阅读说明：本技术 制备铼螯合mag3寡核苷酸的新方法 (Novel method for preparing rhenium chelated MAG3 oligonucleotide ) 是由 E·D·丰德 D·J·汉森 J·霍恩舍梅尔 于 2020-04-28 设计创作，主要内容包括：本发明涉及一种制备式(III)化合物：以及铼螯合MAG3寡核苷酸的新方法。(The present invention relates to a process for preparing a compound of formula (III):)

1. A process for preparing a compound of formula (III), said process comprising the steps of:

a) reacting a compound of formula (I):

wherein R is H or a thiol protecting group,

with a precursor of a rhenium complex in a solvent to give a compound of formula (II):

b) adding N-hydroxysuccinimide and a coupling agent to give a compound of formula (III):

wherein steps a) and b) are carried out in a one-pot reaction.

2. The method according to claim 1, further comprising step c): adding an aminooligonucleotide and a base to give a compound of formula (IV):

3. the process of claim 2, wherein steps a), b) and c) are carried out in a one-pot reaction.

4. The method according to any one of claims 1 to 3, wherein the precursor of the rhenium complex is selected from the group consisting of: ReOCl₃(PPh₃)₂、Re(NPh)Cl₃(PPh₃)₂、ReO₂(PPh₃)₂I and Re (NPh) Cl₃(PPh₃)₂。

5. The method of claim 4, wherein the precursor of the rhenium complex is ReOCl₃(PPh₃)₂。

6. The process according to any one of claims 1 to 5, wherein R is a thiol protecting group selected from the group consisting of: acetyl (Ac), benzoyl (Bz), benzyl (Bn), beta-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 ethers such as Trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS), Triisopropylsilyloxymethyl (TOM) and Triisopropylsilyl (TIPS) ethers, and methyl ether and Ethoxyethyl Ether (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 process according to any one of claims 1 to 7, wherein the solvent in step a) is a mixture of a polar aprotic solvent and a polar protic solvent.

9. The process of claim 8, wherein the volume ratio of DMF to MeOH is about 1: 1.

10. The process according to any one of claims 1 to 9, wherein a base is further used in step a).

11. The process of claim 10 wherein the base is NaOMe.

12. The method according to any one of claims 1 to 11, comprising the steps of:

a) reacting a compound of formula (I):

wherein R is H, a thiol protecting group selected from the group consisting of acetyl (Ac) and benzoyl (Bz),

and ReOCl₃(PPh₃)₂，

Reaction in DMF: MeOH in a volume ratio of about 1:1 to afford the compound of formula (II):

b) adding N-hydroxysuccinimide and EDC-HCl to give a compound of formula (III):

c) adding an aminooligonucleotide and a base to provide a compound of formula (IV):

wherein the oligonucleotide comprises at least one 2' sugar modified nucleoside or LNA nucleoside and steps a), b) and c) are carried out in a one-pot reaction.

13. The method of any one of claims 1 to 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 any one of claims 1 to 14, wherein the coupling agent is selected from the group consisting of: 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 (tripyrrolidinophosphonium hexafluorophosphate bromide), TBTU (2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethylammonium tetrafluoroborate), TSTU (N, N, N ', N' -tetramethyl-O- (N-succinimidyl) uretetrafluoroborate), TNTU (O- (bicyclo [2.2.1] hept-5-ene-2, 3-dicarboximido) -N, N '-tetramethyluronium tetrafluoroborate), T3P (2,4, 6-tripropyl-1, 3,5,2,4, 6-trioxatriphospha-cyclohexane-2, 4, 6-trioxide), HCTU (O- (6-chlorobenzotriazol-1-yl) -N, N' -tetramethyluronium hexafluorophosphate), TATU, HATU (N- [ (dimethylamino) -1H-1,2, 3-triazolo- [4,5-b ] pyridin-1-ylmethylene ] -N-methylmethanium hexafluorophosphate N-oxide) and DMTMM (4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholine n-ium chloride), CDI (1,1' -carbonyldiimidazole).

