A novel method for preparing rhenium-chelating MAG3 oligonucleotides.

Abstract

The present invention relates to compounds of formula (III) and novel methods for preparing rhenium-chelating MAG3 oligonucleotides. TIFF2022530537000034.tif35128

Description

[Technical field]

[0001] The present invention relates to novel methods for preparing rhenium-chelated MAG3 (mercaptoacetyltriglycine) as well as rhenium-chelated MAG3 oligonucleotides. [Background technology]

[0002] Rhenium (Re) and technetium (Tc) show great potential in nuclear medicine because of the future applications of the rhenium radionuclides 186Re and 188Re in therapeutic nuclear medicine and the technetium radionuclide 99mTc in medical diagnostic imaging. Both 186Re and 188Re radionuclides are gamma and beta-emitters, whereas the 99mTc radionuclide is only a gamma emitter; they exhibit nuclear properties that are favorable for medical imaging and therapy. Rhenium and technetium based radiopharmaceuticals have been widely applied in medical diagnostic imaging and therapeutic nuclear medicine in Europe and other parts of the world.

[0003] MAG3 ligands are well-established imaging ligands that form stable metal complexes with rhenium (Re) or technicium (Tc). Many different biologically active molecules have been functionalized with MAG3, such as sugars, siRNAs, and antisense oligonucleotides, to name a few. The resulting Re or Tc complexes may be of medical importance in the field of radiopharmaceuticals, since radioisotopes such as 186Re or 188Re are used for the diagnosis or treatment of tumors or renal function. However, fundamental research is still needed to attach radiometals to biologically active molecules. Most rhenium(V)-based radiopharmaceuticals lack stability (in physiological conditions) or selectivity (with respect to cell targeting or the preparation of precisely defined drugs), and their synthesis remains to be improved.

[0004] For example, Alsbaiee et al. describe the synthesis of rhenium-chelating MAG3-functionalized rosette nanotubes in Tet Lett 2012, 53, 1645-1651 (Non-Patent Document 1), International Journal of Pharmaceutics, 2002, 248, 173-182 (Non-Patent Document 2). P. Winnard et al. describe the two-step preparation and use of NHS-MAG3 for technetium-99m labeling of DNA in Nuclear Medicine & Biology, Vol. 24, pp. 425-432, 1997 (Non-Patent Document 3).

[0005] Alsbaiee and Winnard do not disclose the synthesis of rhenium-chelating MAG3 oligonucleotides.

[0006] The present inventors have discovered a novel method for the synthesis of rhenium-chelated MAG3 and rhenium-chelated MAG3 oligonucleotides. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Alsbaiee et al., Tet Lett 2012, 53, 1645-1651 [Non-Patent Document 2] Alsbaiee et al., International Journal of Pharmaceutics, 2002, 248, 173-182 [Non-Patent Document 3] P. Winnard et al., Nuclear Medicine & Biology, Vol. 24, pp. 425-432, 1997 Summary of the Invention

[0008] In a first aspect, the present invention relates to a novel method for preparing rhenium-chelating MAG3 compounds of formula (III) in a one-pot reaction: TIFF0007644025000001.tif35128

[0009] In a second aspect, the present invention relates to a novel method for preparing a rhenium-chelating MAG3 oligonucleotide compound of formula (IV): TIFF0007644025000002.tif35128

[0010] In a third aspect, the present invention relates to a novel process for preparing a compound of formula (III) or formula (IV) in a one-pot reaction.

[0011] Other aspects of the invention are described in more detail below.

