, a goat anti-mouse ApoB polyclonal 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 after binding to the ApoB / antibody complex. The absorbance of the colorimetric development of tetramethyl-benzidine (TMB, Sigma) was then measured by a Tecan Safire2 microplate reader (Austria, Europe) at 450 nm.
[0303] In figure 1 several siRNA cholesterol conjugates containing dipeptides were compared with the same cholesterol conjugated siRNA, but without the cleavable motif elaborated earlier in this section. The effect of this siRNA conjugate (pairs of SEQ ID NO 266/154, “non-cleavable control”) on serum ApoB levels was adjusted to 1 in order to assess the influence of conjugates containing dipeptides in relation to the non-cleavable control. Replacing the Phe-Lys motif initially used (siRNA with Title of the compound of Example 16) with the corresponding D-amino acids (siRNA with Title of the compound of Example 14), or just replacing Lys with the unnatural enantiomer (siRNA with Title of compound of Example 30) produced less pronounced reduction in ApoB or equivalent to the non-cleavable control siRNA. Substituting Lys for Gly (siRNA with Title of the compound of Example 23) or Phe for p-Methoxyphenylalanine (siRNA with Title of the compound of Example 13) reduced potency compared to siRNA with Title of the compound of Example 16. Other siRNA conjugates containing dipeptide motifs have been shown to be as effective as the original Phe-Lys containing conjugate.
[0304] Figure 2 summarizes siRNA cholesterol conjugates containing dipeptides that were as effective or improved efficacy compared to the Title siRNA of the compound of Example 16, which consists of the Phe-Lys motif. All of these conjugates were significantly more active compared to the “non-cleavable” cholesterol siRNA conjugate of SEQ ID NO 266/154. The best performances of siRNA cholesterol conjugates containing dipeptides had a modified phenyl ring fluorine in the Phy-Lys motif (siRNA with Example 8 compound title, siRNA with Example 9 compound title) or had phenylalanine replaced with beta-phenylalanine (siRNA with Title of the compound of Example 11), or a derivative thereof (siRNA with Title of the compound of Example 10).
[0305] Since siRNA cholesterol conjugates containing dipeptide with dipeptide motifs consist of D-amino acids and are made equal to the non-cleavable control conjugate, it is conceivable that the other dipeptide sequences are actually cleaved by a protease activity in vivo. However, given the wide acceptance of different amino acids and derivatives thereof, it is likely that more than one enzyme participates in the cleavage reaction, as suggested in the literature (Bioconjugate Chem. 2002,13,855).
[0306] As shown in figure 3, the incorporation of a cleavable dipeptide motif by Cathepsin (in this case Phe-Lys, siRNA with Example 16 compound title) between siRNA and the small cholesterol molecule ligand increases the potency of the conjugate of siRNA compared to the direct cholesterol siRNA conjugate (SEQ ID NO 266/154 pair). In addition, the spacing of the cholesterol ligand and the dipeptide motif by PEG-based ligands decreases the potency proportional to the length of the PEG ligand.
[0307] In figure 4, the polymer dose was kept constant at 15 mg / kg. The siRNA dose was titrated and the effect on the ApoB content in the serum was measured. The dipeptide-containing siRNA cholesterol conjugates that contain the Phe-Lys motif (F-K) were significantly more potent compared to the control conjugate without the dipeptide sequence. EXAMPLE 36 SYNTHESIS OF MODIFIED OLIGORRIBONUCLEOTIDE 2 ’
[0308] Oligoribonucleotides were synthesized according to solid phase phosphoramidite technology. Depending on the scale, both an ABI 394 synthesizer (Applied Biosystems) and an AKTA oligopilot 100 (GE Healthcare, Freiburg, Germany) was used. Syntheses were performed on a solid support made of controlled pore glass (CPG, 520Á, with a load of 75 μmol / g, obtained from Prime Synthesis, Aston, PA, USA). All 2'-modified RNA phosphoramidites, as well as auxiliary 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. 2'-Deoxy-2'-fluoro-phosphoramidites carried out the same protective groups as 2'-O-methyl RNA amidites. the amidites were dissolved in anhydrous acetonitrile (100 mM) and molecular sieves (3A) were added. To generate the 5'-phosphate, 2- [2- (4,4'-Dimethoxytrityloxy) ethylsulfonyl] ethyl- (2- cyanoethyl) - (N, N-diisopropyl) -phosphoramidite from Glen Research (Sterling, Virginia, USA) In order to introduce the C-6 aminolinker at the 5 'end of the oligomers, 6- (Trifluo roacetylamino) -hexyl- (2-cyanoethyl) - (N, N-diisopropyl) -phosphoramidite from Thermo Fisher Scientific (Milwaukee, Wisconsin, USA). The 5 'modifications were introduced without any modification to the synthesis cycle. 5-Ethyl tiotetrazola (ETT, 500 mM in acetonitrile) was used as an activating solution. Coupling times were 6 minutes. In order to introduce phosphorothioate bonds, a 50 mM solution of 3 - ((Dimethylamino-methylidene) amino) -3H-1,2,4-dithiazola-3-thione (DDTT, obtained from AM Chemicals, Oceanside, CA, USA) in anhydrous acetonitrile / pyridine (1: 1 v / v). EXAMPLE 37 CLIPPING AND DEPROTECTION OF OLIGOMER CONNECTED TO THE SUPPORT
[0309] After completion of the solid phase synthesis, the dry 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 OLIGORRIBONUCLEOTIDS
[0310] Crude oligomers were purified by anion exchange HPLC using a column loaded 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 perchlorate of 500 mM sodium. A gradient from B 22% to B 42% was used within 32 column volumes (CV). Appropriate fractions of UV traces were recorded at 280 nm, were grouped and precipitated with 3M NaOAc, pH = 5.2 and 70% ethanol. Finally, the pellet was washed with 70% ethanol. EXAMPLE 39 OLIGORRIBONUCLEOTIDE RINGING TO GENERATE SIRNA
[0311] Complementary strips were mixed by combining equimolar RNA solutions. The mixture was lyophilized and reconstituted with an appropriate volume of ring buffer (100 mM NaCl, 20 mM sodium phosphate, pH 6.8) to achieve the desired concentration. This solution was placed in a 95 ° C water bath that was cooled to room temperature within 3 h. EXAMPLE 40 IN VITRO ACTIVITY OF SIRNAS DISPOSED OF 2'-OH WASTE
[0312] In order to investigate whether siRNAs devoid of any 2'-OH residues showed potent knockdown activity in vitro, a panel of siRNAs targeting EGFP mRNA was tested with different 2 'modification chemicals (pairs of SEQ ID 31 / 32 to 149/150, and see Table 3 for examples). SiRNAs were selected 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 the sense and antisense tape, we cloned each sequence from the corresponding 19mer target site as a separate psiCHECK2 construct (psiCHECK2-AT for antisense activity, psiCHECK2-ST for sense activity) in the multiple cloning region located 3 'from the codon stop translation of synthetic Renilla luciferase. Using Lipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany, cat. No. 11668-019) COS7 cells were cotransfected with the vector construct and 3 nM of the corresponding complementary siRNA to the cloned target site. Successful siRNA-mediated silencing was determined 24 hours after transfection through Renilla luciferase activity normalized to firefly luciferase levels to take into account the transfection efficiency (see Figure 5a for antisense activity and Figure 5b for sense activity ). TABLE 3
[0313] Exemplary siRNA sequences and chemical modifications used for the determination of knockdown activity in vitro dependent on 2 'modifications. Duplicate references and selected examples of variants of corresponding modifications used in this study. Xf indicates a 2'-fluorine modification of the X nucleotide, lowercase letters indicate a 2'-O-methyl change, underlined letters indicate a DNA nucleotide, all other capital letters indicate ribonucleotides. The letter "p" indicates a 5'-phosphate.