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

17. The invention as hereinbefore described.

***

Disclosure of Invention

In a first aspect, the present invention relates to a novel process for the preparation of rhenium chelated MAG3 compounds of formula (III) in a one-pot reaction:

in a second aspect, the present invention relates to a novel process for the preparation of rhenium chelated MAG3 oligonucleotide compounds of formula (IV):

in a third aspect, the present invention relates to a novel process for the preparation of a compound of formula (III) or formula (IV) in a one-pot reaction.

Other aspects of the invention are described in more detail below.

Definition of

The term "coupling agent" denotes a compound capable of covalently coupling two organic molecules, such as a carboxylic acid and an amine, alcohol or thiol. More specifically, in the context of the present invention, a coupling agent denotes a compound capable of facilitating the coupling reaction of a carboxylic acid to an amine. The coupling agent can be used as a water-soluble HCl salt. EDC is one example over other carbodiimides. EDC by-products are water soluble and can be easily removed by water treatment. Examples of further coupling agents are 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 (tripyrrolidinophosphonium hexafluorophosphate bromide), TBTU (2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethylammoniumtetrafluoroborate), TSTU (N, N, N ', N' -tetramethyl-O- (N-succinimidyl) urea tetrafluoroborate), TNTU (O- (bicyclo [2.2.1] hept-5-ene-2, 3-dicarboximido) -N, N '-tetramethyluronium tetrafluoroborate), T3P (2,4, 6-tripropyl-1, 3,5,2,4, 6-trioxatriphospha-cyclohexane-2, 4, 6-trioxide), HCTU (O- (6-chlorobenzotriazol-1-yl) -N, N' -tetramethyluronium hexafluorophosphate), TATU, HATU (N- [ (dimethylamino) -1H-1,2, 3-triazolo- [4,5-b ] pyridin-1-ylmethylene ] -N-methylmethanium hexafluorophosphate N-oxide), DMTMM (4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholine n-ium chloride), CDI (1,1' -carbonyldiimidazole) and the like.

The term "alkyl group"alone or in combination" means a straight or branched alkyl group having 1 to 8 carbon atoms, particularly a straight or branched alkyl group having 1 to 6 carbon atoms, and more particularly a straight or branched alkyl group having 1 to 4 carbon atoms. Straight and branched C₁-C₈Examples of alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, the isomeric pentyl, the isomeric hexyl, the isomeric heptyl and the isomeric octyl, in particular methyl, ethyl, propyl, butyl and pentyl. Specific examples of alkyl groups are methyl, ethyl and propyl.

The term "amino" alone or in combination refers to a primary amino group (-NH)₂) A secondary amino group (-NH-) or a tertiary amino group (-N-).

The term "aminooligonucleotide" is meant to encompass non-aromatic NH₂The oligonucleotide of (1). NH (NH)₂The group may be located, for example, at the 3' or 5' end of the oligonucleotide, e.g.3 ' oligonucleotide-CH₂CH₂CH₂CH₂CH₂CH₂-NH₂-5’

The term "amino protecting group" denotes a protecting group for an amino group. Amines are functional groups that often require protecting groups in organic reactions. Carbamates, e.g. tert-butoxycarbonyl (Boc, e.g. by means of concentrated strong acids (e.g. HCl or CF)₃COOH) or by heating to>Removal at 80 ℃), benzyloxycarbonyl (Cbz, e.g. by hydrogenolysis) or 9-fluorenylmethoxycarbonyl (Fmoc, e.g. by base removal, e.g. piperidine) are commonly used amine protecting groups. Other options of protecting groups with different deprotection conditions may be used, such as described by Peter Wipf and co-workers at pittsburgh university, pa, usa, who developed a useful protecting group for primary, secondary and heterocyclic amines: 2,2,6, 6-tetramethylpiperidin-1-yloxycarbonyl (Tempoc). Acetyl (Ac) is common in oligonucleotide synthesis, serves to protect N4 in cytosine and N6 in the adenine nucleobase, and can be removed by base treatment, most commonly with aqueous or gaseous ammonia or methylamine. Ac is too stable to be easily removed from the aliphatic amide. Benzoyl (Bz) is also common in oligonucleotide synthesis, for protection of N4 in cytosine and N6 in adenine nucleobases, and byAlkali treatment removal, most commonly with aqueous ammonia or gaseous ammonia or methylamine. Bz is too stable to be easily removed from the aliphatic amide. Other commonly used suitable amino protecting groups, such as DMF or iBu, may also be considered by those skilled in the art.

