WO2019224172A1 - Nouveau procédé de fabrication d'allofuranose à partir de glucofuranose - Google Patents

Nouveau procédé de fabrication d'allofuranose à partir de glucofuranose Download PDF

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WO2019224172A1
WO2019224172A1 PCT/EP2019/063044 EP2019063044W WO2019224172A1 WO 2019224172 A1 WO2019224172 A1 WO 2019224172A1 EP 2019063044 W EP2019063044 W EP 2019063044W WO 2019224172 A1 WO2019224172 A1 WO 2019224172A1
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lna
synthesis
formula
added
allofuranose
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PCT/EP2019/063044
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Jacob Ravn
Christoph Rosenbohm
Prasad Reddy
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Roche Innovation Center Copenhagen A/S
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H9/00—Compounds containing a hetero ring sharing at least two hetero atoms with a saccharide radical
    • C07H9/02—Compounds containing a hetero ring sharing at least two hetero atoms with a saccharide radical the hetero ring containing only oxygen as ring hetero atoms
    • C07H9/04—Cyclic acetals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals

Definitions

  • the invention relates to a novel process for making allofuranose from gluco- furanose.
  • Allofuranose and glucofuranose are useful intermediates in the manufacture of synthetic, pharmaceutically active oligonucleotides.
  • Allofuranose in particular is useful in the manufacture of Locked Nucleic Acid (LNA) nucleoside monomers, which are useful as monomers of LNA oligonucleotides.
  • LNA Locked Nucleic Acid
  • oligonucleotides as therapeutic agents has witnessed remarkable progress over recent decades leading to the development of molecules acting by diverse mechanisms including RNase H activating gapmers, splice switching oligonucleotides, microRNA inhibitors, siRNA or aptamers (S. T. Crooke, Antisense drug technology : principles, strategies, and applications, 2nd ed. ed., Boca Raton, FL : CRC Press, 2008).
  • LNA monomer synthesis consists of 15-17 steps. Due to the length of the synthesis it is very important that all steps proceed in the most optimal way.
  • the synthesis steps are optimised on four parameters: 1. Fast reaction time; 2. Employing cheap reagents; 3. Easy to handle; 4. Proceeds in high overall yields.
  • the starting material was l,2:5,6-di-0- isopropylidene-a-D-allofuranose.
  • l,2:5,6-di-0-isopropylidene-a-D-allofuranose is comer- cially available (e.g. at Sigma Aldrich now Merck, CAS Number: 2595-05-03 in smaller quantities). Reducing the cost of the starting material by improving the synthesis is of great value for LNA synthesis.
  • l,2:5,6-di-0-isopropylidene-a-D-allofuranose is normally prepared from the much cheaper l,2:5,6-di-0-isopropylidene-a-D-gIucofuranose in two steps.
  • the first step is oxidation of the secondary alcohol which is subsequently reduced to provide the allo- configuration. Oxidation of secondary hydroxyl groups to their corresponding carbonyl derivatives with dimethyl sulfoxide (DMSO) and acetic anhydride has been described (Albright J.D. and Goldman L., J. Am. Chem. Soc, 1967, 89: 10).
  • Fuertes C. M. and Cesar M. prepared l,2:5,6-di-0-isopropylidene-a-D-allofuranose by a consecutive two step reaction from l,2:5,6-di-0-isopropylidene-a-D-glucofuranose.
  • the yield in the oxidation step was 60%.
  • the subsequent reduction was performed in 75% yield, thus the overall yield in the sequential reactions was 45%.
  • W02002098892 describes the synthesis of allofuranose using glucofuranose as starting material in a one-pot reaction.
  • the oxidation of 1,2: 5,6-di-O- isopropylidene-a-D-glucofuranose is carried out with DMSO/acetic anhydride.
  • the reduction reaction is performed in one pot obtaining what was believed to be high yields of recrystallised and analytical pure l,2:5,6-di-0-isopropylidene-a-D-allofuranose.
  • the invention relates to a method for the synthesis of allofuranose from glucofuranose, said method comprising the steps of a catalytic oxidation of glucosefuranose in a reaction solution comprising hypochlorite salt, and a reduction to produce allofuranose.