[0314] The 5 most powerful modified siRNAs (> 60% knockdown) were found to be designed in an alternating pattern of 2'-fluorine / 2'-O-methyls (2’F / 2’-OMe). Although it exhibits antisense activity, this chemical completely eliminated the activity of the corresponding sense tapes, as shown by the lack or minimal activity of renilla luciferase for all tested 2’F / 2’-OMe variants.
[0315] It has been concluded that this 2'F / 2’-OMe pattern is promoting antisense tape activity for siRNAs although the unwanted off-target effects from the sense tape are completely suppressed. This project is specifically preferentially for siRNAs, which come with the need for protection against 2'O-directed nucleolytic cleavage. EXAMPLE 41 DETECTION OF DNASE II SENSITIVE SITES BY IN VITRO TESTING
[0316] A high performance liquid chromatography (HPLC) in reverse phase (RP) with ion pairing (IP) coupled to mass spectrometry (MS) with electrospray ionization (ESI) or a method based on anion exchange HPLC ( AEX) was established to test the in vitro stability of selected single and double stranded RNAs.
[0317] Description of the method: For stability analysis of a 10 μM RNA solution, both single-stranded and double-stranded, it was incubated at 37 ° C in 5 mM sodium acetate buffer (pH 4.5) containing 0, 8 or 8 units of DNase II (from bovine spleen, Type V, Sigma Aldrich). The incubation reaction was stopped by adding a solution of 100 mM triethyl ammonium acetate (TEAA), shifting the pH to 7 and inactivating the enzyme DNase II. The analysis was performed by both LC / MS combined with UV detection and AEX-HPLC with UV detection. Traces of UV detection at 260 nm were used for quantitative analysis, the MS data served to identify the cleavage site within the RNA sequence.
[0318] A. IP-RP-HPLC was performed using a Waters XBridge C18 column (2.5 x 50 mm, 2.5 μm particle size), column temperature at 65 ° C. Gradient elution was performed using hexafluoroisopropanol (HFIP) 100 mM 16 mM triethylamine in 1% methanol as eluent A and 95% methanol as eluent B. A gradient from B 1% to B 18% in 30 was used minutes.
[0319] B. AEX-HPLC was performed on a Dionex DNA Pac200 column (4 x 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 of 25 to 62% B was used in 18 minutes. TABLE 4
[0320] Duplex and the remaining intact tapes evaluated for their stability against DNase II. Important: lower case letters a, c, g, u, are 2'-O-Methyl nucleotides; Capital letters A, C, G, U, followed by "f" indicate a 2'-fluorine nucleotide. The lower letter “p” indicates a 5'-phosphate. (InvdT) represents an inverted (3-3'-linked) deoxythymidine. Phosphorothioate A bonds are symbolized with a lowercase letter "s". dT is deoxythymidine. (NHC6) is the aminohexyl linker incorporated at the 5 'end of the sense tape.
CONCLUSIONS
[0321] A. RNA strands containing at least one 2'-OH nucleotide (for example, both strands of SEQ ID NO 157/158 pair) are rapidly degraded via a cyclic pentavalent intermediate, leading to cyclic phosphates 2 '-3' in the 5'-cleavage product. The formation of the pentavalent intermediate can be inhibited with the use of nucleotides without a 2'-OH group, such as, for example, 2'-deoxy, 2'-OMe or 2'-F.
[0322] B. In addition, the RNA is degraded via a 5'-exonucleolytic pathway, which is independent of the 2 'modification in the 5'-terminal nucleotides. This degradation pathway can be inhibited with the use of 5'-terminal non-nucleotide components, such as, for example, a C6-aminolinker (for example, SEQ ID NO 160 in the pair of SEQ ID NO 160/159 or SEQ ID NO 165 in the pair of SEQ ID NO 165/166) or a phosphorothioate in the first internucleotide bond (for example, SEQ ID NO 160 in the pair of SEQ ID NO 160/159).
[0323] C. A 5'-phosphate group decreases the kinetics of exonucleolytic cleavage, but cannot fully block starting degradation from that end (for example, SEQ ID NO 160 in the pair of SEQ ID NO 160/159). Most likely, this is due to cleavage of the 5'-phosphate, both by phosphatases and by a phosphatase activity inherent in the enzyme DNase II.
[0324] D. The best protection for RNA strands was achieved with oligonucleotides that do not contain a 2'-OH nucleotide inside the strand, starting with a 2'-OMe nucleotide at the 5 'end connected by a phosphorothioate bond to the second nucleotide ( for example, SEQ ID NO 173 in the pair of SEQ ID NO 173/174). Other terminal non-2'-OH nucleotides also protect against 5'-exo degradation, but to a lesser extent compared to the 2'-OMe modification (refer to Table 9) EXAMPLE 42 KNOCKDOWN IN VIVO DE SIRNAS WASTE WASTE ACTIVITY 2'-OH
[0325] In vivo experiments were conducted with mice injected with siRNAs targeting (FVII) Factor VII (pairs of SEQ ID NO 179/166 and 180/168, see Table 5) co-administered with DPC-GalNac. TABLE 5 A
[0326] SiRNA sequences for in vivo experiments. Important: lower case letters a, c, g, u, are 2'-O-Methyl nucleotides; Capital letters A, C, G, U, followed by "f" indicate a 2'-fluorine nucleotide. The lower letter “p” indicates a 5'-phosphate. (InvdT) represents an inverted (3-3'-linked) deoxythymidine. Phosphorothioate A bonds are symbolized with a lowercase letter "s". dT is deoxythymidine. (NHC6) is the aminohexyl linker incorporated at the 5 'end of the sense tape. GalNAc refers to the structure in formula (IV).