[0012] definition The term "coupling reagent" refers to a compound that allows the covalent bonding of two organic molecules, such as a carboxylic acid and an amine, an alcohol, or a thiol. Furthermore, in the context of the present invention, a coupling reagent refers to a compound that can assist in the coupling reaction in which a carboxylic acid is coupled to an amine. The coupling reagent can be used as a water-soluble HCl salt. EDC is another example of a carbodiimide. The by-products of EDC are water-soluble and can be easily removed by aqueous workup. Examples of other coupling reagents 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 (bromotripyrrolidinophosphonium hexafluorophosphate), TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate), TSTU (N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate), TNTU (O-(bis(trimethylaminopropyl)uronium tetrafluoroborate)), and the like. (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 hexafluorophosphatase), phosphate), TATU, HATU (N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide), DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride), CDI (1,1'-carbonyldiimidazole), etc.

[0013] The term "alkyl", alone or in combination, refers to a straight or branched chain alkyl group having 1 to 8 carbon atoms, in particular a straight or branched chain alkyl group having 1 to 6 carbon atoms, more particularly a straight or branched chain alkyl group having 1 to 4 carbon atoms. Examples of straight and branched chain C1-C8 alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, the isomeric pentyls, isomeric hexyls, isomeric heptyls, and isomeric octyls, in particular methyl, ethyl, propyl, butyl, and pentyl. Particular examples of alkyl are methyl, ethyl, and propyl.

[0014] The term "amino," alone or in combination, means a primary amino group (-NH2), a secondary amino group (-NH-), or a tertiary amino group (-N-).

[0015] The term "amino oligonucleotide" refers to an oligonucleotide that contains a non-aromatic NH2. The NH2 group can be located, for example, at the 3' or 5' end of the oligonucleotide, for example, a 3' oligonucleotide -CH2CH2CH2CH2CH2CH2-NH2-5'.

[0016] The term "amino protecting group" refers to a protecting group for the amino group. Amines are functional groups that often require a protecting group during organic reactions. Carbamates such as t-butoxycarbonyl (Boc, e.g., removed by concentrated strong acid (e.g., HCl or CF3COOH) or by heating to >80°C), benzyloxycarbonyl (Cbz, e.g., removed by hydrogenolysis), or 9-fluorenylmethoxycarbonyl (Fmoc, e.g., removed by base such as piperidine) are commonly used amine protecting groups. Additional options for protecting groups with various deprotection conditions can be used, such as those described by Peter Wipf and coworkers at the University of Pittsburgh, Pennsylvania, USA, who developed a useful protecting group for primary, secondary, and heterocyclic amines: 2,2,6,6-tetramethylpiperidin-1-yloxycarbonyl (Tempoc). Acetyl (Ac) group is common in oligonucleotide synthesis to protect N4 of cytosine and N6 of adenine nucleobase, and can be removed by treating with base, most often aqueous or gaseous ammonia or methylamine. Ac is too stable to be easily removed from aliphatic amides. Benzoyl (Bz) group is also common in oligonucleotide synthesis to protect N4 of cytosine and N6 of adenine nucleobase, and can be removed by treating with base, most often aqueous or gaseous ammonia or methylamine. Bz is too stable to be easily removed from aliphatic amides. Other suitable amino protecting groups commonly used, such as DMF or iBu, can also be considered by those skilled in the art.

[0017] The term "aryl," alone or in combination, denotes a monovalent aromatic carbocyclic mono- or bicyclic ring system containing from 6 to 10 carbon ring atoms, optionally substituted with from 1 to 3 substituents independently selected from halogen, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, and formyl. Examples of aryl include phenyl and naphthyl, especially phenyl.

[0018] The term "chelate" refers to the fact that a compound containing a ligand (e.g., organic) binds to a central metal atom at more than one point. This generally involves the formation or presence of two or more separate coordinate bonds between a polydentate (multiple-bonded) ligand and a single central atom. These ligands are called chelant, chelating agents, chelators, or sequestering agents. This is generally a type of binding of ions and molecules to a metal ion. The compound of formula (II) according to the present invention is an example of a chelate or chelate complex.

