Novel Process for Preparing Rhenium Chelated MAG3 Oligonucleotides

Abstract

The present invention relates to a novel process for preparing a compound of formula (III) and rhenium chelated MAG3 oligonucleotides.

##STR00001##

Claims

1. A method for preparing a compound of formula (III) comprising the step of: a) reacting a compound of formula (I): ##STR00026## wherein R is H or a thiol protecting group, with a precursor of a rhenium complex in a solvent to obtain a compound of formula (II): ##STR00027## b) adding N-hydroxysuccinimide with a coupling reagent to obtain a compound of formula (III): ##STR00028## wherein steps a) and b) are performed in a one pot reaction.

2. The method of claim 1, further comprising the step c) of adding an amino oligonucleotide with a base to obtain a compound of formula (IV): ##STR00029##

3. The method of claim 2, wherein steps a), b) and c) are performed in a one pot reaction.

4. The method of claim 1, wherein the precursor of a rhenium complex is selected from the group consisting of ReOCl.sub.3(PPh.sub.3).sub.2, Re(NPh)Cl.sub.3(PPh.sub.3).sub.2, ReO.sub.2(PPh.sub.3).sub.2I, and Re(NPh)Cl.sub.3(PPh.sub.3).sub.2.

5. The method of claim 4, wherein the precursor of a rhenium complex is ReOCl.sub.3(PPh.sub.3).sub.2.

6. The method of claim 1, wherein R is a thiol protecting group selected from the group consisting of acetyl (Ac), benzoyl (Bz), benzyl (Bn), J-methoxyethoxymethyl ether (MEM), dimethoxytrityl (or bis-(4-methoxyphenyl)phenylmethyl) (DMT), trimethoxytrityl (or tris-(4-methoxyphenyl)phenylmethyl) (TMT), methoxymethyl ether (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl (MMT), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl or triphenylmethyl (Tr), silyl ether (for example trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM) and triisopropylsilyl (TIPS) ethers), and methyl ethers and ethoxyethyl ethers (EE).

7. The method of claim 6, wherein R is a thiol protecting group selected from the group consisting of acetyl (Ac) and benzoyl (Bz).

8. The method of claim 1, wherein the solvent in step a) is a mixture of a polar aprotic solvent and of a polar protic solvent.

9. The method of claim 8, wherein the DMF:MeOH in an about 1:1 volume ratio.

10. The method of claim 1, wherein a base is further used in step a).

11. The method of claim 10, wherein the base is NaOMe.

12. The method of claim 1, comprising the steps of: a) Reacting a compound of formula (I): ##STR00030## wherein R is H or a thiol protecting group selected from the group consisting of acetyl (Ac) and benzoyl (Bz), with ReOCl.sub.3(PPh.sub.3).sub.2, in DMF:MeOH in an about 1:1 volume ratio to obtain a compound of formula (II): ##STR00031## b) Adding N-hydroxysuccinimide with EDC-HCl to obtain a compound of formula (III): ##STR00032## c) Adding an amino oligonucleotide with a base to obtain a compound of formula (IV): ##STR00033## wherein the oligonucleotide comprises at least one 2′ sugar modified nucleoside or LNA nucleoside and wherein steps a), b) and c) are performed in a one pot reaction.

13. The method of claim 12, wherein the oligonucleotide is an antisense oligonucleotide.

14. The method of claim 13, wherein the antisense oligonucleotide comprises at least one 2′ sugar modified nucleoside or LNA nucleoside.

15. The method of claim 1, wherein the coupling reagent is selected from the group consisting of coupling reagents is DCC (N,N′-Dicyclohexylcarbodiimide), DIC (N,N′-Diisopropylcarbodiimide), EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide), EDC-HCl (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl), BOP, PyBOP ((benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), PyBrOP (Bromotripyrrolidinophosphonium hexafluorophosphate), TBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate), TSTU (N,N,N′,N′-Tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate), TNTU (O-(Bicyclo[2.2.1]hept-5-ene-2,3-dicarboximido)-N,N,N′,N′-tetramethyluronium tetrafluoroborate), T3P (2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide), HCTU (O-(6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate), TATU, HATU (N-[(Dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide) and DMTMM (4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride), CDI (1,1′-Carbonyldiimidazole).