[0327] An FVII siRNA with an alternating 2'-OMe / 2'-F pattern on the sense and antisense tape was generated with a 5'-terminal 2'-OMe nucleotide on the antisense and a 5'-terminal 2'-F tape on the sense tape. Both tapes are protected by an inv (dT) on the 3'-terminal ledge. The antisense tape was carrying a 5'-phosphate group to maintain siRNA activity. The sense strand was conjugated to a GalNAc-palmitoyl construct at its 5 'end to target hepatocytes through the asylyloglycoprotein receptor. siRNA (2.5 mg / kg) was co-administered with GalNAc targeting PBAVE (15 mg / kg) in mice.
[0328] FVII mRNA measurements were made from liver homogenates using the QuantiGene 1.0 Branched DNA Assay Kit (bDNA) (Panomics, Fremont, Calif., USA, Cat-No: QG0004).
[0329] At necropsy 1 to 2 g of liver tissue was suddenly frozen in liquid nitrogen. The frozen tissue was made into powder with mortar and pistil on dry ice. 15 to 25 mg of tissue was transferred to a cooled 1.5 mL reaction tube, 1 mL of 1: 3 lysis mixture pre-diluted in MilliQ water and 3.3 μL of Proteinase K (50 μg / μL) was added ) and the tissue was lysed by ultrasound sonication for several seconds at 30 to 50% power (HD2070, Bandelin, Berlin, Germany). Lysates were stored at -80 ° C until analysis. For mRNA analysis, the lysate was thawed and digested with Proteinase K for 15 minutes at 1000 rpm in a Thermomixer at 65 ° C (Thermomixer comfort, Eppendorf, Hamburg, Germany). GAPDH FVII and mRNA levels were determined using reagents from the QuantiGene 1.0 bDNA Assay Kit according to the manufacturer's recommendations. FVII mRNA expression was analyzed using 20 mL of lysate and a mouse FVII probe set. GAPDH mRNA expression was analyzed using 40 μL of lysate and rattus norwegicus probe set which proved to be cross-reactive with mice (probe set sequences, see above). As a test reading, the chemiluminescence signal at the end of the test was measured on a Victor 2 Light luminescence counter (Perkin Elmer, Wiesbaden, Germany), as relative light units (RLU). The signal for FVII mRNA was divided by the signal for GAPDH mRNA from the same lysate. Values are reported as FVII mRNA expression normalized to GAPDH.
[0330] The results demonstrate a 79% FVII mRNA knockdown within 48 hours after dosing, after administration of the SEQ ID NO 179/166 pair. In contrast, the 2'-OH nucleotide carrying the siRNA of SEQ ID NO 180/168 showed no significant knockdown (<25%), as shown in Table 5. TABLE 5 B RESULTS OF KNOCKDOWN IN VIVO STUDIES

