Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines

• US 6,380,368 B1
• Filed: 02/12/1996
• Issued: 04/30/2002
• Est. Priority Date: 11/26/1991
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Wagner, Rick, Pudlo, Jeff, Gutierrez, Arnold J., Mattencci, Mark, Froehler, Brian, Jones, Robert J.
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US08/599,738
• Time in Patent Office: 2,269 Days
• Field of Search: 536/24.31, 536/24.5, 536/28.52, 536/28.53, 536/24.3, 536/24.32, 536/26.1, 435/6
• US Class Current: 536/22.1
• 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 19/20 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: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine

Abstract

Novel oligomers are disclosed which have enhanced ability with respect to forming duplexes or triplexes compared with oligomers containing only conventional bases. The oligomers contain the bases 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine or related analogs. The oligomers of the invention are capable of (i) forming triplexes with various target sequences such as virus or oncogene sequences by coupling into the major groove of a target DNA duplex at physiological pH or (ii) forming duplexes by binding to single-stranded DNA or to RNA encoded by target genes. The oligomers of the invention can be incorporated into pharmaceutically acceptable carriers and can be constructed to have any desired sequence, provided the sequence normally includes one or more bases that is replaced with the analogs of the invention. Compositions of the invention can be used as pharmaceutical agents to treat various diseases such as those caused by viruses and can be used for diagnostic purposes in order to detect viruses or disease conditions.

Claims

1. An oligomer comprising at least three 3′

-5′

linked nucleosides or salts thereof wherein at least one internucleoside linkage is not a phosphodiester linkage and at least one of said nucleosides comprises a base of formula (1) or (2);

wherein;

each X is independently O or S;

R² is C_2-12 1-alkynyl or R² is C_2-12 1-alkenyl optionally substituted with an alkynyl group, or R² is cyano, or R² is a C_2-12 heteroaromatic group containing 5-6 ring atoms in which one to three of the ring atoms independently is nitrogen, oxygen or sulfur, or R² is —

C≡

C—

Z wherein Z is hydrogen or C_1-10 alkyl or C_1-10 haloalkyl with 1 to 6 halogen atoms, or R² is a (1-alkynyl)-heteroaromatic group optionally substituted on a ring carbon by oxygen or C_1-4 alkyl or optionally substituted on a ring nitrogen by C_1-4 alkyl; and

Pr is (H)₂ or a protecting group,

with the proviso that when at least one of said nucleosides of said oligomer comprises 2′

-deoxyuridine 5-substituted by vinyl, 1-butenyl, 1-pentenyl, 1-hexenyl, 1-heptenyl, 1-octenyl, 1-propynyl, 1-butynyl, 1-hexynyl, 1-heptynyl, or 1-octynyl, then the remainder of the nucleosides comprising the oligomer are not solely comprised of phosphodiester linked 2′

-deoxyadenosine, 2′

-deoxyguanosine, 2′

-deoxycytidine, thymidine or a combination thereof.

2. The oligomer of claim 1 wherein X is O.

3. The oligomer of claim 1 comprising 4, 5, 6 or 7 nucleomonomers.

4. The oligomer of claim 1 comprising about 8 to 20 nucleomonomers.

5. The oligomer of claim 1 wherein R² is cyano, C_2-12 1-alkenyl or R² is C_2-12 1-alkynyl or R² is a C_2-12 heteroaromatic or 1-ethynyl-hetreoaromatic group containing 5-6 ring atoms in which one to three of the ring atoms is N, S or O.

6. The oligomer of claim 5 wherein R² is C_2-8 1-alkenyl or R² is C_2-8 1-alkynyl or R² is a C_2-8 heteroaromatic or 1-ethynyl-hetreoaromatic group containing 5-6 ring atoms in which one to three of the ring atoms is N, S or O.

