Pyrimidine derivatives and oligonucleotides containing same

• US 6,028,183 A
• Filed: 11/07/1997
• Issued: 02/22/2000
• Est. Priority Date: 11/07/1997
• Status: Expired

• Current Assignee: Daryl D. Muenchau, ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Original Assignee: Gilead Sciences Inc.
• Inventors: Lin, Kuei-Ying, Matteucci, Mark D.
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US08/966,392
• Time in Patent Office: 837 Days
• Field of Search: 536/22.1, 536/23.1, 536/25.3, 536/25.31, 536/25.32, 536/25.34, 536/25.4, 435/6, 435/87, 435/90, 544/249
• US Class Current: 536/22.1
• CPC Class Codes: C07H 19/06 Pyrimidine 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
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Compounds having structure (1) ##STR1## wherein R¹ is --H a protecting group, a linker or a binding partner; and R² and R³⁴ are as defined in the specification. The invention also provides intermediates and methods make the structure (1) compounds, as well as methods to use the compounds as labels in diagnostic assays and to enhance binding to complementary bases.

Claims

1. A compound having the structure (1):

##STR38## and tautomers, solvates and salts thereof, wherein R¹ is an oligonucleotide, a protecting group, a linker or --H;

R² is A(Z)_X1, wherein A is a spacer and Z independently is a label bonding group optionally bonded to a detectable label, but R² is not amine, protected amine, nitro or cyano;

R²⁷ is independently --CH═

, --N═

, --C(C₁ -C₈ alkyl)═

or --C(halogen)═

, but no adjacent R²⁷ are both --N═

, or two adjacent R²⁷ are taken together to form a ring having the structure, ##STR39## where R^a is independently --CH═

, --N═

, --C(C_1-8 alkyl)═

or --C(halogen)═

, but no adjacent R^a are both --N═

;

R³⁴ is --O--, --S-- or --N(CH₃)--; and

X1 is 1, 2 or 3.

2. The compound of claim 1 wherein R² is --R^2C --R^2D, wherein R^2C is a short spacer chain and R^2D is a hydrogen bond donor moiety or a moiety having a net positive charge of at least about +0.5 at pH 6-8 in aqueous solutions.

3. The compound of claim 1 wherein R² is --R⁶ --(CH₂)_t NR⁵ C(NR⁵)(NR³)₂, --R⁶ --CH₂ --CHR³¹ --N(R³)₂, --R⁶ --(R⁷)_v --N(R³)₂, --R⁶ --(CH₂)_t --N(R³)₂, --(CH₂)_1-2 --O--(CH₂)_t --N(R³)₂, ##STR40## R³ is independently --H, --CH₃, --CH₂ CH₃, --(CH₂)_w --N(R³³)₂ or a protecting group, or both R³ together are a protecting group, or when R² is --R⁶ --(CH₂)_t --N(R³)₂, one R³ is --H, --CH₃, --CH₂ CH₃, a protecting group or --(CH₂)_w --N(R³³)₂ and the other R³ is --H, --CH₃, --CH₂ CH₃, --(CH₂)_w --N(R³³)₂, --CH(N[R³³ ]₂)--N(R³³)₂, ##STR41## R⁵ is independently H or a protecting group;

R⁶ is independently --S--, --NR⁵ --, --O-- or --CH₂ --;

R⁷ is independently linear alkyl having 1, 2, 3 or 4 carbon atoms optionally substituted with one --CH═

CH--, --C.tbd.C-- or --CH₂ --O--CH₂ -- moiety, or R⁷ is cyclic alkyl having 3, 4 or 5 carbon atoms, wherein one of the linear alkyl carbon atoms is optionally substituted with a single --CH₃, --CN, ═

O, --OH or protected hydroxyl, provided that the carbon atoms in any --CH═

CH-- or --CH₂ --O--CH₂ -- moiety are not substituted with ═

O, --OH or protected hydroxyl;

