Backbone-modified oligonucleotide analogs and preparation thereof through radical coupling

• US 5,618,704 A
• Filed: 09/02/1994
• Issued: 04/08/1997
• Est. Priority Date: 07/27/1990
• Status: Expired due to Term

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Cook, Phillip D., Sanghvi, Yogesh S.
• Primary Examiner(s): LOW, CHRISTOPHER S F
• Application Number:
  US08/300,072
• Time in Patent Office: 949 Days
• Field of Search: 536/22.1, 435/91.1, 435/91.21, 435/91.41, 435/91.5, 935/16
• US Class Current: 435/91.5
• CPC Class Codes: C07D 405/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 405/14 containing three or more hetero rings
• CPC Class Codes: C07F 7/1804 Compounds having Si-O-C linkages Si-O-acyl linkages C07F7/1896
• 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 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

Abstract

Methods for preparing oligonucleotide analogs which have improved nuclease resistance and improved cellular uptake are provided. In preferred embodiments, the methods involve radical coupling of 3'"'"'- and 5'"'"'-substituted or 5'"'"'- and 3'"'"'-substituted nucleosidic synthons.

Claims

1. A method for forming a covalent, internucleoside linkage having structure 3'"'"'-CH₂ --R_A --NH--CH₂ -4'"'"', 3'"'"'-CH₂ --NH--R_A --CH₂ -4'"'"', 3'"'"'-CH₂ --CH₂ --NH--R_A -4'"'"', or 3'"'"'-R_A --NH--CH₂ --CH₂ -4'"'"' where R_A is O or NR₁ comprising the steps of:

(a) providing synthons having the structure;

##STR8## wherein Z₁ and Y₂ are selected such that

(i) Z₁ is R_B and Y₂ is CH₂ --R_A --N═

CH₂ ;

or

(ii) Z₁ is CH₂ --R_B and Y₂ is R_A --N═

CH₂ ;

or

(iii) Z₁ is CH₂ --R_A --N═

CH₂ and Y₂ is R_B ;

or

(iv) Z₁ is R_A --N═

CH₂ and Y₂ is CH₂ --R_B ; and

where the synthon bearing said R_B group is a donor synthon and the synthon bearing said N═

CH₂ group is an acceptor synthon;

R₁ is H or alkyl having 1 to 10 carbon atoms;

R_B is a radical generating group selected from I, OC(S)O--C₆ H₅, Se--C₆ H₅, OC(S)O--C₆ F₅, OC(S)O--C₆ Cl₅, OC(S)O--(2,4,6-C₆ Cl₃), Br, NO₂, Cl, OC(S)S--Me, OC(S)O--(p-CH₄ F), bis-dimethylglyoximato-pyridine cobalt, OC(S)C₆ H₅, OC(S)SCH₃, OC(S)-imidazole, and OC(O)O-pyridin-2-thione;

Y₁ and Z₂ are, independently, H, hydroxyl, aminomethyl, hydrazinomethyl, hydroxymethyl, C-formyl, phthalimidohydroxymethyl, aryl-substituted imidazolidino, aminohydroxylmethyl, ortho-methylaminobenzenethio, methylphosphonate, methylalkylphosphonate, a nucleoside, a nucleotide, an oligonucleotide, an oligonucleoside, or a hydroxyl-protected or amine-protected derivative thereof;

B_x1 and B_x2 are, independently, nucleosidic bases;

Q₁ and Q₂ are, independently, O, S, CH₂, CHF or CF₂ ; and

X₁ and X₂ are, independently, H, OH, alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF₃, OCF₃, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino or silyl, an RNA cleaving group said alkyl group having 1 to 10 carbon atoms, said alkaryl and aralkyl group having 7 to 14 carbon atoms, and said alkenyl group having 2 to 10 carbon atoms;

(b) contacting said donor synthon with a radical species for a time and under reaction conditions effective to generate a radical-bearing donor synthon having a radical centered at said Z₁ or Y₂ ; and

(c) contacting said radical-bearing donor synthon with said acceptor synthon for a time and under reaction conditions effective to form said covalent linkage.

2. The method of claim 1 wherein R_A is O.

3. The method of claim 1 wherein R_A is NH or NCH₃.

4. The method of claim 1 wherein Z₁ is CH₂ --R_B and Y₂ is R_A --N═

CH₂.

5. The method of claim 1 wherein Z₁ is R_A --N═

CH₂ and Y₂ is CH₂ --R_B.

6. The method of claim 1 wherein Z₁ is R_B and Y₂ is CH₂ --R_A --N═

CH₂.

7. The method of claim 1 wherein Z₁ is CH₂ --R_A --N═

CH₂ and Y₂ is R_B.

8. The method of claim 1 wherein said radical generating group is I.

9. The method of claim 1 wherein said radical species is bis(trimethylstannyl)-benzopinacolate.

10. The method of claim 1 wherein said contacting step (b) is effected in a solvent selected from benzene, dichlorobenzene, t-butylbenzene, t-butyl alcohol, water, acetic acid, chloroform, and carbon tetrachloride.