[0019] The chelator moieties of the present invention may include any number of combinations of atoms selected from the group consisting of nitrogen, sulfur, oxygen, and phosphorus atoms. In certain embodiments, the chelator moieties include combinations of 3 to 5 such atoms. In some embodiments, the chelator can chelate any number of valent metal ions through coordination to other atoms such as nitrogen, sulfur, oxygen, and / or phosphorus atoms. In certain embodiments, the chelator can chelate 3 to 5 valent metal ions. Metal ions of any valence are contemplated for chelation to the chelators of the present invention. Examples of these valent 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.

[0020] The term "halogen" or "halo", alone or in combination, means fluorine, chlorine, bromine or iodine, particularly fluorine, chlorine or bromine, more particularly fluorine. The term "halo", in combination with another group, means substitution of said group with at least one halogen, particularly 1 to 5 halogens, in particular 1 to 4 halogens, i.e. 1, 2, 3 or 4 halogens.

[0021] The term "thiol" refers to a -SH group.

[0022] A "thiol protecting group" is a protecting group for a thiol group and is also used to protect a hydroxyl group. Examples of hydroxyl 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), tri-iso-propylsilyloxymethyl (TOM) and triisopropylsilyl (TIPS) ethers), methyl ether, and ethoxyethyl ether (EE). Particular examples of hydroxyl protecting groups are DMT and TMT, especially DMT.

[0023] The term "LNA monomer" refers to an LNA nucleotide, meaning a nucleotide wherein the nucleoside is an LNA nucleoside as defined herein.

[0024] The term "one-pot synthesis" refers to a strategy for improving 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 for chemists, as it can save time and resources while increasing chemical yields by avoiding lengthy separation processes and purification of intermediate compounds.

[0025] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the free base or free acid, without being biologically or otherwise undesirable. Salts are formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, especially 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, and the like. In addition, these salts can be prepared by adding 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, and magnesium salts. Salts derived from organic bases include, but are not limited to, 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 resin salts.The oligonucleotides of the present invention may also exist in zwitterionic form.In particular, the particularly preferred pharma- ceutically acceptable salts of the present invention are sodium, lithium, potassium and trialkylammonium salts.

[0026] The term "precursor of a rhenium complex" refers to a rhenium atom capable of chelating a compound of formula (I). An example of a precursor of a rhenium complex is ReOCl3(PPh3)2 (oxotrichlorobis(triphenylphosphine)rhenium(V)), which is commercially available. Other examples are Re(NPh)Cl3(PPh3)2, ReO2(PPh3)2I, or Re(NPh)Cl3(PPh3)2.

[0027] The term "protecting group", alone or in combination, means a group that selectively blocks a reactive site of a multifunctional compound so that a chemical reaction can be selectively carried out at an otherwise unprotected reactive site. The protecting group can be removed. Exemplary protecting groups are an amino protecting group, a carboxy protecting group, or a hydroxy protecting group.

[0028] A "phosphate protecting group" is a protecting group for a phosphate group. Examples of phosphate protecting groups are 2-cyanoethyl and methyl. A particular example of a phosphate protecting group is 2-cyanoethyl.

[0029] The rhenium complexes according to the invention are Re(V) complexes.

[0030] If one of the starting materials or compounds of the present invention contains one or more functional groups that are not stable or are reactive under the reaction conditions of one or more reaction steps, suitable protecting groups (e.g., those described in “Protective groups in Organic Chemistry” by TW Greene and PgM Wuts, 3 rd Protective groups (such as those described in 1999, Ed., Wiley, New York) can be introduced before the critical step applying 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 tert-butoxycarbonyl (Boc), 9-fluorenylmethylcarbamate (Fmoc), 2-trimethylsilylethylcarbamate (Teoc), carbobenzyloxy (Cbz), and p-methoxybenzyloxycarbonyl (Moz).

[0031] The compounds described herein may contain several asymmetric centers and may exist in the form of optically pure enantiomers, mixtures of enantiomers, e.g. racemates, mixtures of diastereoisomers, racemates of diastereoisomers, or mixtures of diastereoisomers racemates.