R⁸ is linear alkylene having 1 or 2 carbon atoms wherein one alkylene carbon atom is optionally substituted with a single --CH₃, --CN, ═

O, --OH or protected hydroxyl, or R⁸ is absent;

R²⁸ is independently --CH₂ --, --CH(CH₃)--, --CH(OCH₃)--, --CH(OR⁵)-- or --O--, but both are not --O--;

R²⁹ is independently --N--, --N(CH₃)--, --CH--, --C(CH₃)--, but both are not --N(CH₃)--;

R³⁰ is --H or --N(R³)₂ ;

R³¹ is the side chain of an amino acid;

R³³ is independently --H, --CH₃, --CH₂ CH₃ or a protecting group;

R³⁵ is H, C₁ -C₄ alkyl or a protecting group;

R³⁶ is --H, --CH₃, --CH₂ CH₃, a protecting group or an optionally protected monosaccharide;

t is 1, 2, 3 or 4, but when R⁶ is --O--, --S-- or --NR⁵ --, t is 2, 3 or 4;

v is independently 0, 1 or 2; and

w is independently 1 or 2.

4. The compound of claim 3 wherein R² is --CH₂ --(CH₂)_t N(R³)₂, --NR⁵ --(CH₂)_t N(R³)₂, --S--(CH₂)_t N(R³)₂, --O--(CH₂)_t N(R³)₂, --O--(CH₂)_t NR⁵ C(NR⁵)(NR³)₂, --(CH₂)_1-2 --O--(CH₂)_t N(R³)₂, --R⁶ --CH₂ --CHR³¹ --N(R³)₂, --R⁶ --(R⁷)_v --N(R³)₂, --R⁶ --(CH₂)_t --NR⁵ C(NR⁵)(NR³)₂, or --CH₂ (CH₂)_t NR⁵ C(NR⁵)(NR³)₂.

5. The compound of claim 4 wherein t is 2.

6. The compound of claim 5 wherein R³ independently is --H, --CH₃ --C₂ H₅ or a protecting group.

7. The compound of claim 6 wherein R² is --O--(CH₂)₂ --NH₂, --O--(CH₂)₃ --NH₂, --O--(CH₂)₂ --N(CH₃)₂, --O--(CH₂)₃ --N(CH₃)₂, --O--(CH₂)₂ --NHCH₃, --O--(CH₂)₃ --NHCH₃, --O--CH₂ --CH(CH₃)--NH₂, --CH₂ --O--(CH₂)₂ --NH₂, --CH₂ --O--(CH₂)₃ --NH₂ or --(CH₂)₂ --O--(CH₂)₂ --NH₂.

8. The compound of claim 3 wherein t is 2 or 3.

9. The compound of claim 1 wherein R¹ comprises --H, an optionally protected monosaccharide, hydroxyl, phosphate or hydrogen phosphonate.

10. The compound of claim 1 wherein R¹ is optionally protected 2'"'"'-deoxy-R²¹ -substituted ribose, 2'"'"'-deoxyribose or ribose, wherein R²¹ is H, --OH, halogen or a moiety that enhances the nuclease stability of an oligonucleotide containing the optionally protected 2'"'"'-deoxy-R²¹ -substituted ribose, 2'"'"'-deoxyribose or ribose.

11. The compound of claim 1 having the structure designated by the numbers selected from the group consisting of (104), (105), (133), (134), (111), (112), (113), (115), (135), (136), (137), (138), (139), (120), (121), (121A), (143), (122), (123), (125), or (126):

##STR42## wherein R¹ is an optionally protected monosaccharide;

R^2A is --OH;

R⁵ is independently --H or a protecting group;

R⁶ is --O--, --S--, --NH-- or --CH₂ --,

R²¹ is H, --OH, halogen or a moiety that enhances the nuclease stability of an oligonucleotide;

R²⁴ is a halogen;