The term "aryl" alone or in combination, refers to a monovalent aromatic carbocyclic monocyclic or bicyclic ring system comprising 6 to 10 carbon ring atoms, said system optionally substituted with 1 to 3 substituents independently selected from: halogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxy, alkoxycarbonyl, alkylcarbonyl and formyl. Examples of aryl groups include phenyl and naphthyl, especially phenyl.

The term "chelate" refers to the fact that a compound containing a ligand (e.g., an organic) is bound to a central metal atom at two or more points. It typically involves the formation or presence of two or more separate coordination bonds between a multidentate (multi-bond) ligand and a single central atom. These ligands are known as chelating agents (chelans, chelators, chelating agents or sequencing agents). It is a type of ion and molecule that is typically bound to a metal ion. The compounds of formula (II) according to the invention are examples of chelates or chelate complexes.

The chelator moiety of the present invention may comprise any number of combinations of atoms selected from the group consisting of nitrogen atoms, sulfur atoms, oxygen atoms and phosphorus atoms. In certain particular embodiments, the chelator moiety comprises a combination of three to five such atoms. In some embodiments, the chelating agent is capable of chelating metal ions of any valence by coordination with other atoms, such as nitrogen, sulfur, oxygen, and/or phosphorus atoms. In certain embodiments, the chelating agent is capable of chelating a tri-to pentavalent metal ion. Any valency of the metal ion is contemplated as being chelated to the chelating agent of the present invention. Examples of such valence metal ions include, but are not limited to, 186Re, 188Re, Tc-99m, Cu-60, Cu-61, Cu-62, In-111, Tl-201, Ga-67, and Ga-68.

The term "halogen" or "halo", alone or in combination, refers to fluorine, chlorine, bromine or iodine and especially to fluorine, chlorine or bromine, more especially to fluorine. The term "halo" in combination with another group refers to said group substituted with at least one halogen, especially one to five halogens, especially one to four halogens (i.e. one, two, three or four halogens).

The term "thiol" denotes the-SH group.

"thiol protecting groups" are protecting groups for thiol groups and are also used to protect hydroxyl groups. Examples of hydroxy protecting groups are acetyl (Ac), benzoyl (Bz), benzyl (Bn), β -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 ethers (e.g. Trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), Triisopropylsilyloxymethyl (TOM) and Triisopropylsilyl (TIPS) ether), Methyl ether and Ethoxyethyl Ether (EE). Specific examples of hydroxy protecting groups are DMT and TMT, especially DMT.

The term "LNA monomer" refers to an LNA nucleotide, referring to a nucleotide whose nucleoside is an LNA nucleoside as defined herein.

The term "one-pot synthesis" denotes a strategy to increase the efficiency of chemical reactions whereby reactants are subjected to successive chemical reactions in only one reactor without intermediate separation or purification steps. This is highly desirable by chemists because avoiding lengthy separation processes and purification of intermediates can save time and resources while increasing chemical yields.

The term "pharmaceutically acceptable salts" refers to those salts that retain the biological effectiveness and properties of the free base or free acid, and which are not biologically or otherwise undesirable. These salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid (particularly hydrochloric acid) and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine. In addition, these salts can be prepared by addition of an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium salts. Salts derived from organic bases include, but are not limited to, salts formed with: primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins. The oligonucleotides of the invention may also be present in zwitterionic form. Particularly preferred pharmaceutically acceptable salts of the invention are the sodium, lithium, potassium and trialkylammonium salts.

The term "precursor of a rhenium complex" denotes a rhenium atom capable of chelating a compound of formula (I). An example of a precursor for the rhenium complex is commercially available ReOCl₃(PPh₃)₂(oxotrichlorobis (triphenylphosphine) rhenium (V)). Other examples are Re (NPh) Cl₃(PPh₃)₂、ReO₂(PPh₃)₂I or Re (NPh) Cl₃(PPh₃)₂。

The term "protecting group" used alone or in combination denotes a group that selectively blocks a reactive site in a polyfunctional compound, thereby allowing a chemical reaction to be selectively performed at another unprotected reactive site. The protecting group may be removed. Exemplary protecting groups are amino protecting groups, carboxyl protecting groups, or hydroxyl protecting groups.