  • the inventors were surprised by the following findings about the method of the invention. Firstly, it achieves a high yield when compared to methods of the prior art. Secondly, it is cheap, for instance, it allows for the use of very low amounts of TEMPO as a catalyst. Thirdly, it allows to avoid noxious oxidations by using NaOCl(aq) instead. Finally, the reduction step according to the method of the invention requires no
  • allofuranose signifies allofuranose wherein 4 alcohol moieties are protected except the alcohol on position 3. Allofuranose is also known as (3R, 4S, 5R)-5-[(lR)-l,2-dihydroxyethyl]oxolane-2,3,4-triol or D-allofuranose:
  • glucofuranose signifies glucofuranose wherein 4 alcohol moieties are protected except the alcohol on position 3.
  • Glucofuranose is also known as D-glucose or D-glucofuranose.
  • Glucoruranose according to the invention has the following chemical structure:
  • alkyl signifies a straight-chain or branched- chain alkyl group with 1 to 8 carbon atoms, particularly a straight or branched-chain alkyl group with 1 to 6 carbon atoms and more particularly a straight or branched-chain alkyl group with 1 to 4 carbon atoms.
  • straight-chain and branched-chain Ci-Cs alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert.
  • cycloalkyl signifies a cycloalkyl ring with 3 to 8 carbon atoms and particularly a cycloalkyl ring with 3 to 6 carbon atoms.
  • cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl, more particularly cyclopropyl and cyclobutyl.
  • a particular example of “cycloalkyl” is cyclopropyl.
  • alkoxy signifies a group of the formula alkyl- O- in which the term "alkyl” has the previously given significance, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec.butoxy and tert.butoxy.
  • Particular “alkoxy” are methoxy and ethoxy.
  • Methoxyethoxy is a particular example of
  • alkenyl signifies a straight-chain or branched hydrocarbon residue comprising an olefinic bond and up to 8, preferably up to 6, particularly preferred up to 4 carbon atoms.
  • alkenyl groups are ethenyl, 1- propenyl, 2-propenyl, isopropenyl, l-butenyl, 2-butenyl, 3-butenyl and isobutenyl.
  • alkynyl signifies a straight-chain or branched hydrocarbon residue comprising a triple bond and up to 8, particularly 2 carbon atoms.
  • halogen or“halo”, alone or in combination, signifies fluorine, chlorine, bromine or iodine and particularly fluorine, chlorine or bromine, more particularly fluorine.
  • halo in combination with another group, denotes the substitution of said group with at least one halogen, particularly substituted with one to five halogens, particularly one to four halogens, i.e. one, two, three or four halogens.
  • haloalkyl denotes an alkyl group substituted with at least one halogen, particularly substituted with one to five halogens, particularly one to three halogens.
  • haloalkyl include monofluoro-, difluoro- or trifluoro- methyl, -ethyl or -propyl, for example 3,3,3-trifluoropropyl, 2-fluoroethyl, 2,2,2- trifluoroethyl, fluoromethyl or trifluoromethyl. Fluoromethyl, difluoromethyl and trifluoromethyl are particular“haloalkyl”.
  • halocycloalkyl denotes a cycloalkyl group as defined above substituted with at least one halogen, particularly substituted with one to five halogens, particularly one to three halogens.
  • Particular example of“halocycloalkyl” are halocyclopropyl, in particular fluorocyclopropyl, difluorocyclopropyl and
  • carbonyl alone or in combination, signifies the -C(O)- group.
  • amino alone or in combination, signifies the primary amino group (- NH 2 ), the secondary amino group (-NH-), or the tertiary amino group (-N-).
  • alkylamino alone or in combination, signifies an amino group as defined above substituted with one or two alkyl groups as defined above.
  • sulfonyl alone or in combination, means the -SO2 group.
  • cabamido alone or in combination, signifies the -NH-C(0)-NH 2 group.
  • aryl denotes a monovalent aromatic carbocyclic mono- or bicyclic ring system comprising 6 to 10 carbon ring atoms, optionally substituted with 1 to 3 substituents independently selected from halogen, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl,
  • alkoxycarbonyl alkylcarbonyl and formyl.