R²⁷ is independently --CH═

, --N═

, --C(C₁ -C₈ alkyl)═

or --C(halogen ═

, but no adjacent R²⁷ are both --N═

, or two adjacent R²⁷ are taken together to form a ring having the structure, ##STR43## where R^a is independently --CH═

, --N═

, --C(C_1-8 alkyl)═

or --C(halogen)═

, but no adjacent R^a are both --N═

;

R³⁴ is --O--, --S-- or --N(CH₃)--;

R³⁷ is --O--, --CH₂ -- or --CF₂ --;

R⁴⁷ is --O-- or --S--;

R⁵⁰ is --CH₂ --, --C(O)--, --(CH₂)₂ --O--(CH₂)₂ --, --(CH₂)₂ --NR⁵ --(CH₂)₂ --, --(CH₂)₂ --S--(CH₂)₂ --, --CH(N(R⁵)₂)--, --CH(COOR⁵)-- or --C(CH₃)--, --C(C₂ C₅)-- but adjacent moieties are not C(O);

R⁵² is --(CHR^52A)--(R^52B)--CHR^52A --, --CHR^52A --O--CHR^52A --, --CHR^52A --S--CHR^52A --, --CHR^52A --NR⁵ --CHR⁵² A--, C₁ -C₁₀ alkylene optionally substituted with 1 or 2 moieties selected from the group consisting of C₁ -C₆ alkyl, --OR⁵, ═

O, --NO₂, --N₃, --CN, --COOR⁵, or --N(R⁵)₂, wherein any heteroatom is separated from the nitrogen atoms that R⁵² is linked to by one methylene and one or more --CHR^52A --;

R^52A is --H or C₁ -C₆ alkyl;

R^52B is a bond;

R⁵⁹ is --R⁶ --R⁶⁰ --;

R⁶⁰ is --(CH₂)_Z3 --(R⁶¹)_Z1 --(CH₂)_Z2 --;

R⁶¹ is --O--, --S--, --NR⁵ --, --C(O)--, --CH₂ --O--CH₂ --, --CH₂ --NR⁵ --CH₂ -- or CH₂ --S--CH₂ --;

Z1 is 0 or 1;

Z2 is 1, 2 or 3;

Z3 is 1, 2 or 3;

Y is 1, 2, 3 or 4;

CBZ is carboxybenzoyl;

Fmoc is 9-fluorenylmethoxycarbonyl;

iPr is isopropyl; and

Ac is acetyl.

12. The compound of claim 1 wherein R¹ is an oligonucleotide having the structure (2):

##STR44## wherein D is --OH, protected --OH, an oligonucleotide coupling group or a solid support;

D¹ is an oligonucleotide coupling group, --OH, protected --OH or a solid support, wherein D¹ is bonded to one 2'"'"' or 3'"'"' position in the oligonucleotide of structure (2) and the adjacent 2'"'"' or 3'"'"' position in structure (2) is substituted with R²¹, provided that D and D¹ are not both an oligonucleoide coupling group or they are not both a solid support;

R⁴ is independently a phosphodiester linkage or a phosphodiester substitute linkage, wherein R⁴ is bonded to one 2'"'"' or 3'"'"' position in the structure (2) oligonucleotide and the adjacent 2'"'"' or 3'"'"' position in structure (2) is substituted with R²¹ ;

R²¹ is independently --H, --OH, halogen or a moiety that enhances the oligonucleotide against nuclease cleavage;

R³⁷ is independently --O--, --CH₂ --, --CF₂ --;

n is an integer from 0 to 98; and

B independently is a purine or pyrimidine base or a protected derivative thereof, provided that at least one B is a base of structure (3) ##STR45##