"phosphate protecting group" is a protecting group for a phosphate group. Examples of phosphate protecting groups are 2-cyanoethyl and methyl. A specific example of a phosphate protecting group is 2-cyanoethyl.

According to the invention, the rhenium complex is a Re (V) complex.

If one of the starting materials or compounds of the invention contains one or more functional Groups which are unstable or reactive under the reaction conditions of one or more reaction steps, suitable protecting Groups can be introduced before the critical step (as described, for example, in "Protective Groups in Organic Chemistry" of T.W.Greene and P.G.M.Wuts, 3 rd edition, 1999, Wiley, New York), using methods well known in the art. Such protecting groups can be removed at a later stage of the synthesis using standard methods described in the literature. Examples of protecting groups are t-butyloxycarbonyl (Boc), 9-fluorenylmethylcarbamate (Fmoc), 2-trimethylsilylethylcarbamate (Teoc), benzyloxycarbonyl (Cbz), and p-methoxybenzyloxycarbonyl (Moz).

The compounds described herein may contain several asymmetric centers and may exist as optically pure enantiomers, mixtures of enantiomers such as racemates, mixtures of diastereomers, diastereomeric racemates or mixtures of diastereomeric racemates.

Oligonucleotides

As used herein, the term "oligonucleotide" is defined as a molecule comprising two or more covalently linked nucleosides as is commonly understood by a skilled artisan. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are usually prepared in the laboratory by solid phase chemical synthesis followed by purification. When referring to the sequence of an oligonucleotide, reference is made to the nucleobase portion of a covalently linked nucleotide or nucleoside or a modified sequence or order thereof. The oligonucleotides of the invention are artificial and chemically synthesized and are usually purified or isolated. The oligonucleotides of the invention may comprise one or more modified nucleosides or nucleotides.

Antisense oligonucleotides

The term "antisense oligonucleotide" as used herein is defined as an oligonucleotide capable of modulating the expression of a target gene by hybridizing to a target nucleic acid, particularly to a contiguous sequence on the target nucleic acid. Antisense oligonucleotides are not substantially double-stranded and are therefore not sirnas or shrnas. Preferably, the antisense oligonucleotides of the invention are single stranded. It will be appreciated that single stranded oligonucleotides of the invention may form hairpin or intermolecular duplex structures (duplexes between two molecules of the same oligonucleotide) provided that the degree of internal or inter-self complementarity is less than 50% across the full length of the oligonucleotide

Sugar modification

Oligomers of the invention may comprise one or more nucleosides having a modified sugar moiety (i.e., a modification of the sugar moiety) when compared to the ribose sugar moiety found in DNA and RNA.

Many modified nucleosides have been prepared with ribose moieties, the primary purpose being to improve certain properties of the oligonucleotides, such as affinity and/or nuclease resistance.

Such modifications include those in which the ribose ring structure is modified, for example, by substitution with: a hexose ring (HNA) or a bicyclic ring (LNA) typically having a double-base bridge between the C2 and C4 carbons on the ribose ring or a non-linked ribose ring typically lacking a bond between the C2 and C3 carbons (e.g., UNA). Other sugar-modified nucleosides include, for example, bicyclic hexose nucleic acids (WO 2011/017521) or tricyclic nucleic acids (WO 2013/154798). Modified nucleosides also include nucleosides in which the sugar moiety is replaced with a non-sugar moiety, for example in the case of Peptide Nucleic Acid (PNA) or morpholino nucleic acid.

Sugar modifications also include modifications made by changing the substituents on the ribose ring to groups other than hydrogen or to the 2' -OH group naturally present in DNA and RNA nucleosides. For example, substituents may be introduced at the 2', 3', 4 'or 5' positions.

2' sugar modified nucleosides.

A 2' sugar modified nucleoside is a nucleoside having a substituent other than H or-OH at the 2' position (a 2' substituted nucleoside) or a 2' linked diradical comprising a bridge capable of being formed between the 2' carbon and the second carbon in the ribose ring, such as a LNA (2' -4' diradical bridged) nucleoside.