16. The method of claim 15, wherein the coupling reagent is EDC-HCl (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl).

17. The method of claim 14, wherein the LNA nucleoside is beta-D-oxy-LNA, (S)-6′-methyl-beta-D-oxy-LNA ((S)-cET) and ENA.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0050] In one aspect, the invention relates to a method for preparing a compound of formula (III) comprising the step of: [0051] a) Reacting a compound of formula (I):

##STR00009## [0052] wherein R is H or a thiol protecting group, [0053] with a precursor of a rhenium complex in a solvent to obtain a compound of formula (II):

##STR00010## [0054] b) Adding N-hydroxysuccinimide with a coupling reagent to obtain a compound of formula (III):

##STR00011## [0055] wherein steps a) and b) are performed in a one pot reaction.

[0056] In an embodiment of the method of the invention, there is a further step c) of adding an amino oligonucleotide with a base to obtain a compound of formula (IV):

##STR00012##

[0057] In an embodiment of the method of the invention, steps a) and b) are performed in a one pot synthesis. In an embodiment, steps a), b) and c) are performed in a one pot synthesis.

[0058] In an embodiment of the method of the invention, the precursor of a rhenium complex is selected from the group consisting of ReOCl.sub.3(PPh.sub.3).sub.2, Re(NPh)Cl.sub.3(PPh.sub.3).sub.2, ReO.sub.2(PPh.sub.3).sub.2I, and Re(NPh)Cl.sub.3(PPh.sub.3).sub.2.

[0059] In an embodiment of the method of the invention the precursor of a rhenium complex is ReOCl.sub.3(PPh.sub.3).sub.2.

[0060] In an embodiment of the method of the invention R is H.

[0061] In an embodiment of the method of the invention R is a thiol protecting group selected from the group consisting of acetyl (Ac), benzoyl (Bz), benzyl (Bn), (3-methoxyethoxymethyl ether (MEM), dimethoxytrityl (or bis-(4-methoxyphenyl)phenylmethyl) (DMT), trimethoxytrityl (or tris-(4-methoxyphenyl)phenylmethyl) (TMT), methoxymethyl ether (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl (MMT), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl or triphenylmethyl (Tr), silyl ether (for example trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM) and triisopropylsilyl (TIPS) ethers), and methyl ethers and ethoxyethyl ethers (EE).

[0062] In an embodiment of the method of the invention R is a thiol protecting group selected from the group consisting of acetyl (Ac) and benzoyl (Bz).

[0063] In an embodiment of the method of the invention the solvent in step a) is a mixture of a polar aprotic solvent and of a polar protic solvent. In an embodiment of the method of the invention the solvent is a mixture of DMF and MeOH. In an embodiment of the method of the invention, the solvent is a mixture of DMF and MeOH of an about 1:1 volume ratio.

[0064] In an embodiment of the method of the invention a base is further used in step a). In an embodiment of the method of the invention the base is NaOMe.

[0065] In an embodiment, the method of the invention comprises the steps of: [0066] c) Reacting a compound of formula (I):

##STR00013## [0067] wherein R is H or a thiol protecting group selected from the group consisting of acetyl (Ac) and benzoyl (Bz), [0068] with ReOCl.sub.3(PPh.sub.3).sub.2, [0069] in DMF:MeOH in an about 1:1 volume ratio to obtain a compound of formula (II):

##STR00014## [0070] d) Adding N-hydroxysuccinimide with EDC-HCl to obtain a compound of formula (III):

##STR00015## [0071] c) Adding an amino oligonucleotide with a base to obtain a compound of formula (IV):

##STR00016## [0072] wherein the oligonucleotide comprises at least one 2′ sugar modified nucleoside or LNA nucleoside

[0073] In an embodiment, the method of the invention the oligonucleotide is an antisense oligonucleotide. In an embodiment, the method of the invention the antisense oligonucleotide contains a 2′ sugar modified nucleoside or LNA nucleoside as defined herein.

[0074] MAG3 is commercially available from usual vendors, e.g. Sigma Aldrich or ABX. The compound of formula (I) can be commercially available, for example as NHS-MAG3 acetyl protected from various vendors such as Sigma Aldrich (Nature protocols 2007-4-21, Yi Wang et al.)