[0032] Oligonucleotides The term "oligonucleotide" as used herein is defined as a molecule that contains two or more covalently linked nucleosides as commonly understood by those skilled in the art. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are usually made in the laboratory by solid-phase chemical synthesis followed by purification. When referring to the sequence of an oligonucleotide, the reference is to the sequence or order of the nucleobase moieties of the covalently linked nucleotides or nucleosides, or to modifications thereof. The oligonucleotides of the present invention are artificial, chemically synthesized, and usually purified or isolated. The oligonucleotides of the present invention may contain one or more modified nucleosides or nucleotides.

[0033] Antisense oligonucleotides The term "antisense oligonucleotide" as used herein is defined as an oligonucleotide that can regulate the expression of a target gene by hybridizing to a target nucleic acid, particularly a continuous sequence on the target nucleic acid.Antisense oligonucleotides are not double-stranded in nature, and therefore are not siRNA or shRNA.Preferably, the antisense oligonucleotides of the present invention are single-stranded.It is understood that the single-stranded oligonucleotides of the present invention can form hairpin or intermolecular duplex structures (duplexes between two molecules of the same oligonucleotide), as long as the degree of complementarity within or between themselves is less than 50% over the entire length of the oligonucleotide.

[0034] sugar modification Oligomers of the invention may contain one or more nucleosides which have modified sugar moieties, ie, sugar moieties which are modified compared to the ribose sugar moiety found in DNA and RNA.

[0035] A number of nucleosides with modifications of the ribose sugar moiety have been produced with the primary goal of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0036] Such modifications include those in which the ribose ring structure has been modified, for example, by replacing it with a hexose ring (HNA), or a bicyclic ring, typically having a biradical bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring, typically lacking a bond between the C2 and C3 carbons (e.g., UNA). Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acids (WO 2011 / 017521) or tricyclic nucleic acids (WO 2013 / 154798). Modified nucleosides also include nucleosides in which the sugar moiety has been replaced with a non-sugar moiety, for example, in the case of peptide nucleic acids (PNAs) or morpholino nucleic acids.

[0037] 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 that occurs naturally in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4', or 5' position.

[0038] 2' sugar modified nucleosides A 2' sugar modified nucleoside is a nucleoside that has a substituent other than H or -OH at the 2' position (2' substituted nucleoside) or that contains a 2' linked biradical that can form a bridge between the 2' carbon and a second carbon on the ribose ring, such as an LNA (2'-4' biradical bridged) nucleoside.

[0039] Indeed, much attention has been paid to the development of 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 oligonucleotides with enhanced binding affinity and / or increased nuclease resistance.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. Further examples can be found, for example, in Freier & Altmann; Nucl.Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr.Opinion in Drug Development, 2000, 3(2), 293-213 and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are illustrative examples of some 2'-substituted modified nucleosides. TIFF0007644025000003.tif70128

[0040] In the context of the present invention, 2' modifications do not include 2' bridging molecules such as LNA.

[0041] Locked Nucleic Acid Nucleosides (LNA Nucleosides) "LNA nucleosides" are 2'-modified nucleosides that contain a biradical bond (also called a "2'-4' bridge") linking the C2' and C4' of the ribose sugar ring of the nucleoside, which restricts or fixes the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNAs). Fixation of the ribose conformation is associated with improved hybridization affinity (stabilization of the duplex) when LNA is incorporated into an oligonucleotide of a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complementary duplex.

[0042] Non-limiting, exemplary LNA nucleosides are described 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.

[0043] The 2'-4' bridge can be oriented below the plane of the ribose ring (β-D-configuration) or above the plane of the ring (α-L-configuration), as shown in Formula (A) and Formula (B), respectively.

[0044] It will be appreciated that unless otherwise specified, LNA nucleosides can be either β-D or α-L stereoisoforms.