EXAMPLE 43 DISTRIBUTION OF SIRNAS FABRIC DISPOSED OF 2'-OH WASTE
[0331] The concentration of siRNA in liver tissue samples was determined using a patented oligonucleotide detection method as described in WO2010043512. Briefly, siRNA quantification is based on hybridization of fluorescently labeled PNA probe (Atto-425) (Atto425-OO-GCAAAGGCGTGCCAACT, obtained from Panagene Inc., Korea) complementary to the siRNA duplex antisense tape, followed by separation based on siRNA duplex in AEX-HPLC. Quantification was done by fluorescence detection against an external calibration curve that was generated from a series of dilutions of the two FVII siRNAs used in the in vivo experiment (see example 42). For plasma samples between 0.2 to 2 μL and for tissues, approximately 1mg aliquots were injected into the HPLC system.
[0332] Liver tissue analysis of stabilized siRNA without 2'-OH nucleotide showed high concentrations of intact liver antisense tape in the ug / g range, but approximately 95% was present in the inactive 5'-dephosphorylated form (see table IR.04). The resulting RNA with a 2'-OMe terminal nucleotide is not prone to cytoplasm refosphorylation by phosphokinase hClp1 (see below). In contrast, the siRNA antisense tape containing 2'-OH was completely degraded in the tissue within the first 6 hours after dosing. TABLE 6 ANALYSIS OF STABILIZED SIRNA LIVER TISSUE THAT CONTAINS NO 2'-OH NUCLEOTIDE

* BDL = below the detection limit EXAMPLE 44 KNOCKDOWN IN VITRO ACTIVITY OF SIRNAS WITH OPTIMIZED 5 'END

