Carbohydrate conjugates as delivery agents for oligonucleotides

• US 8,450,467 B2
• Filed: 12/14/2011
• Issued: 05/28/2013
• Est. Priority Date: 12/04/2007
• Status: Active Grant

• Current Assignee: Alnylam Pharmaceuticals Incorporated
• Original Assignee: Alnylam Pharmaceuticals Incorporated
• Inventors: Manoharan, Muthiah, Rajeev, Kallanthottathil G., Jayaprakash, Narayanannair K., Maier, Martin
• Primary Examiner(s): LEWIS, PATRICK T
• Application Number:
  US13/326,203
• Publication Number:
  US 20120136042A1
• Time in Patent Office: 531 Days
• Field of Search: None
• US Class Current: 536/18.7
• CPC Class Codes: A61K 31/70 Carbohydrates; Sugars; Derivatives thereof sorbitol A61K31/047
• CPC Class Codes: A61K 31/7004 Monosaccharides having only carbon, hydrogen and oxygen atoms
• CPC Class Codes: A61K 31/7052 having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 47/16 containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
• CPC Class Codes: A61K 47/22 Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
• CPC Class Codes: A61K 47/28 Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
• CPC Class Codes: A61K 47/543 Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61K 47/554 the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
• CPC Class Codes: A61K 47/56 the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
• CPC Class Codes: Y02P 20/55 : Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The present invention provides iRNA agents comprising at least one subunit of the formula (I):

• • wherein:
  • A and B are each independently for each occurrence O, N(R^N) or S;
  • X and Y are each independently for each occurrence H, OH, a hydroxyl protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, —P(Z′)(Z″)O-nucleoside, —P(Z′)(Z″)O-oligonucleotide, a lipid, a PEG, a steroid, a lipophile, a polymer, —P(Z′)(Z″)O-Linker-OP(Z′″)(Z″″)O-oligonucleotide, a nucleotide, an oligonucleotide, —P(Z′)(Z″)-formula (I), —P(Z′)(Z″)— or -Linker-R;
  • R is L^G, -Linker-L^G, or has the structure shown below:

• • L^G is independently for each occurrence a carbohydrate, e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide;
  • R^N is independently for each occurrence H, methyl, ethyl, propyl, isopropyl, butyl, or benzyl; and
  • Z′, Z″, Z′″ and Z″″ are each independently for each occurrence O or S.

Claims

1. A process of making a ligand-containing conjugate having the structure shown in formula (I):

2. A process of making a ligand-containing conjugate:

3. The process of claim 2, wherein the coupling step is carried out in the presence of succinic anhydride/DMAP in DCM, and HBTU/DIEA in DMF.

4. The process of claim 2, wherein the reacting step comprises:

reacting the hydroxyproline compound 113 with the tether precursor to form a hydroxyproline compound 115

5. The process of claim 4, wherein the step of reacting the hydroxyproline compound 113 with the tether precursor is carried out in the presence of HBTU/DIEA in DMF, or TBTU/HOBt/DIEA in DMF.

6. The process of claim 4, wherein the hydrolyzing step is carried out in the presence of LiOH in THF/DCM/Water, followed by neutralization with acetic acid.

7. The process of claim 4, wherein the step of reacting the hydroxyproline compound 116 with the ligand moiety compound 110 is carried out in the presence of HBTU/DIEA in DMF, or TBTU/HOBt/DIEA in DMF.

8. The process of claim 2, further comprising the steps of:

reacting a GalNAc acid

9. The process of claim 8, wherein the step of reacting the GaNAc acid with the branched linker compound 108 is carried out in the presence of HBTU/DIEA/HOBt in DMF.

10. The process of claim 8, wherein the hydrogenating step is carried out with H₂ in the presence of Pd/C, methanol, and acetic acid.

11. The process of claim 8, further comprising the steps of:

reacting a branched linker compound 106

12. The process of claim 11, wherein the step of reacting the branched linker compound 106 with the linker compound is carried out in the presence of HBTU/DIEA in DMF.

13. The process of claim 11, wherein the deprotecting step is carried out in the presence of TFA/DCM.

14. A process of synthesizing a ligand-conjugated oligonucleotide, comprising the step of:

conjugating a ligand-containing conjugate having the structure shown in formula (I) to an oligonucleotide;

15. The process of claim 14, wherein the ligand-containing conjugate has the structure of

16. The process of claim 15, wherein the ligand-containing conjugate is

17. The process of claim 15, wherein the ligand-containing conjugate is

18. The process of claim 14, wherein at least one ligand-containing conjugate is conjugated to the 3′

-end of the oligonucleotide.

19. The process of claim 18, wherein the ligand-containing conjugate contains a solid support, wherein the conjugating step is carried out by growing the oligonucleotide to the ligand-containing conjugate.

20. The process of claim 14, wherein at least one ligand-containing conjugate is conjugated to the 5′

-end of the oligonucleotide.

21. The process of claim 20, wherein the ligand-containing conjugate contains a phosphoramidite moiety, wherein the conjugating step is carried out by coupling the phosphoramidite of the ligand-containing conjugate to the growing chain of the oligonucleotide.