IP Library › Granted Patent US 7,674,778

Granted Patent B2

US 7,674,778 · App. 11/119,533 · Granted Mar 9, 2010

Oligonucleotides comprising a conjugate group linked through a C5-modified pyrimidine

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Quick Facts

Patent No.: US 7,674,778
App. No.: 11/119,533
Granted: Mar 9, 2010
Kind: B2

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Abstract

One aspect of the present invention relates to a double-stranded oligonucleotide comprising at least one ligand. In certain embodiments, a ligand is bound to only one of the two oligonucleotide strands comprising the double-stranded oligonucleotide. In certain embodiments, both of the oligonucleotide strands of the double-stranded oligonucleotide independently comprise a bound ligand. In certain embodiments, the oligonucleotide strands comprise at least one modified sugar moiety. In certain embodiments, a phosphate linkage in one or both of the strands of the oligonucleotide has been replaced with a phosphorothioate or phosphorodithioate linkage. In a preferred embodiment, the ligand is cholesterol or 5β-cholanic acid. Another aspect of the present invention relates to a single-stranded oligonucleotide comprising at least one ligand. In certain embodiments, the oligonucleotide comprises at least one modified sugar moiety. In certain embodiments, a phosphate linkage of the oligonucleotide has been replaced with a phosphorothioate or phosphorodithioate linkage. In a preferred embodiment, the ligand is cholesterol or 5β-cholanic acid. The ligand improves the pharmacokinetic properties of the oligonucleotide.

Claims (110)

1. A single-stranded siRNA or microRNA represented by formula I:

wherein

X is H, —P(O)(OM) 2 , —P(O)(OM)-O—P(O)(OM) 2 , —P(O)(Oalkyl) 2 , or —P(O)(Oalkyl)-O—P(O)(Oalkyl) 2 ;

M represents independently for each occurrence an alkali metal or a transition metal with an overall charge of +1;

n is 16, 17, 18, 19, 20, 21, 22, 23, or 24;

R 1 , R 2 , and R 5 represent independently for each occurrence H, OH, F, —Oalkyl, —Oallyl, —O(C(R 7 ) 2 ) v OR 7 , —O(C(R 7 ) 2 ) v SR 7 , —O(C(R 7 ) 2 ) v N(R 7 ) 2 , —O(C(R 7 ) 2 ) v C(O)N(R 7 ) 2 , —N(R 7 ) 2 , —S(C 1 -C 6 )alkyl, —O(C(R 7 ) 2 ) v O(C 1 -C 6 )alkyl, —O(C(R 7 ) 2 ) v S(C 1 -C 6 )alkyl, —O(C(R 7 ) 2 ) v O(C(R 7 ) 2 ) v N((C 1 -C 6 )alkyl) 2 , or —O(C(R 7 ) 2 ) v ON((C 1 -C 6 )alkyl) 2 ;

A 1 represents independently for each occurrence:

Z 1 represents independently for each occurrence O or S;

Z 2 represents independently for each occurrence —OH, —OM, —Oalkyl, —Oaryl, —Oaralkyl, —SH, SM, —Salkyl, —Saryl, —Saralkyl, —N(R 3 )R 4 , —(C(R 7 ) 2 ) m N(R 7 ) 2 , —N(R 7 )(C(R 7 ) 2 ) m N(R 7 ) 2 , or alkyl;

R 3 and R 4 are H or alkyl; or R 3 and R 4 taken together form a 3-, 4-, 5-, 6-, or 7-member ring;

R 6 represents independently for each occurrence H, alkyl, or —NHCH 2 CH═CH 2 ;

R 7 represents independently for each occurrence H or alkyl;

A 2 represents independently for each occurrence:

A 3 represents independently for each occurrence

A 4 represents independently for each occurrence the formula:

B 1 represents independently for each occurrence a bond between A 3 and A 4 ;

B 2 represents independently for each occurrence a bond between A 4 and A 5 ;

R represents independently for each occurrence hydrogen or alkyl;

m represents independently for each occurrence 1, 2, 3, 4, 5, 6, 7 or 8;

m 1 represents independently for each occurrence 0, 1, 2, 3, 4, 5, 6, 7 or 8;

v represents independently for each occurrence 1, 2, 3, or 4;

w represents independently for each occurrence 1, 2, or 3 in accord with the rules of valence;

provided that A 5 occurs at least once; and

A 5 represents independently for each occurrence compound C, compound C′, compound D, compound D′ or a radical of a steroid, bile acid, lipid, folic acid, pyridoxal, B12, riboflavin, aromatic compound, polycyclic compound, crown ether, intercalator, cleaver molecule, protein-binding agent, carbohydrate, or an optionally substituted saturated 5-membered ring; wherein compound C is represented by:

and compound C′ is represented by:

wherein for C and C′

R 25 represents independently for each occurrence H, halogen, hydroxyl, thiol, alkoxyl, thioalkyl, azido, —N(R 27 ) 2 , —CO 2 R 27 , —COR 27 , —OC(O)R 27 , alkyl, alkenyl, aryl, or aralkyl;

