MORPHOLINO OLIGONUCLEOTIDE MANUFACTURING METHOD

• US 20160076033A1
• Filed: 11/20/2015
• Published: 03/17/2016
• Est. Priority Date: 05/24/2013
• Status: Active Grant

• Current Assignee: Ajinomoto Co., Inc.
• Original Assignee: Ajinomoto Co., Inc.
• Inventors: Torii, Takayoshi, Takahashi, Daisuke, Katayama, Satoshi
• Granted Patent: US 9,790,497 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C07D 413/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 487/04 Ortho-condensed systems
• CPC Class Codes: C07D 519/00 Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
• 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: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2330/30 chemically synthesised
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Using a morpholino nucleotide wherein 5′-hydroxy group or a hydroxy group present on the substituent of the 5′-hydroxy group is protected by a protecting group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms as a starting material, a method capable of efficiently producing the morpholino oligonucleotide in a high yield by a liquid phase synthesis can be provided.

Claims

1. A morpholino nucleotide represented by formula (I):

2. The morpholino nucleotide according to claim 1, wherein m is 0.

3. The morpholino nucleotide according to claim 1, wherein L is a succinyl group, and R⁵ and/or R⁷ are/is an alkyl group containing 10 to 40 carbon atoms.

4. The morpholino nucleotide according to claim 1, wherein L is a succinyl group, R_a and R_b are both hydrogen atoms, and R⁵ is an alkyl group containing 10 to 40 carbon atoms.

5. The morpholino nucleotide according to claim 1, wherein L is a succinyl group, and R⁵ and/or R⁷ are/is an alkyl group containing 12 to 30 carbon atoms.

6. The morpholino nucleotide according to claim 1, wherein

L is a succinyl group, and

Z—

Y—

is a group selected from the group consisting of

a 3,4,5-tri(octadecyloxy)benzyloxy group,

a 3,5-di(docosyloxy)benzyloxy group,

a 3,5-bis[3′

,4′

,5′

-tri(octadecyloxy)benzyloxy]benzyloxy group,

a 3,4,5-tris[3′

,4′

,5′

-tri(octadecyloxy)benzyloxy]-benzyloxy group,

a 3,4,5-tri(octadecyloxy)benzylamino group,

a 2,4-di(docosyloxy)benzylamino group,

a 3,5-di(docosyloxy)benzylamino group,

a di(4-docosyloxyphenyl)methylamino group,

a 4-methoxy-2-[3′

,4′

,5′

-tri(octadecyloxy)benzyloxy]-benzylamino group,

a 4-methoxy-2-[3′

,4′

,5′

-tri(octadecyloxy)-cyclohexylmethyloxy]benzylamino group,

a 2,4-di(dodecyloxy)benzylamino group,

a phenyl(2,3,4-tri(octadecyloxy)phenyl)methylamino group,

a di[4-(12-docosyloxydodecyloxy)phenyl]methylamino group,

a 3,5-bis[3′

,4′

,5′

-tri(octadecyloxy)benzyloxy]benzylamino group, and

a 3,4,5-tris[3′

,4′

,5′

-tri(octadecyloxy)benzyloxy]-benzylamino group.

7. The morpholino nucleotide according to claim 1, wherein Z—

Y-L- is selected from the group consisting of

a 2-{2,4-di(2′

,3′

-dihydrophytyloxy)benzylaminocarbonyl}-ethylcarbonyl group;

a 3,5-di(2′

,3′

-dihydrophytyloxy)benzylsuccinyl group;

a 4-(2′

,3′

-dihydrophytyloxy)benzylsuccinyl group;

a 2-{1-[(2-chloro-5-(2′

,3′

-dihydrophytyloxy)phenyl)]benzylaminocarbonyl}ethylcarbonyl group;

a 3,4,5-tri(2′

,3′

-dihydrophytyloxy)benzylsuccinyl group;

a 2-{3,4,5-tri(2′

,3′

-dihydrophytyloxy)-benzylaminocarbonyl}ethylcarbonyl group;

a 2-{4-(2′

,3′

-dihydrophytyloxy)benzylaminocarbonyl}-ethylcarbonyl group;

a 2-{2-[3′

,4′

,5′

-tri(2″

,3″

-dihydrophytyloxy)benzyloxy]-4-methoxybenzylaminocarbonyl}ethylcarbonyl group;

a 2-{4-(2′

,3′

-dihydrophytyloxy)-2-methoxybenzylaminocarbonyl}ethylcarbonyl group;

a 4-(2′

,3′

-dihydrophytyloxy)-2-methylbenzylsuccinyl group;