  • aryl examples include phenyl and naphthyl, in particular phenyl.
  • heteroaryl denotes a monovalent aromatic heterocyclic mono- or bicyclic ring system of 5 to 12 ring atoms, comprising 1, 2, 3 or 4 heteroatoms selected from N, O and S, the remaining ring atoms being carbon, optionally substituted with 1 to 3 substituents independently selected from halogen, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl,
  • heteroaryl examples include pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, triazinyl, azepinyl, diazepinyl, isoxazolyl, benzofuranyl, isothiazolyl, benzothienyl, indolyl, isoindolyl, isobenzofuranyl,
  • benzimidazolyl benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, benzooxadiazolyl, benzothiadiazolyl, benzotriazolyl, purinyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, carbazolyl or acridinyl.
  • heterocyclyl signifies a monovalent saturated or partly unsaturated mono- or bicyclic ring system of 4 to 12, in particular 4 to 9 ring atoms, comprising 1, 2, 3 or 4 ring heteroatoms selected from N, O and S, the remaining ring atoms being carbon, optionally substituted with 1 to 3 substituents independently selected from halogen, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl and formyl.
  • Examples for monocyclic saturated heterocyclyl are azetidinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydro-thienyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,1- dioxo-thiomorpholin-4-yl, azepanyl, diazepanyl, homopiperazinyl, or oxazepanyl.
  • bicyclic saturated heterocycloalkyl examples include 8-aza-bicyclo[3.2.l]octyl, quinuclidinyl, 8-oxa-3-aza-bicyclo[3.2.l]octyl, 9-aza-bicyclo[3.3.l]nonyl, 3-oxa-9-aza- bicyclo[3.3.l]nonyl, or 3-thia-9-aza-bicyclo[3.3.l]nonyl.
  • partly unsaturated heterocycloalkyl examples include dihydrofuryl, imidazolinyl, dihydro-oxazolyl, tetrahydro-pyridinyl or dihydropyranyl.
  • salts refers to those salts which retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable.
  • the 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-acetylcystein.
  • salts derived from an inorganic base include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium salts.
  • Salts derived from organic bases include, but are not limited to salts of 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 oligonucleotide of the invention can also be present in the form of zwitterions.
  • Particularly preferred pharmaceutically acceptable salts of the invention are the sodium, lithium, potassium and trialkylammonium salts.
  • protecting group signifies a group which selectively blocks a reactive site in a multifunctional compound such that a chemical reaction can be carried out selectively at another unprotected reactive site.
  • Protecting groups can be removed.
  • Exemplary protecting groups are amino-protecting groups, carboxy-protecting groups or hydroxy-protecting groups.
  • Phosphate protecting group is a protecting group of the phosphate group.
  • phosphate protecting group examples include 2-cyanoethyl and methyl.
  • a particular example of phosphate protecting group is 2-cyanoethyl.
  • “Hydroxyl protecting group” is a protecting group of the hydroxyl group and is also used to protect thiol groups.
  • Examples of hydroxyl protecting groups are acetyl (Ac), benzoyl (Bz), benzyl (Bn), b-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 (T
  • Thiohydroxyl protecting group is a protecting group of the thiohydroxyl group.
  • Examples of thiohydroxyl protecting groups are those of the“hydroxyl protecting group”.
  • one of the starting materials or compounds of the invention contain one or more functional groups which are not stable or are reactive under the reaction conditions of one or more reaction steps
  • appropriate protecting groups as described e.g. in“Protective Groups in Organic Chemistry” by T. W. Greene and P. G. M. Wuts, 3 rd Ed., 1999, Wiley, New York
  • Such protecting groups can be removed at a later stage of the synthesis using standard methods described in the literature.
  • protecting groups are tert-butoxycarbonyl (Boc), 9-fluorenylmethyl carbamate (Fmoc), 2-trimethylsilylethyl carbamate (Teoc), carbobenzyloxy (Cbz) and p-methoxybenzyloxycarbonyl (Moz).
  • the compounds described herein can contain several asymmetric centers and can be present in the form of optically pure enantiomers, mixtures of enantiomers such as, for example, racemates, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates.