In fact, much effort has been expended to develop 2 'modified nucleosides, and many 2' modified nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, 2' modified sugars can provide enhanced binding affinity and/or increased nuclease resistance to oligonucleotides. Examples of 2 'substituted modified nucleosides are 2' -O-alkyl-RNA, 2 '-O-methyl-RNA, 2' -alkoxy-RNA, 2 '-O-methoxyethyl-RNA (MOE), 2' -amino-DNA, 2 '-fluoro-RNA and 2' -F-ANA nucleosides. Other examples can be found, for example: freier and Altmann, nuclear. acid res, 1997, 25, 4429-4443; uhlmann, curr. opinion in Drug Development, 2000, 3(2), 293-; and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. The following are schematic representations of some 2' substituted modified nucleosides.

For the present invention, 2 'modifications do not include 2' bridged molecules such as LNA.

Locked nucleic acid nucleosides (LNA nucleosides)

An "LNA nucleoside" is a 2' -modified nucleoside comprising a diradical of C2' and C4' linking the ribose ring of the nucleoside (also referred to as a "2 ' -4' bridge"), which constrains or locks the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or Bicyclic Nucleic Acids (BNA). When LNA is incorporated into an oligonucleotide of a complementary RNA or DNA molecule, the locking of the ribose conformation is associated with an enhanced affinity for hybridization (duplex stabilization). This can be routinely determined by measuring the melting temperature of the oligonucleotide/complementary duplex.

Non-limiting exemplary LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352, WO 2004/046160, WO 00/047599, WO 2007/134181, WO 2010/077578, WO 2010/036698, WO 2007/090071, WO 2009/006478, WO 2011/156202, WO 2008/154401, WO 2009/067647, WO 2008/150729, Morita et al (Bioorganic & med.chem.lett.12,73-76), Seth et al (j.org.chem.2010, Vol 75(5) pp.1569-81) and Mitsuoka et al (Nucleic Acids Research 2009,37(4), 1225-1238).

The 2'-4' bridge may be located below the plane of the ribose ring (β -D-configuration) or above the plane of the ring (α -L-configuration), as shown in formulas (A) and (B), respectively.

It should be recognized that LNA nucleosides can be in the β -D or α -L stereoisomeric form unless otherwise indicated.

Specific examples of LNA nucleosides of the invention are given in scheme 1 (where B is as defined above).

Scheme 1

Specific LNA nucleosides are β -D-oxy-LNA, 6 '-methyl- β -D-oxy-LNA such as (S) -6' -methyl- β -D-oxy-LNA ((S) -cET) and ENA.

Detailed Description

In one aspect, the present invention relates to a process for preparing a compound of formula (III), comprising the steps of:

a) reacting a compound of formula (I):

wherein R is H or a thiol protecting group,

with a precursor of a rhenium complex in a solvent to give a compound of formula (II):

b) adding N-hydroxysuccinimide and a coupling agent to give a compound of formula (III):

wherein steps a) and b) are carried out in a one-pot reaction.

In one embodiment of the method according to the invention, comprising a further step c), the addition of an amino oligonucleotide and a base, gives a compound of formula (IV):

in one embodiment of the process according to the invention, steps a) and b) are carried out in a one-pot synthesis. In one embodiment, steps a), b) and c) are performed in a one-pot synthesis.

In one embodiment of the method according to the invention, the precursor of the rhenium complex is selected from the group consisting of: ReOCl₃(PPh₃)₂、Re(NPh)Cl₃(PPh₃)₂、ReO₂(PPh₃)₂I and Re (NPh) Cl₃(PPh₃)₂。

In one embodiment of the method according to the invention, the precursor of the rhenium complex is ReOCl₃(PPh₃)₂。

In one embodiment of the process according to the invention, R is H.

In one embodiment of the process according to the invention, R is a thiol protecting group selected from the group consisting of: acetyl (Ac), benzoyl (Bz), benzyl (Bn), beta-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 ethers such as Trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS), Triisopropylsilyloxymethyl (TOM) and Triisopropylsilyl (TIPS) ethers, and methyl ether and Ethoxyethyl Ether (EE).