13. The compound of claim 12 wherein R⁴ is independently 3'"'"'-O--P(S)(S)--O-5'"'"', 3'"'"'-O--P(S)(O)--O-5'"'"', 3'"'"'-O--P(O)(O)--O-5'"'"', 3'"'"'-O--P(Me)(O)--O-5'"'"', 3'"'"'-NH--P(O)(O)--O-5'"'"', 3'"'"'-S--CH₂ --O-5'"'"', 2'"'"'-S--CH₂ --O-5'"'"', 3'"'"'-O--CH₂ --O-5'"'"', 2'"'"'-O--CH₂ --O-5'"'"', 3'"'"'O--P(Me)(S)--O-5'"'"', 3'"'"'-CH₂ --N(CH₃)--O-5'"'"', 2'"'"'-CH₂ --N(CH₃)--O-5'"'"', or 3'"'"'-R³⁸ --P(N₂)(O)--O-5'"'"', wherein R³⁸ independently is --O--, --CH₂ -- or --NH--;

R³⁹ is a protecting group;

R⁴⁰ independently is hydrogen, a protecting group, C₁ -C₁₂ alkyl optionally substituted with one, or two --O--, --C(O)--, --OC(O)--, --C(O)O--, --OR⁴², --SR⁴³, --C(O)NR³⁹ --, --C(O)N(R⁴¹)₂, --NR⁴¹ --, --N(R⁴¹)₂, halo, --CN, or --NO₂ moieties, or both R⁴⁰ together with the nitrogen atom to which they are attached form ##STR46## or both R⁴⁰ together are a protecting group;

R⁴¹ independently is hydrogen, a protecting group, alkyl (C₁ -C₄ or both R⁴¹ together are a protecting group;

R⁴² is hydrogen or a protecting group;

R⁴³ is C_1-6 alkyl or a protecting group; and

R⁴⁵ is --H, a counter ion or ##STR47## R⁴⁶ is alkyl containing 1-8 carbon atoms.

14. The compound of claim 12 wherein R² is --R⁶ --(CH₂)_t NR⁵ C(NR⁵)(NR³)₂, --R⁶ --CH₂ --CHR³¹ --N(R³)₂, --R⁶ --(R⁷)_v --N(R³)₂, --R⁶ --(CH₂)_t --N(R³)₂, --(CH₂)_1-2 --O--(CH₂)_t --N(R³)₂, ##STR48## R³ is independently --H, --CH₃, --CH₂ CH₃, --(CH₂)_w --N(R³³)₂ or a protecting group, or both R³ together are a protecting group, or when R² is --R⁶ --(CH₂)_t --N(R³)₂, one R³ is --H, --CH₂ CH₃, a protecting group or --(CH₂)_w --N(R³³)₂ and the other R³ is --H, --CH₃, --CH₂ CH₃, --(CH₂)_w --N(R³³)₂, --CH(N[R³³ ]₂)--N(R³³)₂, ##STR49## R⁵ is independently H or a protecting group;

R⁶ is independently --S--, --NR⁵ --, --O-- or --CH₂ --;

R⁷ is independently linear alkyl having 1, 2, 3 or 4 carbon atoms optionally substituted with one --CH═

CH--, --C.tbd.C-- or --CH₂ --O--CH₂ -- moiety, or R⁷ is cyclic alkyl having 3, 4 or 5 carbon atoms, wherein one of the linear alkyl carbon atoms is optionally substituted with a single --CH₃, --CN, ═

O, --OH or protected hydroxyl, provided that the carbon atoms in any --CH═

CH-- or --CH₂ --O--CH₂ -- moiety are not substituted with ═

O, --OH or protected hydroxyl;

R⁸ is linear alkylene having 1 or 2 carbon atoms wherein one alkylene carbon atom is optionally substituted with a single --CH₃, --CN, ═

O, --OH or protected hydroxyl, or R⁸ is absent;

R²⁸ is independently --CH₂ --, --CH(CH₃)--, --CH(OCH₃)--, --CH(OR⁵)-- or --O--, but both are not --O--;

R²⁹ is independently --N--, --N(CH₃)--, --CH--, --C(CH₃)--, but both are not --N(CH₃)--;

R³⁰ is --H or --N(R³)₂ ;

R³¹ is the side chain of an amino acid;

R³³ is independently --H, --CH₃, --CH₂ CH₃ or a protecting group;

R³⁵ is H, C₁ -C₄ alkyl or a protecting group;

R³⁶ is --H, --CH₃, --CH₂ CH₃, a protecting group or an optionally protected monosaccharide;

t is 1, 2, 3 or 4, but when R⁶ is --O--, --S-- or --NR⁵ --, t is 2, 3 or 4;

v is independently 0, 1 or 2; and

w is independently 1 or 2.