  • oligonucleotide as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification. When referring to a sequence of the oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. The
  • oligonucleotide of the invention is man-made, and is chemically synthesized, and is typically purified or isolated.
  • the oligonucleotide of the invention may comprise one or more modified nucleosides or nucleotides.
  • Antisense oligonucleotide as used herein is defined as oligonucleotides capable of modulating expression of a target gene by hybridizing to a target nucleic acid, in particular to a contiguous sequence on a target nucleic acid.
  • oligonucleotides are not essentially double stranded and are therefore not siRNAs or shRNAs.
  • the antisense oligonucleotides of the present invention are single stranded. It is understood that single stranded oligonucleotides of the present invention can form hairpins or intermolecular duplex structures (duplex between two molecules of the same oligonucleotide), as long as the degree of intra or inter self complementarity is less than 50% across of the full length of the oligonucleotide
  • sequence refers to the region of the sequence
  • oligonucleotide which is complementary to the target nucleic acid.
  • the term is used interchangeably herein with the term“contiguous nucleobase sequence” and the term “oligonucleotide motif sequence”.
  • all the nucleotides of the oligonucleotide constitute the contiguous nucleotide sequence.
  • the oligonucleotide comprises the contiguous nucleotide sequence, such as a F-G-F’ gapmer region, and may optionally comprise further nucleotide(s), for example a nucleotide linker region which may be used to attach a functional group to the contiguous nucleotide sequence.
  • the nucleotide linker region may or may not be complementary to the target nucleic acid.
  • Nucleotides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention include both naturally occurring and non-naturally occurring nucleotides.
  • nucleotides such as DNA and R A nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which is absent in nucleosides).
  • Nucleosides and nucleotides may also interchangeably be referred to as“units” or“monomers”.
  • modified nucleoside or“nucleoside modification” as used herein refers to nucleosides modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the (nucleo)base moiety.
  • the modified nucleoside comprise a modified sugar moiety.
  • modified nucleoside may also be used herein interchangeably with the term “nucleoside analogue” or modified“units” or modified“monomers”. Nucleosides with an unmodified DNA or RNA sugar moiety are termed DNA or RNA nucleosides herein. Nucleosides with modifications in the base region of the DNA or RNA nucleoside are still generally termed DNA or RNA if they allow Watson Crick base pairing. Nucleobase
  • nucleobase includes the purine (e.g. adenine and guanine) and pyrimidine (e.g. uracil, thymine and cytosine) moieties present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridization.
  • pyrimidine e.g. uracil, thymine and cytosine
  • nucleobase also encompasses modified nucleobases which may differ from naturally occurring nucleobases, but are functional during nucleic acid hybridization.
  • nucleobase refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants. Such variants are for example described in Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1.
  • the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5 -methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5- thiazolo-uracil, 2-thio-uracil, 2’thio-thymine, inosine, diaminopurine, 6-aminopurine, 2- aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine.
  • a nucleobase selected from isocytosine, pseudoisocytosine, 5 -methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil,
  • the nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C or U, wherein each letter may optionally include modified nucleobases of equivalent function.
  • the nucleobase moieties are selected from A, T, G, C, and 5-methyl cytosine.
  • 5-methyl cytosine LNA nucleosides may be used.
  • modified oligonucleotide describes an oligonucleotide comprising one or more sugar-modified nucleosides and/or modified intemucleoside linkages.
  • chimeric oligonucleotide is a term that has been used in the literature to describe oligonucleotides with modified nucleosides.
  • a stereodefined oligonucleotide is an oligonucleotide wherein at least one of the intemucleoside linkages is a stereodefined intemucleoside linkage.
  • a stereodefined phosphorothioate oligonucleotide is an oligonucleotide wherein at least one of the intemucleoside linkages is a stereodefined phosphorothioate
  • Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A) - thymine (T)/uracil (U).
  • oligonucleotides may comprise nucleosides with modified nucleobases, for example 5-methyl cytosine is often used in place of cytosine, and as such the term complementarity encompasses Watson Crick base-paring between non-modified and modified nucleobases (see for example Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).
  • the term“% complementary” as used herein, refers to the proportion of nucleotides in a contiguous nucleotide sequence in a nucleic acid molecule (e.g.