[0045] Specific examples of LNA nucleosides of the present invention are shown in Scheme 1, where B is as defined above. Scheme 1 TIFF0007644025000004.tif93128TIFF0007644025000005.tif213128TIFF0007644025000006.tif209128TIFF0007644025000007.tif33128

[0046] Particular LNA nucleosides are β-D-oxy-LNA, 6'-methyl-β-D-oxy LNA, for example (S)-6'-methyl-β-D-oxy-LNA ((S)-cET), and ENA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Detailed Description of the Invention In one aspect, the present invention relates to a process for preparing a compound of formula (III), the process comprising: a) a compound of formula (I): TIFF0007644025000008.tif20128 [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): Steps to get TIFF0007644025000009.tif32128, b) Adding N-hydroxysuccinimide along with a coupling reagent to give a compound of formula (III): Steps to get TIFF0007644025000010.tif35128 Including, wherein steps a) and b) are carried out in a one-pot reaction.

[0048] In one embodiment of the method of the invention there is a further step c) in which an amino oligonucleotide is added together with a base to obtain a compound of formula (IV): TIFF0007644025000011.tif35128

[0049] In one embodiment of the process of the present invention, steps a) and b) are carried out in a one-pot synthesis. In one embodiment, steps a), b) and c) are carried out in a one-pot synthesis.

[0050] In one embodiment of the method of the present 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.

[0051] In one embodiment of the method of the present invention, the precursor to the rhenium complex is ReOCl3(PPh3)2.

[0052] In one embodiment of the method of the present invention, R is H.

[0053] In one 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), 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 ether (e.g., trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM) and triisopropylsilyl (TIPS) ethers), and methyl ether and ethoxyethyl ether (EE).

[0054] In one embodiment of the method of the present invention, R is a thiol protecting group selected from the group consisting of acetyl (Ac) and benzoyl (Bz).

[0055] In one embodiment of the process of the present 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 of the present invention, the solvent is a mixture of DMF and MeOH. In one embodiment of the process of the present invention, the solvent is a mixture of DMF and MeOH in a volume ratio of about 1:1.

[0056] In one embodiment of the process of the invention a base is further used in step a). In one embodiment of the process of the invention the base is NaOMe.

[0057] In one embodiment, the method of the invention comprises: c) A compound of formula (I): TIFF0007644025000012.tif20128 [wherein R is H or a thiol protecting group selected from the group consisting of acetyl (Ac) and benzoyl (Bz)] With ReOCl3(PPh3)2, Reaction in about a 1:1 volume ratio of DMF:MeOH affords a compound of formula (II): Steps to get TIFF0007644025000013.tif32128, d) adding N-hydroxysuccinimide along with EDC-HCl to obtain the compound of formula (III): TIFF0007644025000014.tif35128c) Adding an amino oligonucleotide together with a base to give a compound of formula (IV): Steps to get TIFF0007644025000015.tif35128 Including, Here, the oligonucleotide comprises at least one 2' sugar modified nucleoside or an LNA nucleoside.

[0058] In one embodiment, in the methods of the invention, the oligonucleotide is an antisense oligonucleotide.In one embodiment, in the methods of the invention, the antisense oligonucleotide comprises a 2' sugar modified nucleoside or an LNA nucleoside as defined herein.

[0059] MAG3 is commercially available from conventional suppliers, such as, for example, Sigma Aldrich or ABX. Compounds of formula (I) can be commercially available as protected NHS-MAG3 acetyl from various suppliers, such as, for example, Sigma Aldrich (Nature protocols 2007-4-21, Yi Wang et al.).

[0060] Unless otherwise stated, all starting products, reagents, and solvents are commercially available.

[0061] In step b) a solvent is used, which can be a polar aprotic solvent, a polar protic solvent, or a mixture thereof.

[0062] Examples of polar aprotic solvents are DCM (dichloromethane), N-methylpyrrolidone, THF (tetrahydrofuran), ETOAc (ethyl acetate), acetone, DMF (dimethylformamide), MeCN (acetonitrile), DMSO (dimethylsulfoxide), and PC (propylene carbonate), or mixtures thereof.