15. The compound of claim 14 wherein R² is --CH₂ --(CH₂)_t N(R³)₂, --NR⁵ --(CH₂)_t N(R³)₂, --S--(CH₂)_t N(R³)₂, --O--(CH₂)_t N(R³)₂, --O--(CH₂)_t NR⁵ C(NR⁵)(NR³)₂, --(CH₂)_1-2 --O--(CH₂)_t N(R³)₂, --R⁶ --CH₂ --CHR³¹ --N(R³)₂, --R⁶ --(R⁷)_v --N(R³)₂, --R⁶ --(CH₂)_t --NR⁵ C(NR⁵)(NR³)₂, or --CH₂ (CH₂)_t NR⁵ C(NR⁵)(NR³)₂.

16. The compound of claim 15 wherein t is 2 or 3.

17. The compound of claim 16 wherein R³ independently is --H, --CH₃, --C₂ H₅ or a protecting group.

18. The compound of claim 17 wherein R² is --O--(CH₂)₂ --NH₂, --O--(CH₂)₃ --NH₂, --O--(CH₂)₂ --N(CH₃)₂, --O--(CH₂)₃ --N(CH₃)₂, --O--(CH₂)₂ --NHCH₃, --O--(CH₂)₃ --NHCH₃, --O--CH₂ --CH(CH₃)--NH₂, --CH₂ --O--(CH₂)₂ --NH₂, --CH₂ --O--(CH₂)₃ --NH₂ or --(CH₂)₂ --O--(CH₂)₂ --NH₂.

19. The compound of claim 12 wherein R²¹ is independently --H, --OH, halogen, protected hydroxyl, --O-methyl, --O-ethyl, --O-n-propyl, --O-allyl, --O--(CH₂)₂ OH, --O--(CH₂)₃ OH, --O--(CH₂)₂ F, --O--(CH₂)_s R⁶⁵, --O--(CH₂)₂ --[O--(CH₂)₂ ]_r R⁶⁵, --O--(CH₂)_r --O--(CH₂)_r --O--(CH₂)_r --R⁶⁵), --NH-methyl, --NH-ethyl, --NH-n-propyl, --NH--(CH₂)₂ OH, --NH--(CH₂)₃ OH, --NH--(CH₂)_s R⁶⁵, --S-methyl, --S-ethyl, --S-n-propyl, --S-allyl, --S--(CH₂)₂ OH, --S--(CH₂)₃ OH --S--(CH₂)₂ F, --S--(CH₂)_s R⁶⁵, or --S--(CH₂)₂ --[O--(CH₂)₂ ]_r R⁶⁵, wherein

R⁶⁵ is --H, --F, --OH, --OCH₃, --NH₂, --SH, protected hydroxyl, protected amino or protected thiol;

r is 1, 2, 3 or 4; and

s is 2, 3, 4, 5, 6, 7 or 8.

20. The compound of claim 19 wherein R²¹ is independently --H, --OH, --F, protected hydroxyl, --OCH₃, --O--CH₂ CH₃, --O--CH₂ CH₂ OH, --O--CH₂ CH₂ F, --O--CH₂ CH₂ CH₃, --O--CH₂ CH₂ CH₂ OH, --O--CH₂ CH₂ CH₂ F, --O--CH₂ CF₂ H, --O--CH₂ CF₃ or --O--CH₂ CH₂ --O--CH₃.