In one embodiment of the process according to the invention, R is a thiol protecting group selected from the group consisting of acetyl (Ac) and benzoyl (Bz).

In one embodiment of the process according to the invention, the solvent in step a) is a mixture of a polar aprotic solvent and a polar protic solvent. In one embodiment of the process according to the invention, the solvent is a mixture of DMF and MeOH. In one embodiment of the process according to the invention, the solvent is a mixture of DMF and MeOH in a volume ratio of about 1: 1.

In one embodiment of the process according to the invention, a base is further used in step a). In one embodiment of the process according to the invention, the base is NaOMe.

In one embodiment, the method of the present invention comprises the steps of:

c) reacting a compound of formula (I):

wherein R is H or a thiol protecting group selected from the group consisting of acetyl (Ac) and benzoyl (Bz),

and ReOCl₃(PPh₃)₂，

Reaction in DMF: MeOH in a volume ratio of about 1:1 to afford the compound of formula (II):

d) adding N-hydroxysuccinimide and EDC-HCl to give a compound of formula (III):

c) adding an aminooligonucleotide and a base to provide a compound of formula (IV):

wherein the oligonucleotide comprises at least one 2' sugar modified nucleoside or LNA nucleoside

In one embodiment, the oligonucleotide in the methods of the invention is an antisense oligonucleotide. In one embodiment, in the methods of the invention, the antisense oligonucleotide comprises a 2' sugar modified nucleoside or LNA nucleoside as defined herein.

MAG3 is commercially available from common suppliers, such as Sigma Aldrich or ABX. The compounds of formula (I) are commercially available, for example in acetyl protected form NHS-MAG3 from different suppliers, for example Sigma Aldrich (Nature protocols 2007-4-21, Yi Wang et al).

All starting products, reagents and solvents are commercially available unless otherwise indicated.

In step b), a solvent is used. It may be a polar aprotic solvent, a polar protic solvent or a mixture thereof.

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

In one or all embodiments of the process according to the invention, the polar aprotic solvent is DMF.

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

In one or all embodiments of the process according to the invention, the polar protic solvent is MeOH.

In one or all embodiments of the process according to the invention, the solvent may be a mixture of a polar aprotic solvent and a polar protic solvent, e.g. DMF: MeOH, e.g. in a volume ratio of about 1:1 DMF: MeOH.

In step c), a solvent may be used. In one embodiment, N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride is used.

The coupling agent of step b) may be selected from the group consisting of: 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 (tripyrrolidinophosphonium hexafluorophosphate bromide), TBTU (2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethylammonium tetrafluoroborate), TSTU (N, N, N ', N' -tetramethyl-O- (N-succinimidyl) uretetrafluoroborate), TNTU (O- (bicyclo [2.2.1] hept-5-ene-2, 3-dicarboximido) -N, N '-tetramethyluronium tetrafluoroborate), T3P (2,4, 6-tripropyl-1, 3,5,2,4, 6-trioxatriphospha-cyclohexane-2, 4, 6-trioxide), HCTU (O- (6-chlorobenzotriazol-1-yl) -N, N' -tetramethyluronium hexafluorophosphate), TATU, HATU (N- [ (dimethylamino) -1H-1,2, 3-triazolo- [4,5-b ] pyridin-1-ylmethylene ] -N-methylmethanium hexafluorophosphate N-oxide), DMTMM (4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholine n-ium chloride), CDI (1,1' -carbonyldiimidazole) and the like.

In one embodiment of the process according to the invention, the coupling agent is EDC-HCl (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide-HCl).

The process of the invention can be carried out by the following non-limiting examples:

1. a process for preparing a compound of formula (III), the process comprising the steps of:

a) reacting a compound of formula (I):

wherein R is H or a thiol protecting group,

with a precursor of a rhenium complex in a solvent to give a compound of formula (II):

b) adding N-hydroxysuccinimide and a coupling agent to give a compound of formula (III):

wherein steps a) and b) are carried out in a one-pot reaction.

2. The method according to example 1, further comprising the step of c) adding an amino oligonucleotide and a base to obtain a compound of formula (IV):

3. the process according to example 2, wherein steps a), b) and c) are carried out in a one-pot synthesis.