  • oligonucleotide which, at a given position, are complementary to (i.e. form Watson Crick base pairs with) a contiguous nucleotide sequence, at a given position of a separate nucleic acid molecule (e.g. the target nucleic acid).
  • the percentage is calculated by counting the number of aligned bases that form pairs between the two sequences (when aligned with the target sequence 5’-3’ and the oligonucleotide sequence from 3’-5’), dividing by the total number of nucleotides in the oligonucleotide and multiplying by 100.
  • a nucleobase/nucleotide which does not align (form a base pair) is termed a mismatch.
  • insertions and deletions are not allowed in the calculation of %
  • insertions and deletions are not allowed in the calculation of %
  • hybridizing or“hybridizes” as used herein is to be understood as two nucleic acid strands (e.g. an oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands thereby forming a duplex.
  • the affinity of the binding between two nucleic acid strands is the strength of the hybridization. It is often described in terms of the melting temperature (T m ) defined as the temperature at which half of the oligonucleotides are duplexed with the target nucleic acid. At physiological conditions T m is not strictly proportional to the affinity (Mergny and Lacroix,
  • AG° is the energy associated with a reaction where aqueous concentrations are 1M, the pH is 7, and the temperature is 37°C.
  • the hybridization of oligonucleotides to a target nucleic acid is a spontaneous reaction and for spontaneous reactions AG° is less than zero.
  • AG° can be measured experimentally, for example, by use of the isothermal titration calorimetry (ITC) method as described in Hansen et al., 1965, Chem. Comm. 36- 38 and Holdgate et al., 2005, Drug Discov Today. The skilled person will know that commercial equipment is available for AG° measurements.
  • AG° can also be estimated numerically by using the nearest neighbor model as described by SantaLucia, 1998, Proc Natl Acad Sci USA.
  • oligonucleotides of the present invention hybridize to a target nucleic acid with estimated AG° values below -10 kcal for oligonucleotides that are 10-30 nucleotides in length.
  • the degree or strength of hybridization is measured by the standard state Gibbs free energy AG°.
  • the oligonucleotides may hybridize to a target nucleic acid with estimated AG° values below the range of -10 kcal, such as below -15 kcal, such as below -20 kcal and such as below -25 kcal for oligonucleotides that are 8-30 nucleotides in length.
  • the oligonucleotides hybridize to a target nucleic acid with an estimated AG° value of -10 to -60 kcal, such as -12 to -40, such as from -15 to -30 kcal or-l6 to -27 kcal such as -18 to -25 kcal.
  • the oligomer of the invention may comprise one or more nucleosides which have a modified sugar moiety, i.e. a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.
  • a modified sugar moiety i.e. a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.
  • Numerous nucleosides with modification of the ribose sugar moiety have been made, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or nuclease resistance.
  • Such modifications include those where the ribose ring structure is modified, e.g. by replacement with a hexose ring (HNA), or a bicyclic ring, which typically have a biradical bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g. UNA).
  • HNA hexose ring
  • LNA ribose ring
  • UNA unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons
  • Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO
  • Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.
  • PNA peptide nucleic acids
  • Sugar modifications also include modifications made via altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2’ -OH group naturally found in DNA and RNA nucleosides. Substituents may, for example be introduced at the 2’, 3’, 4’ or 5’ positions.
  • a 2’ sugar modified nucleoside is a nucleoside which has a substituent other than H or -OH at the 2’ position (2’ substituted nucleoside) or comprises a 2’ linked biradical capable of forming a bridge between the 2’ carbon and a second carbon in the ribose ring, such as LNA (2’ - 4’ biradical bridged) nucleosides.
  • the 2’ modified sugar may provide enhanced binding affinity and/or increased nuclease resistance to the oligonucleotide.
  • 2’ substituted modified nucleosides are 2’-0-alkyl-RNA, 2’-0-methyl-RNA, 2’-alkoxy-RNA, 2’-0-methoxyethyl-RNA (MOE), 2’-amino-DNA, 2’-fluoro-RNA and 2’- F-ANA nucleoside. Further examples can be found in e.g. Freier & Altmann; Nucl.
