Phosphorodiamidate backbone linkage for oligonucleotides
This invention relates to antisense oligonucleotides comprising at least one N3′→P5′ phosphorodiamidate linkage (NPN) in the backbone, useful for modulating gene expression involved in the pathogenesis of a disease. Compounds useful as building blocks of said antisense oligonucleotides and methods of preparing building block compounds including NPN linkages are provided.
1. A compound of formula (II):
wherein
R 1 is an acid labile amino protecting group;
R 3 is independently selected from hydrogen, hydroxyl, and —O—R 3a ;
wherein R 3a is C 1-6 alkyl or C 1-6 alkyl substituted with —NR 3b R 3c , imidazolyl, —(CH 2 ) a O(CH 2 ) b NR 3b R 3c , or —(CH 2 ) a ONR 3d (CH 2 ) b NR 3b R 3c ;
wherein R 3b is hydrogen or C 1-2 alkyl;
R 3c is hydrogen or C 1-2 alkyl;
R 3d is hydrogen or C 1-2 alkyl;
a is an integer selected from one to 4;
b is an integer selected from one to 4;
R 4 is C 1-6 alkyl or C 1-6 alkyl substituted with —NR 4a R 4b ;
wherein R 4a is hydrogen or C 1-2 alkyl and R 4b is hydrogen or C 1-2 alkyl;
R 5 is C 1-6 alkyl or C 1-6 alkyl substituted with —NR 5a R 5b ;
wherein R 5a is hydrogen or C 1-2 alkyl and R 5b is hydrogen or C 1-2 alkyl; or
R 4 and R 5 taken together with the nitrogen to which they are attached form a monocyclic heterocyclyl or a monocyclic heterocyclyl substituted with C 1-6 alkyl, sperminyl or spermidinyl;
R 6 is a leaving group;
W is independently selected from O, S, and Se; and
B 1 is a heterocyclic base moiety or a protected heterocyclic base moiety;
or a salt thereof.
2. The compound of claim 1 , or a salt thereof, wherein W is O.
3. The compound of claim 1 , or a salt thereof, wherein B 1 is selected from purinyl and pyrimidinyl.
4. The compound of claim 1 , or a salt thereof, wherein B 1 is selected from 4-benzoyl-1-cytosinyl, 6-benzoyl-9-adeninyl, 6-dimethylformamidino-9-adeninyl, 2-isobutyryl-9-guaninyl, 2-dimethylformamidino-9-guaninyl, 9-adeninyl, 9-guaninyl, 1-cytosinyl, 1-thyminyl and 1-uracilyl.
5. The compound of claim 1 , or a salt thereof, wherein B 1 is selected from 9-adeninyl, 9-guaninyl, 1-cytosinyl, 1-thyminyl and 1-uracilyl.
6. The compound of claim 1 , or a salt thereof, wherein R 3 is hydrogen.
7. The compound of claim 1 , or a salt thereof, wherein R 4 and R 5 are methyl.
8. The compound of claim 1 , or a salt thereof, wherein R 1 is selected from trityl, dimethoxytrityl and methoxytrityl.
9. The compound of claim 1 , or a salt thereof, wherein R 6 is selected from chloro, iodo, bromo, fluoro, methanesulfonyloxy, tosyloxy, triflyloxy, nitro-phenylsulfonyloxy and bromo-phenylsulfonyloxy.
10. The compound of claim 1 , or a salt thereof, wherein R 6 is halo.
11. The compound of claim 1 , or a salt thereof, wherein R 1 is selected from trityl, dimethoxytrityl and methoxytrityl and R 6 is halo.
12. The compound of claim 1 , or a salt thereof, wherein the compound is selected from:
13. A method of preparing a compound of formula (II):
wherein
R 1 is an acid labile amino protecting group;
R 3 is independently selected from hydrogen, hydroxyl, and —O—R 3a ;
wherein R 3a is C 1-6 alkyl or C 1-6 alkyl substituted with —NR 3b R 3c , imidazolyl, —(CH 2 ) a O(CH 2 ) b NR 3b R 3c , or —(CH 2 ) a ONR 3d (CH 2 ) b NR 3b R 3c ;
wherein R 3b is hydrogen or C 1-2 alkyl;
R 3c is hydrogen or C 1-2 alkyl;
R 3d is hydrogen or C 1-2 alkyl;
a is an integer selected from one to 4;
b is an integer selected from one to 4;
R 4 is C 1-6 alkyl or C 1-6 alkyl substituted with —NR 4a R 4b ;
wherein R 4a is hydrogen or C 1-2 alkyl and R 4b is hydrogen or C 1-2 alkyl;
R 5 is C 1-6 alkyl or C 1-6 alkyl substituted with —NR 5a R 5b ;
wherein R 5a is hydrogen or C 1-2 alkyl and R 5b is hydrogen or C 1-2 alkyl; or
R 4 and R 5 taken together with the nitrogen to which they are attached form a monocyclic heterocyclyl or a monocyclic heterocyclyl substituted with C 1-6 alkyl, sperminyl, or spermidinyl;
R 6 is a leaving group;
W is independently selected from O, S, and Se; and
B 1 is a heterocyclic base moiety or a protected heterocyclic base moiety;
or a salt thereof;
wherein the method comprises:
a) contacting a compound of formula (A)
with a phosphorylating reagent having the formula:
14. The method of claim 13 , wherein R 4 and R 5 are methyl.
15. The method of claim 13 , wherein R 1 is selected from trityl, dimethoxytrityl and methoxytrityl.
16. The method of claim 13 , wherein R 6 is selected from chloro, iodo, bromo, fluoro, methanesulfonyloxy, tosyloxy, triflyloxy, nitro-phenylsulfonyloxy and bromo-phenylsulfonyloxy.
17. The method of claim 13 , wherein R 6 is halo.
18. The method of claim 14 , wherein the phosphorylating agent is dimethylamino-phosphorodichioridate.
19. The method of claim 14 , wherein the phosphorylating agent is thiophosphorodichloridate or selenophosphorodichloridate.