Gapped oligomers having site specific chiral phosphorothioate internucleoside linkages

• US 6,867,294 B1
• Filed: 11/12/1999
• Issued: 03/15/2005
• Est. Priority Date: 07/14/1998
• Status: Expired due to Term

• Current Assignee: Ionis Pharmaceuticals, Inc.
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Sanghvi, Yogesh S., Manoharan, Muthiah
• Primary Examiner(s): Wilson, James O.
• Assistant Examiner(s): Owens, Jr., Howard V.
• Application Number:
  US09/438,989
• Time in Patent Office: 1,950 Days
• Field of Search: 536/24.5, 536/24.3, 536/24.31, 536/24.32, 536/24.33, 536/25.3, 536/25.31, 536/26.31, 536/22.1
• US Class Current: 536/24.5
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• 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

Novel chiral compounds that mimic and/or modulate the activity of wild-type nucleic acids are disclosed. In general, the compounds are phosphorothioate oligonucleotides wherein the 5′, and the 3′-terminal internucleoside linkages are chirally Sp and internal internucleoside linkages are chirally Rp.

Claims

1. An oligomeric compound of the formula:

5′

-(Nu₁—

L₁)_n—

Y—

(L₂-Nu₂)_p—

3′

wherein;

each Nu₁ and Nu₂, independently, has the formula;

wherein

Bx is a heterocyclic base moiety;

Lx is hydrogen, a protecting group or a substituent group;

one of R₁₂, R₁₃ and R₁₄ is hydroxyl, a protected hydroxyl, a covalent attachment to a solid support, a nucleoside, an oligonucleoside, a nucleotide, an oligonucleotide, a conjugate group or an optionally protected substituent group;

another of R₁₂, R₁₃ and R₁₄ is hydrogen, hydroxyl, a protected hydroxyl or an optionally protected substituent group;

the remaining of R₁₂, R₁₃ and R₁₄, of Nu₁, is L₁;

the remaining of R₁₂, R₁₃ and R₁₄, of Nu₂, is L₂;

each L₁ and each L₂ is, independently, a phosphodiester, phosphorodithioate;

chiral Sp phosphorothioate;

phosphoramidate;

thiophosphoramidate;

phosphonate;

methylene phosphonate;

phosphotriesters;

thionoalkylphosphonate;

thionoalkylphosphotriester;

boranophosphate;

boranothiophosphate;

thiodiester;

thionocarbamate;

siloxane;

carbamate;

sulfamate;

morpholino sulfamide;

sulfonamide;

sulfide;

sulfonate;

N,N′

-dimethylhvdrazine;

thioformacetal;

formacetal;

thioketal;

ketal;

amine (—

NH—

CH₂—

CH₂—

);

hydroxylamine;

hydroxylimine;

hydrazinyl;

amide (—

CH₂—

N(JJ)—

C(O)—

) and (—

CH₂—

C(O)—

N(JJ)—

);

oxime (—

CH₂—

O—

N═

CH—

);

or alkylphosphorus (—

C(JJ)₂—

P(═

O)(OJJ)—

C(JJ)₂—

C(JJ)₂—

) internucleoside linkage, wherein each JJ is, independently, hydrogen or C₁ to C₁₀ alkyl wherein at least one of L₁ and L₂ is other than phosphodiester;

Y has the formula;

wherein;

each Rp is a chiral Rp phosphorothioate internucleotide linkage; and

each n, m and p is, independently, from 1 to 100;

where the sum of n, m and p is from 3 to about 200;

wherein the oligomeric compound comprises from 5 to about 50 nucleosides.

2. The oligomeric compound of claim 1 wherein at least one Nu₁ or at least one Nu₂ comprises a substituent group.

3. The oligomeric compound of claim 2 wherein at least one Nu₁ and at least one Nu₂ independently comprise a substituent group.

4. The oligomeric compound of claim 1 wherein each Nu₁ and each Nu₂ independently comprises a substituent group.

5. The oligomeric compound of claim 2 wherein said substituent group is covalently attached to the 2′

, 3′

or 5′

-position of said Nu₁ or Nu₂.

6. The oligomeric compound of claim 5 wherein said substituent group is covalently attached to the 2′

-position of said Nu₁ or Nu₂.

7. The oligomeric compound of claim 1 wherein each of said substituent groups is, independently, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, C₅-C₂₀ aryl, O-alkyl, O-alkenyl, O-alkynyl, O-alkylamino, O-alkylalkoxy, O-alkylaminoalkyl, O-alkyl imidazole, thiol, S-alkyl, S-alkenyl, S-alkynyl, NH-alkyl, NH-alkenyl, NH-alkynyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido, halogen keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, heterocycle, carbocycle, polyamine, polyamide, polyalkylene glycol, and polyether;

or each substituent group has one of formula I or II;

wherein;

Z₀ is O, S or NH;

J is a single bond, O or C(═

O);

E is C₁-C₁₀ alkyl, N(R₁)(R₂), N(R₁)(R₅), N═

C(R₁)(R₂), N═

C(R₁)(R₅) or has one of formula III or IV;

each R₆, R₇, R₈, R₉ and R₁₀ is, independently, hydrogen, C(O)R₁₁, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;

or optionally, R₇ and R₈, together form a phthalimido moiety with the nitrogen atom to which they are attached;

or optionally, R₉ and R₁₀, together form a phthalimido moiety with the nitrogen atom to which they are attached;

each R₁₁ is, independently, substituted or unsubstituted C₁-C₁₀ alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl;

R₅ is T-L,

T is a bond or a linking moiety;

L is a chemical functional group, a conjugate group or a solid support material;

each R₁ and R₂ is, independently, H, a nitrogen protecting group, substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, wherein said substitution is OR₃, SR₃, NH₃⁺, N(R₃)(R₄), guanidino or acyl where said acyl is an acid amide or an ester;

or R₁ and R₂, together, are a nitrogen protecting group or are joined in a ring structure that optionally includes an additional heteroatom selected from N and O;

or R₁, T and L, together, are a chemical functional group;

each R₃ and R₄ is, independently, H, C₁-C₁₀ alkyl, a nitrogen protecting group, or R₃ and R₄, together, are a nitrogen protecting group;

or R₃ and R₄ are joined in a ring structure that optionally includes an additional heteroatom selected from N and O;

Z₄ is OX, SX, or N(X)₂;

each X is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl, C(═

NH)N(H)R₅, C(═

O)N(H)R₅ or OC(═

O)N(H)R₅;

R₅ is H or C₁-C₈ alkyl;

Z₁, Z₂ and Z₃ comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;

Z₅ is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(R₁)(R₂) OR₁, halo, SR₁ or CN;

each q₁ is, independently, an integer from 1 to 10;

each q₂ is, independently, 0 or 1;

q₃ is 0 or an integer from 1 to 10;

q₄ is an integer from 1 to 10;

q₅ is from 0, 1 or 2; and

provided that when q₃ is 0, q₄ is greater than 1.

8. The oligomeric compound of claim 1 wherein at least one R₁₄ is L₁ or L₂.

9. The oligomeric compound of claim 1 wherein at least one R₁₄ is L₁ and at least one R₁₄ is L₂.

10. The oligomeric compound of claim 1 comprising from 8 to about 30 nucleosides.

11. The oligomeric compound of claim 1 comprising from 15 to about 25 nucleosides.