  • LNA nucleosides Locked Nucleic Acid Nucleosides
  • A“LNA nucleoside” is a 2’-modified nucleoside which comprises a biradical linking the C2’ and C4’ of the ribose sugar ring of said nucleoside (also referred to as a “2’- 4’ bridge”), which restricts or locks the conformation of the ribose ring.
  • These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature.
  • BNA bicyclic nucleic acid
  • the locking of the conformation of the ribose is associated with an enhanced affinity of hybridization (duplex stabilization) when the LNA is incorporated into an oligonucleotide for a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide/complement duplex.
  • Non limiting, exemplary LNA nucleosides are disclosed in WO 99/014226, WO
  • R a and R b are independently selected from hydrogen, halogen, hydroxyl, cyano, thiohydroxyl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkoxyalkyl, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, formyl, aryl, heterocyclyl, amino, alkylamino, carbamoyl, alkylaminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, alkylcarbonylamino, carbamido, alkanoyloxy, sulfonyl, alkylsulfonyloxy, nitro, azido, thiohydroxylsulfidealkylsulfanyl, aryloxycarbonyl, aryloxy, arylcarbonyl, hetero
  • X a is oxygen, sulfur or -NR C ; R c , R d and R e are independently selected from hydrogen and alkyl; and n is 1 , 2 or 3.
  • Y is -CR a R b -, -CR a R b -CR a R b - or - CR a R b CR a R b CR a R b - , particularly -CH 2 -CHCH 3 -, -CHCH 3 -CH 2 -, -CH2-CH2- or -CH2- CH2-CH2-.
  • R a and R b are independently selected from the group consisting of hydrogen, halogen, hydroxyl, alkyl and alkoxyalkyl, in particular hydrogen, halogen, alkyl and alkoxyalkyl.
  • R a and R b are independently selected from the group consisting of hydrogen, fluoro, hydroxyl, methyl and -CH2-0-CH 3 , in particular hydrogen, fluoro, methyl and -CH2-0-CH 3 .
  • one of R a and R b of -X-Y- is as defined above and the other ones are all hydrogen at the same time.
  • R a is hydrogen or alkyl, in particular hydrogen or methyl.
  • R b is hydrogen or or alkyl, in particular hydrogen or methyl.
  • R a and R b are hydrogen.
  • R a and R b are hydrogen.
  • one of R a and R b is methyl and the other one is hydrogen.
  • R a and R b are both methyl at the same time.
  • -X-Y- is -0-CR a R b - wherein R a and R b are independently selected from the group consisting of hydrogen, alkyl and alkoxyalkyl, in particular hydrogen, methyl and -CH2-O-CH3.
  • -X-Y- is -O-CH2- or -0-CH(CH 3 )-, particularly -O-CH2-
  • the 2’- 4’ bridge may be positioned either below the plane of the ribose ring (beta- D- configuration), or above the plane of the ring (alpha-L- configuration), as illustrated in formula (A) and formula (B) respectively.
  • the LNA nucleoside according to the invention is in particular of formula (Bl) or (B2)
  • W is oxygen, sulfur, -N(R a )- or -CR a R b -, in particular oxygen;
  • B is a nucleobase or a modified nucleobase
  • Z is an intemucleoside linkage to an adjacent nucleoside or a 5'-terminal group
  • Z* is an intemucleoside linkage to an adjacent nucleoside or a 3'-terminal group
  • R 1 , R 2 , R 3 , R 5 and R 5* are independently selected from hydrogen, halogen, alkyl, haloalkyl, alkenyl, alkynyl, hydroxy, alkoxy, alkoxyalkyl, azido, alkenyloxy, carboxyl, alkoxycarbonyl, alkylcarbonyl, formyl and aryl; and X, Y, R a and R b are as defined above.
  • R a is hydrogen or alkyl, in particular hydrogen or methyl.
  • R b is hydrogen or alkyl, in particular hydrogen or methyl.
  • one or both of R a and R b are hydrogen.
  • only one of R a and R b is hydrogen.
  • one of R a and R b is methyl and the other one is hydrogen.
  • R a and R b are both methyl at the same time.
  • R a is hydrogen or alkyl, in particular hydrogen or methyl.