21. The compound of claim 12 wherein B independently are selected from the group consisting of a base of structure (3), guanosine, adenine, thymine, uracil, cytosine, 5-methylcytosine, 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine, 5-(1-butynyl)uracil therefor 5-(1-butynyl)cytosine.

22. The compound of claim 12 wherein D¹ is H-phosphonate, a methylphosphonamidite, a β

-cyanoethylphosphoramidite or a phosphoramidite.

23. A compound having the structure (4) ##STR50## and tautomers, solvates and salts thereof wherein, R¹, R² and R²⁷ have the meanings given in claim 1;

R²⁴ is a halogen;

R²⁵ is --SH, --OH, ═

S or ═

O.

24. The compound of claim 23 wherein R¹ is --H or an optionally protected monosaccharide.

25. The compound of claim 24 wherein the optionally protected monosaccharide is 2'"'"'-deoxy-R²¹ -substituted ribose, wherein R²¹ is H, --OH, halogen or a moiety that enhances the nuclease stability of an oligonucleotide containing the optionally protected 2'"'"'-deoxy-R²¹ -substituted ribose, 2'"'"'-deoxyribose or ribose.

26. The compound of claim 25 wherein R²¹ is --H, --OH, --F, protected hydroxyl, --OCH₃, --O--CH₂ CH₃, --O--CH₂ CH₂ OH, --O--CH₂ CH₂ F, --O--CH₂ CH₂ CH₃, --O--CH₂ CH₂ CH₂ OH, --O--CH₂ CH₂ CH₂ F, --O--CH₂ CF₂ H, --O--CH₂ CF₃ or --O--CH₂ CH₂ --O--CH₃.

27. The compound of claim 1 having the structure (1):

##STR51## and tautomers, solvates and salts thereof, wherein R¹ is a protecting group, an oligonucleotide, a nucleic acid, a polysaccharide, an optionally protected monosaccharide, hydroxyl, phosphate, hydrogen phosphonate, halo, azido, protected hydroxyl, or --H;

R² is A(Z)_X1, but R² is not amine, protected amine, nitro or cyano;

R⁵ is H or a protecting group;

R²⁷ is independently --CH═

, --N═

, --C(C₁ -C₈ alkyl)═

or --C(halogen)═

, but no adjacent R²⁷ are both --N═

, or two adjacent R²⁷ are taken together to form a ring having the structure, ##STR52## R³⁴ is --O--, --S-- or --N(CH₃)--;

R^a is independently --CH═

, --N═

, --C(C_1-8 alkyl)═

or --C(halogen)═

, but no adjacent R^a are both --N═

;

A is a backbone chain of 2-16 carbon atoms, any 1, 2 or 3 of which are optionally replaced with N, O or S atoms, wherein the backbone chain is optionally substituted independently with 1, 2 or 3 of the following;

C₁ -C₈ alkyl, --OR⁵, ═

O, --NO₂, --N₃, --COOR⁵, --N(R⁵)₂, or --CN groups, C₁ -C₈ alkyl substituted with --OH, ═

O, --NO₂, --N₃, --COOR⁵, --N(R⁵)₂, or --CN groups, or any of the foregoing in which --CH₂ -- is replaced with --O--, --NH-- or --N(C₁ -C₈ alkyl);

X1 is 1, 2 or 3;

Y is H, 2-hydroxypyridine, N-hydroxysuccinimide, p-nitrophenyl, acylimidazole, maleimide, trifluoroacetate, an imido, a sulfonate, an imine 1,2-cyclohexanedione, glyoxal or an alpha-halo ketone; and

Z independently is --NH₂, --CHO, --SH, --CO₂ Y, OY.

28. The compound of claim 27 wherein Z is bonded to a detectable label.

29. The compound of claim 27 wherein R¹ is an oligonucleotide.

30. The compound of claim 27 wherein R¹ is an optionally protected monosaccharide.