R 26 represents independently for each occurrence alkyl alkenyl, aryl, or aralkyl;

R 27 represents independently for each occurrence H, alkyl, alkenyl, aryl, or aralkyl;

R 28 represents independently for each occurrence —OC(O)R 27 , —N(R 27 )C(O)R 27 , —N(R 27 ) 2 , —CO 2 R 27 , —COR 27 , alkyl, alkenyl, aryl, or aralkyl; and

p represents independently for each occurrence 1, 2, 3, 4, 5, 6, or 7; and

compound D is represented by:

and compound D′ is represented by:

wherein for D and D′

R 29 represents independently for each occurrence H, halogen, hydroxyl, thiol, alkoxyl, thioalkyl, azido, —N(R 30 ) 2 , —CO 2 R 30 , —COR 30 , —OC(O)R 30 , alkyl, alkenyl, aryl, or aralkyl;

R 30 represents independently for each occurrence H, alkyl, alkenyl, aryl, or aralkyl;

R 31 represents independently for each occurrence alkyl, alkenyl, aryl, or aralkyl; and

t represents independently for each occurrence 1, 2, 3, 4, 5, 6, or 7.

2. The single-stranded siRNA or microRNA of claim 1 , wherein n is 18, 19, 20, 21, or 22.

3. The single-stranded siRNA or microRNA of claim 1 , wherein n is 20.

4. The single-stranded siRNA or microRNA of claim 1 , wherein A 5 occurs at least twice.

5. The single-stranded siRNA or microRNA of claim 1 , wherein A 5 represents independently for each occurrence compound C, compound D, or a radical of cholesterol, 5β-cholanic acid, progesterone, aldosterone, dehydroaldosterone, isoandrosterone, esterone, estradiol, ergosterol, dehydroergosterol, lanosterol, 4-cholesten-3-one, guggulsterone, testosterone, nortestosterone, formestane, hydroxyecdysone, ketoestriol, corticosterone, dienestrol, dihydroxypregnanone, pregnanone, copornmon, equilenin, equilin, estriol, ethinylestradiol, mestranol, moxestrol, mytatrienediol, quinestradiol, quinestrol, helvolic acid, protostadiene, fuisidic acid, cycloartenol, tricallol, cucurbitanin cedrelone, euphol, dammerenediol, parkeol, dexametasone, methylprednisolone, prednisolone, hydrocortisone, parametasone, betametasone, cortisone, fluocinonide, fluorometholone, halcinonide, budesonide, or any one of them further substituted with one or more of hydroxyl, halogen, amino, alkylamino, alkyl, carboxylic acid, ester, amide, carbonyl, alkoxyl, or cyano.

6. The single-stranded siRNA or microRNA of claim 1 , wherein A 5 represents independently for each occurrence a radical of cholesterol or 5β-cholanic acid.

7. The single-stranded siRNA or microRNA of claim 1 , wherein A 5 represents independently for each occurrence:

wherein R 20 represents independently for each occurrence hydroxyl, amino, halogen, alkoxyl, alkyl, aminoalkyl, azido, carbonyl, acyl, or acyloxy; Z 3 represents independently for each occurrence O, S, or NR; and y represents independently for each occurrence 0, 1, 2, 3, 4, 5, or 6 in accord with the rules of valence.

8. A double-stranded siRNA or microRNA comprising a first strand and a second strand, wherein said first strand and said second strand are represented independently by formula II:

X is H, —P(O)(OM) 2 , —P(O)(OM)-O—P(O)(OM) 2 , —P(O)(Oalkyl) 2 , or —P(O)(Oalkyl)-O—P(O)(Oalkyl) 2 ;

M represents independently for each occurrence an alkali metal or a transition metal with an overall charge of +1;

n is 16, 17, 18, 19, 20, 21, 22, 23, or 24;

R 1 , R 2 , and R 5 represent independently for each occurrence H, OH, F, —Oalkyl, —Oallyl, —O(C(R 7 ) 2 ) v OR 7 , —O(C(7) 2 ) v SR 7 , —O(C(R 7 ) 2 ) v N(R 7 ) 2 , —O(C(R 7 ) 2 ) v C(O)N(R 7 ) 2 , —N(R 7 ) 2 , —S(C 1 -C 6 )alkyl, —O(C(R 7 ) 2 ) v O(C 1 -C 6 )alkyl, —O(C(R 7 ) 2 ) v S(C 1 -C 6 )alkyl, —O(C(R 7 ) 2 ) v O(C(R 7 ) 2 ) v N((C 1 -C 6 )alkyl) 2 , or —O(C(R 7 ) 2 ) v ON((C 1 -C 6 )alkyl) 2 ;