[0333] An additional in vitro selection for FVII siRNAs was conducted in order to identify the siRNAs that can be intracellularly (re) phosphorylated at the 5 'antisense end to result in the competent RNAi species. All siRNAs from that selection are shown in the Table 7. The alternating 2'-OMe / 2'-F modification pattern was identical to the 1st generation project (without any 2'-OH residues), with the exception of several modifications to the first two nucleotides at the 5 'end of the antisense tape . The two 5'-terminal nucleotides of the antisense tape were generated as modified 2'-F or 2'-deoxy nucleotides in various combinations, with and without an additional 5'-phosphate or 5'-phosphotioate. All siRNAs were selected in response to the dose (24 nM to 0.00037 nM in 4-fold dilutions) for knockdown activity after transfection of primary mouse hepatocytes (30,000 cells per well, 96-well plate format) using Lipofectamine 2000 according to the manufacturer's instructions. Two siRNAs were active comparable to the parental duplex (pair of SEQ ID NO 182/168); comparable active siRNAs: pairs of SEQ ID NO 181/186 and 181/185), in terms of IC50 values, one with a 2'F 5'-terminal and a phosphate group and one with two 2'-deoxy 5 'nucleotides -terminal and a 5'-phosphorothioate (see Table 7 for IC 50 values). Both are approximately 5 to 6 times more active compared to siRNA (pair of SEQ ID NO 181/166) Petition 870200130839, of 10/16/2020, p. 109/302 100/146 used in the first animal experiment with the terminal 2'-OMe nucleotide TABLE 7 IC 50 VALUES EXAMPLE 45 5 'Phosphorylation IN VITRO OF SIRNAS WITH 5' OPTIMIZED TERMINATIONS
[0334] All siRNAs without a 5'-phosphate or 5'-phosphorothioate listed in Table 7 were evaluated for phosphorylation by hClp1 in an extract from HeLa S100 cells.
[0335] Phosphorylation 5 'was analyzed from the extracts of HeLa S100 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). Immediately after the incubation of 1 μM of siRNAs in HeLa S100 extract containing 5mM ATP, the solution was analyzed by both IP-RP-HPLC and AEX-HPLC under denaturation conditions by injection of 5 μL of sample solution: A. IP -RP-HPLC was performed using a Waters XBridge C18 column (2.5 x 50 mm, 2.5 μm of particle size), column temperature at 65 ° C. Gradient elution was performed using hexafluoroisopropanol (HFIP) 100 mM 16 mM triethylamine in 1% methanol as eluent A and 95% methanol as eluent B. A gradient from B 1% to B 18% in 30 was used minutes. B. AEX-HPLC was performed on a Dionex DNA Pac200 column (4 x 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 of 25 to 62% B was used in 18 minutes.
[0336] The 5 'phosphorylation ratio is calculated for each siRNA strand from the UV trace at 260 nm using the following equation (PA is the peak area):% (5' phosphorylation) = 100 * PA [5 'phosphorylated tape] / (PA [5' phosphorylated tape] + PA [standard tape]) In Table 8, it is shown that the antisense tape of a siRNA cannot be phosphorylated 5 ', when a 2'-OMe nucleotide is located at the 5 'termination (SEQ ID NO 181/196 pair and SEQ ID NO 181/195 pair). In contrast, the antisense tape is susceptible to 5 'phosphorylation, when a 2'-F, 2'-deoxy or 2'OH nucleotide is incorporated at the 5' termination (SEQ ID NO 181/195 pair, SEQ ID NO pair 181/192, SEQ ID NO 181/197 pair, SEQ ID NO 181/199 pair and SEQ ID NO 182/168 pair). The two siRNAs, which were comparable active in the in vitro assay as the parental SEQ ID NO pair 182/168 (SEQ ID NO 181/186 and 181/185 pairs), are susceptible to 5 'phosphorylation since the synthetic group 5'-phosphate / 5'-PTO introduced synthetically is cleaved in vivo, for example, by phosphatases. TABLE 8
[0337] Percentage of 5'-phosphorylated tape after 4 hours of incubation in S100 HeLa cell extract. Important: lower case letters a, c, g, u, are 2'-O-Methyl nucleotides; Capital letters A, C, G, U, followed by "f" indicate a 2'-fluorine nucleotide. (InvdT) represents an inverted (3-3'-linked) deoxythymidine. Phosphorothioate A bonds are symbolized with a lowercase letter "s". dT is deoxythymidine.
EXAMPLE 46 DNASE II IN VITRO STABILITY OF SIRNAS WITH OPTIMIZED 5 'END
[0338] All antisense tapes were selected for DNAse II stability, as described in example 41. The two antisense tapes present in the siRNAs that were active comparable to the parental duplex (SEQ ID NO 186 and pair of SEQ ID NO 185, one with a 5'-terminal 2'-F and a phosphate group and one with two terminals 5 'nucleotides 2'-deoxy and a 5'-phosfortioate are stable towards DNAse II cleavage II (> 70% intact tape after 20 incubation hours.) TABLE 9 IN VITRO SIRNAS STABILITY DIRECTED TO DNASE II AFTER 20 HOURS OF INCUBATION
EXAMPLE 47 KNOCKDOWN IN VIVO SIRNAS ACTIVITY WITH OPTIMIZED 5 'END
[0339] In order to assess whether in vitro enhancement by transferring 5 'ends optimized for the in vivo situation, additional experiments were performed on mice with selected GalNAc-palmitoyl conjugates of siRNAs (see Table 10). The siRNAs were applied under identical conditions as described for the first mouse experiment (example 42, from this patent application).
[0340] For the measurement of FVII levels, plasma samples from mice were prepared by blood collected (9 volumes) by submandibular bleeding in a tube microcentrifuge containing 0.109 mol / L of sodium citrate anticoagulant (1 volume), following procedures pattern. Plasma FVII activity was measured using a chromogenic method using a BIOPHEN VII kit (Hyphen BioMed / Aniara, Mason, OH) following the manufacturer's recommendations. The absorbance of colorimetric development was measured using a Tecan Safire2 microplate reader at 405 nm.
[0341] The siRNAs under investigation showed improved in vivo activity, correlating fully with the results of in vitro selection. Serum FVII activity was reduced by more than 80% for both siRNAs 48 hours after dosing, compared to 49% with the use of the first generation of stable siRNA DNase II (see Table 10). This result clearly highlights the importance of a 5'-terminal nucleotide in the antisense strip that can be phosphorylated effectively, in case in vivo phosphatases cleave the synthetically generated 5'-phosphate or 5'-phosphotioate group. In the case of a 5'-terminal 2'-OMe nucleotide as used in the first project 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 phosphate of synthesis in vivo would lead to a strong reduction in the potency of the corresponding siRNA. TABLE 10
[0342] In vivo knockdown activity of siRNAs with optimized 5 'ends. Important: lower case letters a, c, g, u are 2'-O-methyl nucleotides; Capital letters A, C, G, U, followed by "f" indicate a 2'-fluorine nucleotide. The lower letter “p” indicates a 5'-phosphate. (InvdT) represents an inverted (3-3'-linked) deoxythymidine. Phosphorothioate A bonds are symbolized with a lowercase letter "s". (NHC6) is the aminohexyl linker incorporated at the 5 'end of the sense tape. GalNAc refers to the structure in formula (IV).
EXAMPLE 48 KNOCKDOWN IN VITRO SIRNAS ACTIVITY WITH OPTIMIZED 3 'END
[0343] To further increase the activity of stable DNase II siRNAs, a SAR study of the 3 'overhang was performed. Various combinations of invdT, dTinvdT or dTsdT in both the 3 'overhang of the sense tape and in the antisense were applied to siRNAs targeting Aha1 and siRNAs EGFP (see Tables 11 and 12, respectively) and were matched for the composition of both ends 3 'in more powerful siRNAs. All siRNAs were selected in response to the dose (24 nm at 0.00037 nM in 4-fold dilutions) for knockdown activity after transfection of primary mouse hepatocytes (30,000 cells / well, 96-well plate format) using Lipofectamine2000 according to the manufacturer's instructions. TABLE 11 KNOCKDOWN IN VITRO SIRNAS ACTIVITY FOR EGFP WITH 3 'DIFFERENT EXTREMITIES