  • R b is hydrogen or alkyl, in particular hydrogen or methyl.
  • one or both of R a and R b are hydrogen.
  • only one of R a and R b is hydrogen.
  • one of R a and R b is methyl and the other one is hydrogen.
  • R a and R b are both methyl at the same time.
  • R a is hydrogen or alkyl, in particular hydrogen or methyl.
  • R b is hydrogen or alkyl, in particular hydrogen or methyl.
  • one or both of R a and R b are hydrogen.
  • only one of R a and R b is hydrogen.
  • one of R a and R b is methyl and the other one is hydrogen.
  • R a and R b are both methyl at the same time.
  • R 1 , R 2 , R 3 , R 5 and R 5* are independently selected from hydrogen and alkyl, in particular hydrogen and methyl.
  • R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen at the same time.
  • R 1 , R 2 , R 3 are all hydrogen at the same time, one of R 5 and R 5* is hydrogen and the other one is as defined above, in particular alkyl, more particularly methyl.
  • R 5 and R 5* are independently selected from hydrogen, halogen, alkyl, alkoxyalkyl and azido, in particular from hydrogen, fluoro, methyl, methoxy ethyl and azido.
  • one of R 5 and R 5* is hydrogen and the other one is alkyl, in particular methyl, halogen, in particular fluoro, alkoxyalkyl, in particular methoxyethyl or azido; or R 5 and R 5* are both hydrogen or halogen at the same time, in particular both hydrogen of fluoro at the same time.
  • W can advantageously be oxygen, and -X-Y- advantageously -O-CH2-.
  • -X-Y- is -O-CH2-
  • W is oxygen
  • R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen at the same time.
  • LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352 and WO 2004/046160 which are all hereby incorporated by reference, and include what are commonly known in the art as beta-D-oxy LNA and alpha-L-oxy LNA nucleosides.
  • -X-Y- is -S-CH2-
  • W is oxygen
  • R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen at the same time.
  • -X-Y- is -NH-CH2-
  • W is oxygen
  • R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen at the same time.
  • -X-Y- is -O-CH2CH2- or - OCH2CH2CH2-
  • W is oxygen
  • R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen at the same time.
  • LNA nucleosides are disclosed in WO 00/047599 and Morita et al, Bioorganic & Med.Chem. Lett. 12, 73-76, which are hereby incorporated by reference, and include what are commonly known in the art as 2’-0-4’C-ethylene bridged nucleic acids (ENA).
  • -X-Y- is -O-CH2-
  • W is oxygen
  • R 1 , R 2 , R 3 are all hydrogen at the same time
  • one of R 5 and R 5* is hydrogen and the other one is not hydrogen, such as alkyl, for example methyl.
  • R 5 and R 5* is hydrogen and the other one is not hydrogen, such as alkyl, for example methyl.
  • -X-Y- is -0-CR a R b -, wherein one or both of R a and R b are not hydrogen, in particular alkyl such as methyl, W is oxygen, R 1 , R 2 , R 3 are all hydrogen at the same time, one of R 5 and R 5* is hydrogen and the other one is not hydrogen, in particular alkyl, for example methyl.
  • R a and R b are not hydrogen, in particular alkyl such as methyl
  • W is oxygen
  • R 1 , R 2 , R 3 are all hydrogen at the same time
  • one of R 5 and R 5* is hydrogen and the other one is not hydrogen, in particular alkyl, for example methyl.
  • Such bis modified LNA nucleosides are disclosed in WO 2010/077578 which is hereby incorporated by reference.
  • -X-Y- is -0-CHR a -
  • W is oxygen
  • R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen at the same time.
  • R a is in particular C1-C6 alkyl, such as methyl.
  • -X-Y- is -0-CH(CH2-0-CH3)- (“2’ O-methoxyethyl bicyclic nucleic acid”, Seth et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81).
  • -X-Y- is -0-CH(CH 2 CH3)-; In another particular embodiment of the invention, -X-Y- is -0-CH(CH 2 -0-CH3)-,
  • W is oxygen and R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen at the same time.
  • LNA nucleosides are also known in the art as cyclic MOEs (cMOE) and are disclosed in WO 2007/090071.