[0075] Unless stated otherwise, all starting products, reagents and solvents are commercially available.

[0076] In step b) a solvent is used. It can be a polar aprotic solvent, a polar protic solvent or mixtures thereof.

[0077] Examples of polar aprotic solvents are DCM (dichloromethane), N-methylpyrrolidone, THF (tetrahydrofurane), ETOAc (ethyl acetate), acetone, DMF (dimethylformamide), MeCN (acetonitrile), DMSO (dimethyl sulfoxide) and PC (propylene carbonate) or mixtures thereof.

[0078] In one or all embodiments of the method of the invention, the polar aprotic solvent is DMF.

[0079] Examples of polar protic solvents are formic acid, n-butanol, IPA (isopropanol), EtOH (ethanol), MeOH (methanol), AcOH (acetic acid) and water or mixtures thereof.

[0080] In one or all embodiments of the method of the invention, the polar protic solvent is MeOH.

[0081] In one or all embodiments of the method of the invention, the solvent can be a mixture of a polar aprotic solvent and of a polar protic solvent, for example DMF:MeOH, for example DMF:MeOH in an about 1:1 volume ratio.

[0082] In step c) a solvent can be used. In an embodiment, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride is used.

[0083] The coupling reagent of step b) can be selected from the group consisting of coupling reagents is DCC (N,N′-Dicyclohexylcarbodiimide), DIC (N,N′-Diisopropylcarbodiimide), EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide), EDC-HCl (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl), BOP, PyBOP ((benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), PyBrOP (Bromotripyrrolidinophosphonium hexafluorophosphate), TBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate), TSTU (N,N,N′,N′-Tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate), TNTU (O-(Bicyclo[2.2.1]hept-5-ene-2,3-dicarboximido)-N,N,N′,N′-tetramethyluronium tetrafluoroborate), T3P (2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide), HCTU (O-(6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate), TATU, HATU (N-[(Dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide) and DMTMM (4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholiniumchloride), CDI (1,1′-Carbonyldiimidazole).

[0084] In an embodiment of the method of the invention, the coupling reagent is EDC-HCl (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl).

[0085] The method of the invention can be implemented in the following non-limiting embodiments:

[0086] 1. A method for preparing a compound of formula (III) comprising the step of: [0087] a) Reacting a compound of formula (I):

##STR00017## [0088] wherein R is H or a thiol protecting group, [0089] with a precursor of a rhenium complex in a solvent to obtain a compound of formula (II):

##STR00018## [0090] b) Adding N-hydroxysuccinimide with a coupling reagent to obtain a compound of formula (III):

##STR00019## [0091] wherein steps a) and b) are performed in a one pot reaction.

[0092] 2. The method of embodiment 1, further comprising the step c) of adding an amino oligonucleotide with a base to obtain a compound of formula (IV):

##STR00020##

[0093] 3. The method of embodiment 2, wherein steps a), b) and c) are performed in a one pot synthesis.

[0094] 4. The method of any one of embodiments 1 to 3, wherein the precursor of a rhenium complex is selected from the group consisting of ReOCl.sub.3(PPh.sub.3).sub.2, Re(NPh)Cl.sub.3(PPh.sub.3).sub.2, ReO.sub.2(PPh.sub.3).sub.2I, and Re(NPh)Cl.sub.3(PPh.sub.3).sub.2.

[0095] 5. The method of embodiment 4, wherein the precursor of a rhenium complex is ReOCl.sub.3(PPh.sub.3).sub.2.

[0096] 6. The method of any one of embodiments 1 to 5, wherein R is H.

[0097] 7. The method of any one of embodiments 1 to 5, wherein R is a thiol protecting group selected from the group consisting of acetyl (Ac), benzoyl (Bz), benzyl (Bn), (3-methoxyethoxymethyl ether (MEM), dimethoxytrityl (or bis-(4-methoxyphenyl)phenylmethyl) (DMT), trimethoxytrityl (or tris-(4-methoxyphenyl)phenylmethyl) (TMT), methoxymethyl ether (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl (MMT), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl or triphenylmethyl (Tr), silyl ether (for example trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM) and triisopropylsilyl (TIPS) ethers), and methyl ethers and ethoxyethyl ethers (EE).