TABLE 12 KNOCKDOWN IN VITRO SIRNAS ACTIVITY DIRECTED TO AHA1 WITH DIFFERENT 3 'ENDS


[0344] It was found that 2 nucleotide siRNAs with dTsdT protrusions on the antisense strip always performed better than those with a single invdT protrusion at the 3 'end of the antisense (although the sense tapes were the same). Even more beneficial was the combination with a modified sense tape with a unique invdT overhang as a 3 'overhang. EXAMPLE 49 KNOCKDOWN IN VITRO SIRNAS ACTIVITY IN NON-HUMAN PRIMATES PREPARATION OF DPCS AND DOSAGE
[0345] DPCs were prepared covalently by “149 RAFT” binding polymers for coagulation Factor VII directed to siRNA (siF7) at a ratio of 4: 1 p: p (polymer: siRNA) through a disulfide bond and, in then modifying the polymer-siRNA conjugate with a 2: 1 w: w CDM-PEG: CDM-NAG mixture at a 7x w: w ratio (CDM: polymer). Cynomolgous monkeys were dosed with 1 mg / kg of DPC (polymer weight) and 0.25 mg / kg of the indicated siRNA. One animal received DPC containing siF7 pair of SEQ ID NO 151/152, two animals received DPC containing siF7 pair of SEQ ID NO 253/254), No. 1 and No. 2), and two animals received DPC containing pair of SEQ ID NO NO 251/255, No. 1 and No. 2). The F7 values were normalized to the average of the two pre-dose values. Animals that received DPCs containing SEQ ID NO 253/254 pair or SEQ ID NO 251/255 pair had higher F7 knockdown levels and more PT than the animal that received SEQ ID NO 251/252 pair. DPC INJECTION PROCEDURE
[0346] For each injection procedure, the 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 transferred to a procedure room. In the procedure room, the animals were placed on a heating pad covered with water and the injection site was scraped and prepared with an antiseptic. An intravenous catheter (gauge 20 to 22) was inserted into the systemic vein (cephalic or small saphenous vein) and the CPP solution was infused (2 mL / kg) slowly over 1 to 2 minutes. A pulse oximeter was used to monitor heart rate and oxygen saturation during and immediately after the injection procedure. Each injection procedure took about 20 minutes to complete. After the injection, the catheter was removed and gentle pressure was applied to the puncture site of the vein. The animals were returned to their cages and given an IM injection of the reversal drug atipamezole (Antisedan) (0.10 to 0.15 mg / kg). The animals were monitored until normal activity was restored. BLOOD COLLECTION PROCEDURE
[0347] Blood samples (1 to 5 mL) were obtained for measurement of gene inhibition (F7 activity, clotting time), blood chemistry, and liver damage markers (CBC, chemical panel, ALT, cytokines , complement). For these blood collection procedures, the 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, the animals were transferred to a portable procedure table and a 22 gauge needle and syringe were used to collect blood from the femoral vein. Immediately after collecting blood, pressure was applied to the vein puncture site and the blood was divided into the appropriate sample tubes for each blood test. The animals were then given an IM injection of the reversal drug atipamezole (Antisedan) (0.10 to 0.15 mg / kg) and returned to their cages. No more than 20% of the total blood volume was taken in any 30-day period (estimated blood volume = 60 mL / kg). Each blood collection procedure took about 10 minutes to complete. FACTOR VII ACTIVITY MEASUREMENTS (F7)
[0348] Blood samples were prepared from non-human primates by filling separator tubes with whole blood and allowing the blood to clot at room temperature for at least 20 minutes. After coagulation, the blood tubes were centrifuged for 3 minutes at 9000 rpm, aliquoted in Eppendorf tubes and stored at -20 ° C until analyzed. Serum FVII activity was measured using a chromogenic method using a BIOPHEN VII kit (Hyphen BioMed / Aniara, Mason, OH) following the manufacturer's recommendations. The absorbance of colorimetric development was measured using a Tecan Safire2 microplate reader at 405 nm. COAGULATION TESTS (PROTIME, PARTIAL PROTIME AND FIBROGEN)
[0349] Blood samples were prepared from non-human primates by completely filling tubes of sodium citrate (BD Vacutainer) with whole blood and mixing gently to prevent clots. The tubes were transported to a clinical testing laboratory within an hour and clotting assays were performed within 4 hours from the time of collection. TABLE 13
[0350] FVII SiRNA used for NHP experiment: Important: lower case letters a, c, g, u, are 2'-O-Methyl nucleotides; Capital letters A, C, G, U, followed by "f" indicate a 2'-fluorine nucleotide. The lower letter “p” indicates a 5'-phosphate. (InvdT) represents an inverted (3-3'-linked) deoxythymidine. Phosphorothioate A bonds are symbolized with a lowercase letter "s". dT is deoxythymidine.