7. The oligomer of claim 6 wherein R² is selected from the group consisting of phenylethynyl, 2-, 3-, and 4-pyridine-ethynyl, 2-, 4- and 5-pyrimidine-ethynyl, triazine-ethynyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 2-, 4- and 5-oxazolyl-ethynyl, 2-, 4- and 5-thiazolyl-ethynyl, 1-methyl-2-imidazolyl, 2- and 4-imidazolyl, 2-, 4- and 5-oxazolyl, 2-, 4- and 5-imidazolyl-ethynyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2- and 3-thienyl-ethynyl, 2- and 3-furanyl-ethynyl, 2- and 3-pyrrolyl-ethynyl, 2- and 3-thienyl, 2-, 4-, and 5-oxazolyl, 2- and 3-furanyl, 2- and 3-pyrrolyl, propenyl, vinyl and —

C≡

C—

Z where Z is H, alkyl (C_1-10), haloalkyl (C_1-10 with 1 to 6 halogen atoms) or heteroalkyl (C_1-10 with 1 to 3 heteroatoms).

8. The oligomer of claim 1 wherein R² is selected from the group consisting of 1-propynyl, 1-propenyl, 3-buten-1-ynyl, 3-methyl-1-butynyl, 3,3-dimethyl-1-butynyl, 1,3-pentadiynyl, 1-butynyl, ethynyl, vinyl, bromovinyl, phenylethynyl, 2-, 3-, and 4-pyridine-ethynyl, 2-, 4- and 5-pyrimidine-ethynyl, triazine-ethynyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 2-, 4- and 5-oxazolyl-ethynyl, 2-, 4- and 5-thiazolyl-ethynyl, 1-methyl-2-imidazolyl, 2- and 4-imidazolyl, 2-, 4- and 5-oxazolyl, 2-, 4- and 5-imidazolyl-ethynyl, 2-pyridinyl, 3-pyridinyl, 4-pyridinyl, 2- and 3-thienyl-ethynyl, 2- and 3-furanyl-ethynyl, 2- and 3-pyrrolyl-ethynyl, 2- and 3-thienyl, 2-, 4-, and 5-oxazolyl, 2- and 3-furanyl, and 2- and 3-pyrrolyl.

9. The oligomer of claim 1 complexed with a cationic lipid.

10. An oligomer of claim 1 comprising a positive modification comprising at least one base of formula (1) or (2) and a negative modification, with respect to the binding affinity of the oligomer to a complementary nucleic acid sequence, wherein the positive modification counteracts the effect of the negative modification to a degree that is more than additive with respect to the binding affinity.

11. The oligomer of claim 10 wherein the positive modification R² is cyano, C_2-12 1-alkenyl or 1-alkynyl or is a C_2-12 heteroaromatic or 1-ethynyl-heteroaromatic group containing 5-6 ring atoms in which one to three of the ring atoms is independently N, S or O.

12. The oligomer of claim 11 wherein the heterocycle base modification R² is C_2-8 1-alkenyl or 1-alkynyl or is a C_2-8 heteroaromatic or 1-ethynyl-heteroaromatic group containing 5 to 6 ring atoms in which one ring atom is N and optionally in which a second ring atom is N, S or O and each X is O.

19. A duplex wherein one of the two oligomers of the duplex is comprised of an oligomer of claim 1.

21. A triplex wherein one of the three oligomers of the triplex is comprised of the oligomer of claim 1.

13. An oligomer of the formula (16):

wherein

each R¹ is independently H, PO₃<sup>−

2</sup>, or a blocking group;

each R³ is independently selected from the group consisting of H, OH, F, NH₂, OCH₃, OC₂H₅, OC₃H₇, SCH₃, SC₂H₅, SC₃H₇, OC₃H₅, and SC₃H₅;

each X¹ is independently a substitute linkage selected from the group consisting of —

P(S)(O)—

, —

P(O)(O)—

, —

P(Me)(O)— and

—

P(Me)(S)—

wherein at least one of said linkages is not —

P(O)(O)—

;

n is an integer from 1 to 98; and

B is a purine or pyrimidine base, provided that at least one B is of formula (1) or (2);

 

wherein

each X is independently O or S;