a 2-{4-(2′

,3′

-dihydrophytyloxy)-2-methylbenzylaminocarbonyl}ethylcarbonyl group;

a 4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]-benzylsuccinyl group;

a 2-{4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]-benzylaminocarbonyl}ethylcarbonyl group;

a 4-(3,7,11-trimethyldodecyloxy)benzylsuccinyl group;

a 2-{4-(3,7,11-trimethyldodecyloxy)benzylaminocarbonyl}-ethylcarbonyl group;

a 2-{3,5-di(2′

,3′

-dihydrophytyloxy)benzylaminocarbonyl}-ethylcarbonyl group;

a 2-{1-[2,3,4-tri(2′

,3′

-dihydrophytyloxy)phenyl]-benzylaminocarbonyl}ethylcarbonyl group;

a 2-{1-[4-(2′

,3′

-dihydrophytyloxy)phenyl]-4′

-(2′

,3′

-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group;

a 3,4,5-tris[3,4,5-tri(2′

,3′

-dihydrophytyloxy)benzyl]-benzylsuccinyl group; and

a 2-{3,4,5-tris[3,4,5-tri(2′

,3′

-dihydrophytyloxy)benzyl]-benzylaminocarbonyl}ethylcarbonyl group.

8. The morpholino nucleotide according to claim 1, wherein P¹ is a trityl group, a monomethoxytrityl group, or a dimethoxytrityl group.

9. A method of producing an n+p-mer morpholino oligonucleotide, comprising:

(1) condensing (i) a p-mer morpholino oligonucleotide, wherein p is any integer of one or more, and wherein a 5′

-hydroxy group is activated (thio)phosphated or activated (thio)phosphoramidated, and a morpholine ring nitrogen atom is protected by a temporary protecting group removable under acidic conditions, with (ii) an n-mer morpholino oligonucleotide, wherein n is an integer of one or more, and wherein a 5′

-hydroxy group or, when the 5′

-hydroxy group has a substituent having a hydroxy group, the hydroxy group present on the substituent is protected by a protecting group having an alkyl group containing not less than 10 and not more than 300 carbon atoms and/or an alkenyl group containing not less than 10 and not more than 300 carbon atoms, and the morpholine ring nitrogen atom is not protected, by a (thio)phosphoramidate bond or (thio)phosphorodiamidate bond via the morpholine ring nitrogen atom,

to obtain a reaction mixture comprising said n+p-mer morpholino oligonucleotide.

10. The method according to claim 9, wherein p is 1.

11. The method according to claim 9, comprising treating said reaction mixture with a quenching agent after completion of said condensing.

12. The method according to claim 9, further comprising:

(1′

) removing said temporary protecting group of said morpholine ring nitrogen atom by reacting, before said condensing (1), the n-mer morpholino oligonucleotide wherein the 5′

-hydroxy group or, when the 5′

-hydroxy group has a substituent having a hydroxy group, the hydroxy group present on the substituent is protected by a protecting group having an alkyl group containing not less than 10 and not more than 300 carbon atoms and/or an alkenyl group containing not less than 10 and not more than 300 carbon atoms, and the morpholine ring nitrogen atom is protected by a temporary protecting group removable under acidic conditions, with an acid in a non-polar solvent.

13. The according to claim 12, wherein said temporary protecting group is removed in the presence of a cation scavenger.

14. The method according to claim 12, further comprising neutralizing with an organic base after said (1′

) removing said temporary protecting group of the morpholine ring nitrogen atom.

15. The method according to claim 9, wherein said protecting group having an alkyl group containing not less than 10 and not more than 300 carbon atoms and/or an alkenyl group containing not less than 10 and not more than 300 carbon atoms is a group represented by formula (II):

Z—

Y-L- 

 

(II)

wherein

L is a single bond, or a group represented by formula (a1);

16. The method according to claim 15, further comprising:

(2) obtaining said morpholino oligonucleotide by adding a polar solvent to said reaction mixture obtained in step (1) and/or (1′

) and collecting a precipitate thereof by solid-liquid separation.

17. The method according to claim 12, wherein said non-polar solvent is selected from the group consisting of a halogenated solvent, an aromatic solvent, an ester solvent, an aliphatic solvent, a non-polar ether solvent, and a combination thereof.

18. The method according to claim 16, wherein said polar solvent is an alcohol solvent or a nitrile solvent.

19. The method according to claim 9, further comprising:

(3) removing all protecting groups of said n+p-mer morpholino oligonucleotide.

20. The method according to claim 9, wherein said temporary protecting group removable under acidic conditions is a trityl group, a dimethoxytrityl group, or a monomethoxytrityl group.