[0351] Changing a single nucleotide (invdT) -3'-boss on both tapes to an asymmetric siRNA design with a 3 '- (invdT) boss on the sense tape and a dTsdT boss on the antisense tape, but otherwise the pattern of constant modification leads to a more pronounced reduction in FVII in serum and a significantly prolonged duration of this effect in non-human primates (see figure 6a). This observation is supported by an expected biological consequence, that is, a more pronounced effect on the prothrombin time that corresponds, to some extent, to a reduction in Factor 7 (see figure 6b). EXAMPLE 50 KNOCKDOWN IN VIVO SIRNAS ACTIVITY WITH CLIVABLE RNA BINDERS
[0352] In Table 14, in vivo efficacy based on the inhibition of FVII protein in serum was compared with the use of cholesterol or the siRNA GalNAc-palmitoil conjugate in the same sequence context in mice. The in vivo experiment was conducted as described in example 42. Inhibition of FVII was strongly decreased for cholesterol-conjugated siRNAs not containing 2'-OH nucleotide compared to the GalNAc-palmitoil conjugated counterparts (SEQ ID NO 179/166 versus 179/190, SEQ ID NO 257/264 pair versus SEQ ID NO 179/262 pair, SEQ ID NO 257/263 pair versus SEQ ID NO 179/163 pair and SEQ ID NO 257/166 pair versus ( pair of SEQ ID NO 179/166) Unlike a siRNA containing 2'-OH the cholesterol conjugate leads to higher FVII inhibition compared to the GalNAc-palmitoil derivative (pair of SEQ ID NO 180/168 versus pair of SEQ ID NO 258/168).
[0353] The small molecule ligands of GalNAc-palmitoyl and cholesterol used in the described in vivo experiment are linked to siRNA via a non-cleavable ligand to the 5 'end of the sense strand. In case the sense strand exhibits 2'-OH nucleotides the linker is still cleavable by nucleases (for example, DNase II in the endosomal or lysosomal compartment). The cleavage reaction releases the free siRNA which is then released into the cytoplasm by the endosomal disrupting activity of the delivery polymer.
[0354] For siRNAs without a 2'-OH nucleotide on the sense strand, the ligands are stably connected to the duplex, as a non-enzymatic mechanism (nuclease / protease / esterase, etc.) or chemical that triggers ligand cleavage. Therefore, fully stable cholesterol-conjugated siRNA can be trapped in cell membranes due to the interaction of the lipophilic cholesterol ligand membrane. Even high concentrations of siRNA in the tissue are not sufficient to effectively release siRNA into the cytoplasm. In contrast, the smaller 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 effective compared to a cholesterol conjugate to the same siRNA.
[0355] Developing cleavable ligand constructs would help to circumvent the issue of membrane entrapment for siRNA cholesterol conjugates with stability. With the use of disulfide, chemical ligand is described as an attractive possibility to introduce a defined cleavage site, but cleavage is probably more restricted to the reducing environment of specific organelles within the cell (PNAS, 2006, 103, 13872). Cleavage is expected to be slow in the endosomal / lysosomal compartment and most of the cholesterol-disulfide conjugated siRNA can still be trapped in the membrane, as described for non-cleavable cholesterol conjugates TABLE 14

[0356] In addition, for the well-established bisulfide cleavable chemical linker another possibility is the generation of defined cleavage sites using 2'-OH nucleotides in certain positions. The introduction of 2'-OH nucleotides in selective positions is a new approach to achieve cleavage of RNA strand conjugates. 2'-OH nucleotides can be implemented by adding single strand protrusions with at least one 2'-OH nucleotide at the 3 'or 5' end of the RNA strand or using 2'-OH nucleotides within the duplex region of a siRNA. The enzymatic activity of nucleases present in the endosome / lysosome cleaves selectively at these positions. In a first project, cholesterol was connected to the sense strand via a single strand protrusion containing 3 2'-OH (AUC) nucleotides at the 5 'termination.
[0357] Cholesterol conjugated siRNAs comparing various cleavable chemical ligands are shown in Table 15. All siRNAs have identical sequence context, only the chemical ligand has been changed. Cholesterol was connected to the sense strand through a single strand protrusion comprised of 3 2'-OH (AUC) nucleotides for the 5 'termination. When co-administered with a polymer that distributes this siRNA (SEQ ID NO 260/263 pair) it leads to 77% negative FVII modulation in the mouse serum, compared to just 60% when using identical siRNA with a stable bound cholesterol (SEQ ID 257/263 pair). The same siRNA with a cholesterol conjugated via a ligand according to formula Ia to the 5 'end of the sense strand (SEQ ID NO 261/263 pair) leads to a 93% reduction in factor VII activity in serum. All results were achieved by co-administering 15 mg / kg of a delivery polymer with 2.5 mg / kg of cholesterol conjugated siRNA in mice. TABLE 15 IN VIVO COMPARISON OF VARIOUS CHEMICAL BINDERS FOR CHOLESE CONJUGATED TO CHOLESTEROL

