Backbone modified oligonucleotide analogs and preparation thereof through reductive coupling

• US 6,025,482 A
• Filed: 09/14/1998
• Issued: 02/15/2000
• Est. Priority Date: 07/27/1990
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Cook, Phillip Dan, Sanghvi, Yogesh S.
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US09/152,958
• Time in Patent Office: 519 Days
• Field of Search: 536/23.1, 536/25.3, 536/25.32, 536/25.33, 536/25.34, 536/25.6, 536/27.14, 536/28.2, 536/4.1, 536/26.8, 536/26.7, 536/26.26, 536/22.1
• US Class Current: 536/23.1
• CPC Class Codes: A61K 31/70 Carbohydrates; Sugars; Derivatives thereof sorbitol A61K31/047
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• 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 reductive coupling of 3'"'"'- and 4'"'"'- substituted or 4'"'"'- and 3'"'"'-substituted nucleosidic synthons.

Claims

1. A method for forming a covalent linkage having structure CH═

N--R_A --CH₂, CH₂ --R_A --N═

CH, or R_A --N═

CH--CH₂ where R_A is O or NR₁, comprising the steps of;

(a) providing synthons having structure;

##STR4## (b) contacting said synthons for a time and under reaction conditions effective to form said covalent linkage;

wherein;

Z₁ and Y₂ are selected such that

(i) Z₁ is C(O)H and Y₂ is CH₂ R_A NH₂ ;

or

(ii) Z₁ is CH₂ R_A NH₂ and Y₂ is C(O)H;

(iii) Z₁ is CH₂ C(O)H and Y₂ is R_A NH₂ ;

or

(iv) Z₁ is R_A NH₂ and Y₂ is H(O)CCH₂ ;

R₁ and R₂ are the same or different and are H;

alkyl or substituted alkyl having 1 to about 10 carbon atoms;

alkenyl or substituted alkenyl having 2 to about 10 carbon atoms;

alkynyl or substituted alkynyl having 2 to about 10 carbon atoms;

alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl having 7 to about 14 carbon atoms;

alicyclic;

heterocyclic;

a reporter molecule;

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 or substituted alkyl having 1 to about 10 carbon atoms;

alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl having 7 to about 14 carbon atoms;

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 substituted silyl.

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 C(O)H and Y₂ is CH₂ R_A NH₂.

5. The method of claim 1 wherein Z₁ is CH₂ R_A NH₂ and Y₂ is C(O)H.

6. The method of claim 1 wherein Z₁ is CH₂ C(O)H and Y₂ is R_A NH₂.

7. The method of claim 1 wherein Z₁ is R_A NH₂ and Y₂ is H(O)CCH₂.

8. A method for forming a covalent linkage having structure CH₂ --NR₁ --R_A --CH₂, CH₂ --R_A --NR₁ --CH₂, R_A --NR₁ --CH₂ --CH₂, or NR₁ --R_A --CH₂ --CH₂, where R_A is O or NR₂, comprising the steps of:

(a) providing synthons having structures;

##STR5## (b) contacting said synthons for a time and under reaction conditions effective to form an intermediate linkage; and

(c) reducing said intermediate linkage to form said covalent linkage;

wherein;

Z₁ and Y₂ are selected such that

(i) Z₁ is C(O)H and Y₂ is CH₂ R_A NH₂ ;

or

(ii) Z₁ is CH₂ R_A NH₂ and Y₂ is C(O)H;

(iii) Z₁ is CH₂ C(O)H and Y₂ is R_A NH₂ ;

or

(iv) Z₁ is R_A NH₂ and Y₂ is H(O)CCH₂ ;

R₁ and R₂ are, independently, H;

alkyl or substituted alkyl having 1 to about 10 carbon atoms;

alkenyl or substituted alkenyl having 2 to about 10 carbon atoms;

alkynyl or substituted alkynyl having 2 to about 10 carbon atoms;

alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl having 7 to about 14 carbon atoms;

alicyclic;

heterocyclic;

a reporter molecule;

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 or substituted alkyl having 1 to about 10 carbon atoms;

alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl having 7 to about 14 carbon atoms;

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 substituted silyl.

9. The method of claim 8 wherein R_A is O.

10. The method of claim 8 wherein R_A is NH or NCH₃.

11. The method of claim 8 wherein Z₁ is C(O)H and Y₂ is CH₂ R_A NH₂.

12. The method of claim 8 wherein Z₁ is CH₂ R_A NH₂ and Y₂ is C(O)H.

13. The method of claim 8 wherein Z₁ is CH₂ C(O)H and Y₂ is R_A NH₂.

14. The method of claim 8 wherein Z₁ is R_A NH₂ and Y₂ is H(O)CCH₂.

15. A compound having structure:

##STR6## wherein;

Z₁ is C(O)H, CH₂ C(O)H, CH₂ R_A NH₂, or R_A NH₂ ;

R_A is O or NR₁ ;

Y₁ is hydroxymethyl, a nucleoside, a nucleotide, an oligonucleotide, an oligonucleoside, or a hydroxyl-protected or amine-protected derivative thereof;

R₁ is H;

alkyl or substituted alkyl having 1 to about 10 carbon atoms;

alkenyl or substituted alkenyl having 2 to about 10 carbon atoms;

alkynyl or substituted alkynyl having 2 to about 10 carbon atoms;

alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl having 7 to about 14 carbon atoms;

alicyclic;

heterocyclic;

a reporter molecule;

B_X1 is a nucleosidic base;

Q₁ is O, S, CH₂, CHF or CF₂ ; and

X₁ is H;

OH;

alkyl or substituted alkyl having 1 to about 10 carbon atoms;

alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl having 7 to about 14 carbon atoms;

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 substituted silyl.

16. The compound of claim 15 wherein R_A is O.

17. The compound of claim 15 wherein R_A is NH.

18. The compound of claim 15 wherein Z₁ is C(O)H or CH₂ C(O)H.

19. The compound of claim 15 wherein Z₁ is CH₂ R_A NH₂ or R_A NH₂.

20. A compound having structure:

##STR7## wherein;

Y₂ is C(O)H, CH₂ C(O)H, CH₂ R_A NH₂, or R_A NH₂ ;

R_A is O or NR₂ ;

Z₂ is hydroxymethyl, a nucleoside, a nucleotide, an oligonucleotide, an oligonucleoside, or a hydroxyl-protected or amine-protected derivative thereof;

R₁ is H;

alkyl or substituted alkyl having 1 to about 10 carbon atoms;

alkenyl or substituted alkenyl having 2 to about 10 carbon atoms;

alkynyl or substituted alkynyl having 2 to about 10 carbon atoms;

alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl having 7 to about 14 carbon atoms;

alicyclic;

heterocyclic;

a reporter molecule;

B_X1 is a nucleoside base;

Q₁ is O, S, CH₂, CHF or CF₂ ; and

X₂ is H;

OH;

alkyl or substituted alkyl having 1 to about 10 carbon atoms;

alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl having 7 to about 14 carbon atoms;

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 substituted silyl.

21. The compound of claim 20 wherein R_A is O.

22. The compound of claim 20 wherein R_A is NH.

23. The compound of claim 20 wherein Y₂ is C(O)H or CH₂ C(O)H.