R² is C_2-12 1-alkynyl, or R² is C_2-12 1-alkenyl optionally substituted with an alkynyl group, or R² is cyano, or R² is a C_2-12 heteroaromatic group containing 5-6 ring atoms in which one to three of the ring atoms is nitrogen, oxygen or sulfur, or R² is —

C≡

C—

Z wherein Z is hydrogen or C_1-10 alkyl or C_1-10 haloalkyl with 1 to 6 halogen atoms, or R² is a (1-alkynyl)-heteroaromatic group optionally substituted on a ring carbon by oxygen or C_1-4 alkyl or optionally substituted on a ring nitrogen by C_1-4 alkyl; and

Pr is H₂ or a protecting group and with the proviso that when at least one of said nucleomonomers of said oligomer comprises 2′

-deoxyuridine 5-substituted by vinyl, 1-butenyl, 1-pentenyl, 1-hexenyl, 1-heptenyl, 1-octenyl, 1-propynyl, 1-butynyl, 1-hexynyl, 1-heptynyl, or 1-otynyl, then the remainder of the nucleomonomers comprising said oligomer are not solely comprised of phosphodiester linked 2′

-deoxyadenosine, 2′

-deoxyguanosine, 2′

-deoxycytidine, thymidine or a combination thereof.

14. The oligomer of claim 13 wherein at least one B is 5-propynyluracil, 5-(1-butynyl)uracil, 5-(3-methyl-1-butynyl)uracil, 5-propynylcytosine, 5-(1-butynyl)cytosine or 5-(3-methyl-1-butynyl)cytosine.

15. The oligomer of claim 13 wherein at least one R¹ is H, PO₃⁻

2, DMT, MMT, H-phosphonate, methyl phosphonamidite, methylphosphoramidite, β

-cyanoethylphosphoramidite or alkylphosphoramidite.

16. The oligomer of claim 13 wherein each R³ is independently H, OH, or —

O-allyl.

17. The oligomer of claim 13 complexed with a cationic lipid.

18. The oligomer of claim 14 wherein the cationic lipid is DOTMA.

20. A duplex wherein one of the two oligomers of the duplex is comprised of an oligomer of claim 13.

22. A triplex wherein one of the three oligomers of the triplex is comprised of the oligomer of claim 13.

23. The oligomer of claim 13 wherein Pr is (H)₂.

24. The oligomer of claim 23 wherein n had a value of from 1 to 28.

25. The oligomer of claim 24 wherein R³ is independently selected from the group consisting of hyrogen, fluorine, O-methyl, O-ethyl, O-propyl and O-allyl.

26. The oligomer of claim 25 wherein each X¹ is independently —

P(S)(O)—

or —

P(O)(O)—

.

27. An oligomer comprising at least three 3′

-5′

linked nucleosides or salts thereof wherein at least one of said nucleosides comprises a base of formula (1) or (2);

wherein;

each X is oxygen or sulfur;

R² is C_2-12 1-alkynyl or R² is C_2-12 1-alkenyl optionally substituted with halogen or a group, or R² is cyano, or R² is a C_2-12 heteroaromatic group containing 5-6 ring atoms in which one to three of the ring atoms independently is nitrogen, oxygen or sulfur, or R² is —

C≡

C—

Z wherein Z is hydrogen or C_1-10 alkyl or C_1-10 haloalkyl with 1 to 6 halogen atoms, or R² is a (1-alkynyl)-heteroaromatic group optionally substituted on a ring carbon by oxygen or C_1-4 alkyl or optionally substituted on a ring nitrogen by C_1-4 alkyl; and

Pr is (H)₂ or a protecting group,

with the proviso that when at least one of said nucleosides of said oligomer comprises 2′

-deoxyuridine 5-substituted by vinyl, 1-butenyl, 1-pentenyl, 1-hexenyl, 1-heptenyl, 1-octenyl, 1-propynyl, 1-butynyl, 1-hexynyl, 1-heptynyl, or 1-octynyl, then the remainder of the nucleosides comprising the oligomer are not solely comprised of phosphodiester linked 2′

-deoxyadenosine, 2′

-deoxyguanosine, 2′

-deoxycytidine, thymidine or a combination thereof.