[0063] In one or all embodiments of the process of the present invention, the polar aprotic solvent is DMF.

[0064] Examples of polar protic solvents are formic acid, n-butanol, IPA (isopropanol), EtOH (ethanol), MeOH (methanol), AcOH (acetic acid), and water, or mixtures thereof.

[0065] In one or all embodiments of the methods of the present invention, the polar protic solvent is MeOH.

[0066] In one or all embodiments of the methods of the present invention, the solvent can be a mixture of a polar aprotic solvent and a polar protic solvent, such as DMF:MeOH, for example, about a 1:1 volume ratio of DMF:MeOH.

[0067] In step c), a solvent can be used. In one embodiment, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride is used.

[0068] The coupling reagents for step b) were 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-benzotriazol-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]heptafluorophosphate), and TECHNICAL DATA: -5-ene-2,3-dicarboximide)-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).

[0069] In one embodiment of the method of the invention, the coupling reagent is EDC-HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl).

[0070] The method of the present invention can be carried out in the following non-limiting embodiments:

[0071] 1.a) A compound of formula (I): TIFF0007644025000016.tif20128 [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): Steps to get TIFF0007644025000017.tif32128, b) Adding N-hydroxysuccinimide along with a coupling reagent to give a compound of formula (III): Steps to get TIFF0007644025000018.tif35128 Including, wherein steps a) and b) are carried out in a one-pot reaction; Process for the preparation of compounds of formula (III).

[0072] 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): TIFF0007644025000019.tif35128

[0073] 3. The method according to embodiment 2, wherein steps a), b), and c) are carried out in a one-pot synthesis.

[0074] 4. The method of any 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.

[0075] 5. The method of embodiment 4, wherein the precursor of the rhenium complex is ReOCl3(PPh3)2.

[0076] 6. The method of any one of the preceding embodiments, wherein R is H.

[0077] 7. The method of any of embodiments 1 to 5, wherein R is a thiol protecting group selected from the group consisting of 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), tri-iso-propylsilyloxymethyl (TOM) and triisopropylsilyl (TIPS) ethers), and methyl ether and ethoxyethyl ether (EE).

[0078] 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).

[0079] 9. The method according to any of the preceding claims, wherein the solvent in step a) is a mixture of a polar aprotic solvent and a polar protic solvent.

[0080] 10. The method of embodiment 8, wherein the DMF:MeOH is in a volume ratio of about 1:1.

[0081] 11. The method according to any of the preceding claims, wherein a base is further used in step a).

[0082] 12. The method of embodiment 11, wherein the base is NaOMe.

[0083] 13.a) A compound of formula (I): TIFF0007644025000020.tif20128 [wherein R is a thiol protecting group selected from the group consisting of acetyl (Ac) and benzoyl (Bz)] With ReOCl3(PPh3)2, Reaction in about a 1:1 volume ratio of DMF:MeOH affords a compound of formula (II): Steps to get TIFF0007644025000021.tif33128, b) Adding N-hydroxysuccinimide along with EDC-HCl to give a compound of formula (III): Steps to get TIFF0007644025000022.tif35128, c) adding an amino oligonucleotide together with a base to obtain a compound of formula (IV): Steps to get TIFF0007644025000023.tif35128 Including, 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. A method according to any one of embodiments 1 to 12.

[0084] 14. The method of any of embodiments 1 to 13, wherein the oligonucleotide is an antisense oligonucleotide.

[0085] 15. The method of embodiment 14, wherein the antisense oligonucleotide comprises at least one 2' sugar modified nucleoside or LNA nucleoside.

[0086] 16. Coupling reagents include 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), phosphate), PyBrOP (bromotripyrrolidinophosphonium hexafluorophosphate), TBTU (2-(1H-benzotriazol-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- dicarboximide)-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 16. The method of any of the preceding claims, wherein the coupling reagent is selected from the group consisting of: -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).