[0098] 8. The method of embodiment 6 or 7, wherein R is a thiol protecting group selected from the group consisting of acetyl (Ac) and benzoyl (Bz).

[0099] 9. The method of any one of embodiments 1 to 8, wherein the solvent in step a) is a mixture of a polar aprotic solvent and of a polar protic solvent.

[0100] 10. The method of embodiment 8, wherein the DMF:MeOH in an about 1:1 volume ratio.

[0101] 11. The method of any one of embodiments 1 to 10, wherein a base is further used in step a).

[0102] 12. The method of embodiment 11, wherein the base is NaOMe.

[0103] 13. The method of any one of embodiments 1 to 12 comprising the steps of: [0104] a) Reacting a compound of formula (I):

##STR00021## [0105] wherein R is a thiol protecting group selected from the group consisting of acetyl (Ac) and benzoyl (Bz), [0106] with ReOCl.sub.3(PPh.sub.3).sub.2, [0107] in DMF:MeOH in an about 1:1 volume ratio to obtain a compound of formula (II):

##STR00022## [0108] b) Adding N-hydroxysuccinimide with EDC-HCl to obtain a compound of formula (III):

##STR00023## [0109] c) Adding an amino oligonucleotide with a base to obtain a compound of formula (IV):

##STR00024## [0110] wherein the oligonucleotide comprises at least one 2′ sugar modified nucleoside or LNA nucleoside and wherein steps a), b) and c) are performed in a one pot reaction.

[0111] 14. The method of any one of embodiments 1 to 13, wherein the oligonucleotide is an antisense oligonucleotide.

[0112] 15. The method of embodiment 14, wherein the antisense oligonucleotide comprises at least one 2′ sugar modified nucleoside or LNA nucleoside.

[0113] 16. The method of any one of embodiments 1 to 15 wherein the coupling reagent is selected from the group consisting of coupling reagents is DCC (N,N′-Dicyclohexylcarbodiimide), DIC (N,N′-Diisopropylcarbodiimide), EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide), EDC-HCl (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl), BOP, PyBOP ((benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate), PyBrOP (Bromotripyrrolidinophosphonium hexafluorophosphate), TBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate), TSTU (N,N,N′,N′-Tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate), TNTU (O-(Bicyclo[2.2.1]hept-5-ene-2,3-dicarboximido)-N,N,N′,N′-tetramethyluronium tetrafluoroborate), T3P (2,4,6-Tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide), HCTU (O-(6-Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate), TATU, HATU (N-[(Dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide) and DMTMM (4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholiniumchloride), CDI (1,1′-Carbonyldiimidazole).

[0114] 17. The method of embodiment 16, wherein the coupling reagent is EDC-HCl (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl).

Example

[0115] ##STR00025##

[0116] Degassed (N.sub.2 and/or argon) DMF:MeOH (2.5 mL, ratio 1:1) was added to a flask containing protected Mag-3-COOH (10 mg, 27 umol), ReOCl.sub.3(PPh.sub.3).sub.2 (45 mg, 54 umol), and NaOMe (29.4 mg, 554 umol). The resulting solution was then stirred for 60 min. Hereafter, the reaction mixture was evaporated to approximately 0.5 mL volume using a stream of N.sub.2. Then, N-hydroxysuccinimide (9.4 mg, 82 umol) and EDC-HCl (6.3 mg, 33 umol) was added and the resulting mixture was left to stir overnight. After reacting overnight the mixture was diluted w. 0.5 mL DMSO and stirred for 15 min. Then the amino-modified oligonucleotide (30 mg dissolved in 0.5 mL NaHCO.sub.3 pH=8.5) was added. The resulting mixture was stirred overnight in order to afford the wanted oligonucleotide compound (i.e. amino oligonucleotide conjugated to Mag-3 chelated with rhenium).

[0117] Purification: The oligonucleotide was precipitated from the one-pot reaction by adding a solution of 2% LiClO.sub.4 in acetone to the above reaction mixture. Then the flask was swirled, centrifuged, and decanted. The residue remaining (precipitated oligonucleotide) was then dissolved in water and purified by reverse phase HPLC.