Process for the preparation of 2'-O-alkyl purine phosphoramidites

• US 6,133,438 A
• Filed: 10/02/1998
• Issued: 10/17/2000
• Est. Priority Date: 01/11/1990
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Cook, Phillip Dan, McGee, Daniel Peter Claude, Guinosso, Charles John
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US09/165,680
• Time in Patent Office: 746 Days
• Field of Search: 536/55.3, 536/26.71, 536/26.72, 536/27.81, 536/25.34, 536/28.5, 536/28.51
• US Class Current: 536/25.34
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• 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: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 23/00 Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12 esters with inorganic acids C07H11/00; metal salts, see parent compounds
• CPC Class Codes: C07J 43/003 not condensed
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/3125 Methylphosphonates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/32 of the sugar
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/33 of the base
• CPC Class Codes: C12N 2310/333 Modified A
• CPC Class Codes: C12N 2310/336 Modified G
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2310/3523 : Allyl
• CPC Class Codes: C12N 2310/3527 : Other alkyl chain
• CPC Class Codes: C12N 2310/3531 : Hydrogen
• CPC Class Codes: C12N 2310/3533 : Halogen
• CPC Class Codes: C12N 9/0069 : acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12Q 1/68 : involving nucleic acids
• CPC Class Codes: C12Y 113/11012 : Linoleate 13S-lipoxygenase (1.13.11.12)

Abstract

Processes for preparing 2'"'"'-O-alkylated guanosine, uridine, cytidine, and 2,6-diaminopurine 3'"'"'-O-phosphoramidites include the steps of alkylating nucleoside precursors, adding suitable blocking groups and phosphitylating. For the guanosine 2'"'"'-O-alkylated 3'"'"'-O-phosphoramidites, alkylation is effected on 2,6-diamino-9-(β-D-ribofuranosyl)purine followed by deamination. For uridine 2'"'"'-O-alkylated 3'"'"'-O-phosphoramidites, alkylation is effect on a dialkyl stannylene derivative of uridine. For cytidine 2'"'"'-O-alkylated 3'"'"'-O-phosphoramidites, alkylation is effected directly on cytidine. Alkylation is effected directly upon 2,6-diaminopurine.

Claims

1. A process for preparing a 2'"'"'-O-alkylated cytidine 3'"'"'-O-phosphoramidite comprising the steps of:

alkylating an unblocked cytidine to form a 2'"'"'-O-alkylated cytidine;

blocking the 5'"'"'-hydroxyl moiety of said 2'"'"'-O-alkylated cytidine; and

phosphitylating the 3'"'"'-position of said 5'"'"'-blocked 2'"'"'-O-alkylated cytidine.

2. The process of claim 1 wherein said alkylation is in the presence of base.

3. The process of claim 2 wherein said base is a metal hydride.

4. The process of claim 1 wherein said blocking step adds a dimethoxytrityl group.

5. The process of claim 1 wherein said phosphitylation step is conducted with bis-N,N-diisopropylaminocyanoethylphosphite.

6. The process of claim 5 wherein said phosphitylation step is conducted in the presence of N,N-diisopropylamino-hydrotetrazolide.

7. The process of claim 1 further comprising blocking the N4-amino moiety of said 2'"'"'-O-alkylated cytidine prior to blocking said 5'"'"' hydroxyl moiety.