[0087] 17. The method of embodiment 16, wherein the coupling reagent is EDC-HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl). EXAMPLES

[0088] TIFF0007644025000024.tif55152

[0089] Degassed (N2 and / or argon) DMF:MeOH (2.5 mL, 1:1 ratio) was added to a flask containing protected Mag-3-COOH (10 mg, 27 μmol), ReOCl3(PPh3)2 (45 mg, 54 μmol), and NaOMe (29.4 mg, 554 μmol). The resulting solution was then stirred for 60 minutes. The reaction mixture was then evaporated to a volume of about 0.5 mL using a stream of N2. N-hydroxysuccinimide (9.4 mg, 82 μmol) and EDC-HCl (6.3 mg, 33 μmol) were then added and the resulting mixture was kept stirring overnight. After reacting overnight, the mixture was diluted with 0.5 mL of DMSO and stirred for 15 minutes. The amino-modified oligonucleotide (30 mg dissolved in 0.5 mL of NaHCO3, pH=8.5) was then added. The resulting mixture was stirred overnight to obtain the desired oligonucleotide compound (ie, amino oligonucleotide conjugated to rhenium-chelated Mag-3).

[0090] Purification: The oligonucleotides were precipitated from the one-pot reaction by adding a solution of 2% LiClO4 in acetone to the above reaction mixture. The flask was then spun, centrifuged, and decanted. The remaining residue (precipitated oligonucleotide) was then dissolved in water and purified by reverse phase HPLC.

Claims

1. a) A compound of formula (I): wherein R is H or a thiol protecting group. is reacted with a precursor of a rhenium complex in a solvent to obtain a compound of formula (II): obtaining a b) N-hydroxysuccinimide is added along with a coupling reagent to give a compound of formula (III): obtaining a c) adding an amino oligonucleotide together with a base to obtain a compound of formula (IV): Steps to get Including, wherein steps a), b) and c) are carried out in a one-pot reaction; 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; The solvent in step a) is a mixture of a polar aprotic solvent and a polar protic solvent; Process for the preparation of compounds of formula (IV).

2. The precursor of the rhenium complex is ReOCl ３ (PPh ３ ) ２ The method of claim 1, wherein

3. 3. The method of claim 1 or 2, wherein R is a thiol protecting group selected from the group consisting of 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), tri-iso-propylsilyloxymethyl (TOM) and triisopropylsilyl (TIPS) ethers), and methyl ether and ethoxyethyl ether (EE).

4. 4. The method of claim 3, wherein R is a thiol protecting group selected from the group consisting of acetyl (Ac) and benzoyl (Bz).

5. 5. The process according to any one of claims 1 to 4, wherein the mixture of polar aprotic solvent and polar protic solvent is a mixture of DMF and MeOH, with a volume ratio of DMF:MeOH of 1:

1.

6. 6. The method according to claim 1, wherein a base is further used in step a).

7. The method of claim 6, wherein the base is NaOMe.

8. Coupling reagents include 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-benzotriazol-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-[(dimethyl 8. The method of any one of claims 1 to 7, wherein the aryl group is selected from the group consisting of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride), DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride), and CDI (1,1'-carbonyldiimidazole).

9. The method of claim 8, wherein the coupling reagent is EDC-HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl).

10. a) 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). ReOCl ３ (PPh ３ ) ２ and, Reaction in a 1:1 volume ratio of DMF:MeOH affords a compound of formula (II): obtaining a b) N-hydroxysuccinimide is added along with EDC-HCl to give the compound of formula (III): obtaining a c) adding an amino oligonucleotide together with a base to obtain a compound of formula (IV): Steps to get Including, 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.

10. The method according to any one of claims 1 to 9.

11. 11. The method of any one of claims 1 to 10, wherein the oligonucleotide is an antisense oligonucleotide.

12. The method of claim 11, wherein the antisense oligonucleotide comprises at least one 2' sugar modified nucleoside or LNA nucleoside.

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