4. The method according to any one of embodiments 1 to 3, wherein the precursor of the rhenium complex is selected from the group consisting of: ReOCl₃(PPh₃)₂、Re(NPh)Cl₃(PPh₃)₂、ReO₂(PPh₃)₂I and Re (NPh) Cl₃(PPh₃)₂。

5. The method according to embodiment 4, wherein the precursor of the rhenium complex is ReOCl₃(PPh₃)₂。

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

7. The method according to 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), beta-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 ethers such as Trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS), Triisopropylsilyloxymethyl (TOM) and Triisopropylsilyl (TIPS) ethers, and methyl ether and Ethoxyethyl Ether (EE).

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

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

10. The method of embodiment 8, wherein the volume ratio of DMF to MeOH is about 1: 1.

11. The process according to any one of embodiments 1 to 10, wherein a base is further used in step a).

12. The process according to example 11, wherein the base is NaOMe.

13. The method according to any one of embodiments 1 to 12, comprising the steps of:

a) reacting a compound of formula (I):

wherein R is a thiol protecting group selected from the group consisting of acetyl (Ac) and benzoyl (Bz),

and ReOCl₃(PPh₃)₂，

Reaction in DMF: MeOH in a volume ratio of about 1:1 to afford the compound of formula (II):

b) adding N-hydroxysuccinimide and EDC-HCl to give a compound of formula (III):

c) adding an aminooligonucleotide and a base to provide a compound of formula (IV):

wherein the oligonucleotide comprises at least one 2' sugar modified nucleoside or LNA nucleoside and steps a), b) and c) are carried out in a one-pot reaction.

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

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

16. The method according to any one of embodiments 1 to 15, wherein the coupling agent is selected from the group consisting of: 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 (tripyrrolidinophosphonium hexafluorophosphate bromide), TBTU (2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethylammonium tetrafluoroborate), TSTU ((N, N, N ', N' -tetramethyl-O- (N-succinimidyl) uretetrafluoroborate)), TNTU (O- (bicyclo [2.2.1] hept-5-ene-2, 3-dicarboximido) -N, N '-tetramethyluronium tetrafluoroborate), T3P (2,4, 6-tripropyl-1, 3,5,2,4, 6-trioxatriphospha-cyclohexane-2, 4, 6-trioxide), HCTU (O- (6-chlorobenzotriazol-1-yl) -N, N' -tetramethyluronium hexafluorophosphate), TATU, HATU (N- [ (dimethylamino) -1H-1,2, 3-triazolo- [4,5-b ] pyridin-1-ylmethylene ] -N-methylmethanium hexafluorophosphate N-oxide), DMTMM (4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholine n-ium chloride), CDI (1,1' -carbonyldiimidazole) and the like.

17. The method of example 16, wherein the coupling agent is EDC-HCl (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide-HCl).

Examples of the invention

To be degassed (N)₂And/or argon) DMF MeOH (2.5mL, ratio 1:1) was added to the solution containing protected Mag-3-COOH (10mg,27umol), ReOCl₃(PPh₃)₂(45mg,54umol) and NaOMe (29.4mg, 554 umol). The resulting solution was then stirred for 60 minutes. Thereafter, N is used₂The flow evaporated the reaction mixture to a volume of about 0.5 mL. Then, N-hydroxysuccinimide (9.4mg,82umol) and EDC-HCl (6.3mg,33umol) were added, and the resulting mixture was stirred overnight. After overnight reaction, the mixture was diluted with 0.5mL DMSO and stirred for 15 minutes. Amino-modified oligonucleotide (30mg in 0.5mL NaHCO) was then added₃pH 8.5). The resulting mixture was stirred overnight to provide the desired oligonucleotide compound (i.e., Mag-3 conjugated amino oligonucleotide chelated with rhenium).

And (3) purification: by adding 2% LiClO to the above reaction mixture₄In acetone, to precipitate the oligonucleotide from the one-pot reaction. The flask was then spun, centrifuged and decanted. The remaining residue (precipitated oligonucleotide) was then dissolved in water and purified by reverse phase HPLC.