[0358] These results indicate that the use of a cleavable linker improves the in vivo potency of siRNAs that do not contain 2'-OH nucleotide. The cleavable linker can either be comprised of nucleotides containing 2'-OH, a dipeptide cleavage motif or a disulfide chemical linker. All cleavable ligand constructs improve potency in vivo in a co-administration configuration of cholesterol-conjugated siRNAs with a slow endosomal release delivery polymer. EXAMPLE 51 IN VITRO SERIAL STABILITY OF SIRNAS WITH CLIVABLE BINDERS
[0359] The stability of the cleavable ligand was assessed in an in vitro stability assay. The sense strips of cholesterol conjugates were incubated in 90% mouse serum at 37 ° C for various time points. The incubation reaction was stopped by adding proteinase K in a buffer containing sodium dodecyl sulfate (SDS). The treatment degrades all proteins and enzymes, without interfering with the integrity of the RNA strand. 25 μL of this solution was directly injected into an AEX-HPLC system connected to a UV detector at 260 nm. AEX-HPLC was performed on a Dionex DNA Pac200 column (4 x 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 of B 25 to 62% in 18 minutes was used.
[0360] Cholesterol containing single-stranded RNA elutes from the HPLC column as a broad peak at 260 nm. After cholesterol cleavage, sharp symmetrical peaks are observed with shorter retention times. The cholesterol cleavage rate was determined by the following equation (PA = Peak Area):% (RNA free) = 100 * PA [RNA free] / (PA [RNA lire] + PA [cholesterol conjugated RNA] )
[0361] In vitro, showed that the protrusion (AUC) of the 3nt nucleotide is quantitatively cleaved in less than 1 hour in 90% mouse serum. Cleavage occurs 3 'for the two pyrimidine nucleotides on the overhang, leading to two distinct cleavage metabolites (peak areas of metabolites have been summarized for data evaluation). In contrast, the linker-containing dipeptide according to formula 1a, the disulfide and the stably bound cholesterol are completely stable in mouse serum. EXAMPLE 52 DISTRIBUTION OF SIRNAS FABRIC WITH CLIVABLE BINDERS
[0362] The concentration of siRNA in liver tissue samples was determined using a patented oligonucleotide detection method as described in WO2010043512. Briefly, siRNA quantification is based on hybridization of fluorescently labeled PNA probe (Atto-425) (Atto425-OO-TGAGTTGGCACGCCTTT, obtained from Panagene Inc., Korea) complementary to the siRNA duplex sense tape, followed by separation based on siRNA duplex in AEX-HPLC. Quantification was done by fluorescence detection against an external calibration curve that was generated from a series of dilutions of the two FVII siRNAs used in the in vivo experiment (see example 42). For plasma samples between 0.2 to 2 μL and for tissues, approximately 1mg aliquots were injected into the HPLC system.
[0363] The results of the liver tissue analysis are shown in Table 16. When analyzing the siRNA content, it was found that the sense strand, which is present in the liver tissue, is quantitatively cleaved from cholesterol when used both the dipeptide binding motif as the 5 '3 nt overhang with the unmodified AUC sequence ligand. In contrast, only 15% of the disulfide bound to siRNA that is present in the liver is cleaved from cholesterol in the first 48 hours after dosing, and none of the stable cholesterol is cleaved from the siRNA.
[0364] When comparing the absolute amounts of cholesterol-free siRNA in the liver tissue, similar amounts were found for the disulfide ligand and the RNA AUC ligand, cleverly correlating with equal serum FVII activity 48 hours after dosing. The lower FVII activity achieved with the cholesterol siRNA-bound dipeptide correlates fully with the higher absolute amount of cleaved cholesterol-free siRNA.
[0365] The total amount of siRNA cholesterol conjugate equipped with a ligand (AUC) in the sense strip distributed in the liver is approximately six times less, compared to the stable or disulfide-bound cholesterol and approximately 3 times less in comparison cholesterol siRNA-conjugated dipeptide. The presence of reduced tissue can be attributed to the fact that the AUC ligand is not only a substrate for intracellular nucleases, but also for nucleases present in the 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 in the urine without distribution to the tissue. TABLE 16

[0366] The following tables summarize the siRNAs used in the examples: TABLE 17 MAIN SEQUENCES

TABLE 18 MAPPING OF MAIN SEQUENCES AND MODIFIED SEQUENCES

权利要求:
Claims (11)
[0001]
1. COMPOUND, characterized by being of formula (I):
[0002]
2. COMPOUND, according to claim 1, characterized by having the conformation as shown in formula (Ia):
[0003]
COMPOSITE according to any one of claims 1 to 2, characterized in that Y is - (CH2) 3-.
[0004]
COMPOSITE according to claim 3, characterized in that: Y is - (CH2) 3-; R2 is - (CH2) k-N-C (Ph) 3, in which the phenyl rings are unsubstituted or independently substituted with -O- (C1-4) alkyl; and R3 is -NH-phenyl, in which the phenyl group is further substituted with - (CH2) -O-C (O) -O- (4-nitro-phenyl); n is 0; and R1 and k have the meaning given above.
[0005]
COMPOSITE according to any one of claims 1 to 2, characterized in that Y is -C (O) -N- (CH2-CH2-O) p-CH2-CH2-.
[0006]
A COMPOUND according to claim 5, characterized in that: Y is -C (O) -N- (CH2-CH2-O) p-CH2-CH2-; R2 is - (CH2) k-N-C (Ph) 3, in which phenyl rings are unsubstituted or independently substituted with -O- (C1-4) alkyl; and R3 is -NH-phenyl, in which the phenyl group is further substituted with - (CH2) -O-C (O) -O- (4-nitro-phenyl); n is 0; and R1, k and p have the meaning given above.
[0007]
7. USE OF THE COMPOUND, as defined in any one of claims 1 to 6, characterized in that it is as a binder of a biologically active compound, wherein said binder is covalently bound to the biologically active compound.
[0008]
8. USE, according to claim 7, characterized in that said biologically active compound is a peptide.
[0009]
9. COMPOUND, characterized by being of formula (II):
[0010]
10. COMPOUND, according to claim 9, characterized by having the conformation as shown in formula (IIa):
[0011]
11. PHARMACEUTICAL COMPOSITION, characterized by comprising the compounds, as defined in any one of claims 9 to 10.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-03-26| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-05-14| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-07-21| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-11-17| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-01-05| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 22/12/2011, OBSERVADAS AS CONDICOES LEGAIS. |

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2021-02-09| B16C| Correction of notification of the grant [chapter 16.3 patent gazette]|Free format text: REF. RPI 2609 DE 05/01/2021 QUANTO AO INVENTOR. |

优先权:

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申请号 | 申请日 | 专利标题

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US201061427845P| true| 2010-12-29|2010-12-29|

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US61/427,845|2010-12-29|

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PCT/EP2011/073718|WO2012089602A1|2010-12-29|2011-12-22|Small molecule conjugates for intracellular delivery of biologically active compounds|

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