  • -X-Y- is -0-CH(CH3)- (“2 ⁇ - ethyl bicyclic nucleic acid”, Seth at ai, J. Org. Chem. 2010, Vol 75(5) pp. 1569-81).
  • -X-Y- is -O-CH2-O-CH2- (Seth et al., J. Org. Chem 2010 op. cit.)
  • -X-Y- is -0-CH(CH3)-
  • W is oxygen
  • R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen at the same time.
  • 6’-methyl LNA nucleosides are also known in the art as cET nucleosides, and may be either (S)-cET or (R)-cET diastereoisomers, as disclosed in WO 2007/090071 (beta-D) and WO
  • -X-Y- is -0-CR a R b -, wherein neither R a nor R b is hydrogen, W is oxygen and R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen at the same time.
  • R a and R b are both alkyl at the same time, in particular both methyl at the same time.
  • Such 6’ -di- substituted LNA nucleosides are disclosed in WO 2009/006478 which is hereby incorporated by reference.
  • -X-Y- is -S-CHR a -
  • W is oxygen
  • R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen at the same time.
  • R a is alkyl, in particular methyl.
  • R a and R b are advantagesously independently selected from hydrogen, halogen, alkyl and alkoxyalkyl, in particular hydrogen, methyl, fluoro and methoxymethyl.
  • R a and R b are in particular both hydrogen or methyl at the same time or one of R a and R b is hydrogen and the other one is methyl.
  • Such vinyl carbo LNA nucleosides are disclosed in WO 2008/154401 and WO 2009/067647 which are both hereby incorporated by reference.
  • -X-Y- is -N(OR a )-CH2-
  • W is oxygen
  • R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen at the same time.
  • R a is alkyl such as methyl.
  • LNA nucleosides are also known as N substituted LNAs and are disclosed in WO 2008/150729 which is hereby incorporated by reference.
  • -X-Y- is -0-N(R a )-, -N(R a )-0-, -NR a - CR a R b -CR a R b - or -NR a -CR a R b -, W is oxygen and R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen at the same time.
  • R a and R b are advantagesously independently selected from hydrogen, halogen, alkyl and alkoxyalkyl, in particular hydrogen, methyl, fluoro and methoxymethyl.
  • R a is alkyl, such as methyl
  • R b is hydrogen or methyl, in particular hydrogen.
  • -X-Y- is -0-N(CH3)- (Seth et al, J.
  • R 5 and R 5* are both hydrogen at the same time.
  • one of R 5 and R 5* is hydrogen and the other one is alkyl, such as methyl.
  • R 1 , R 2 and R 3 can be in particular hydrogen and -X-Y- can be in particular -O-CH2- or -0-CHC(R a )3-, such as -0-CH(CH3)-.
  • -X-Y- is -CR a R b -0-CR a R b -, such as - CH2-O-CH2-
  • W is oxygen and R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen at the same time.
  • R a can be in particular alkyl such as methyl, R b hydrogen or methyl, in particular hydrogen.
  • LNA nucleosides are also known as conformationally restricted nucleotides (CRNs) and are disclosed in WO 2013/036868 which is hereby incorporated by reference.
  • -X-Y- is -0-CR a R b -0-CR a R b -, such as - O-CH2-O-CH2-
  • W is oxygen and R 1 , R 2 , R 3 , R 5 and R 5* are all hydrogen at the same time.
  • R a and R b are advantagesously independently selected from hydrogen, halogen, alkyl and alkoxyalkyl, in particular hydrogen, methyl, fluoro and methoxymethyl.
  • R a can be in particular alkyl such as methyl, R b hydrogen or methyl, in particular hydrogen.
  • Such LNA nucleosides are also known as COC nucleotides and are disclosed in Mitsuoka et al, Nucleic Acids Research 2009, 37(4), 1225-1238, which is hereby incorporated by reference.
  • the LNA nucleosides may be in the beta- D or alpha-L stereoisoform.
  • LNA nucleosides are beta-D-oxy-LNA, 6’-methyl-beta-D-oxy LNA such as (S)-6’-methyl-beta-D-oxy-LNA ((S)-cET) and ENA.
  • the RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule.
  • WOO 1/23613 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH.
  • an oligonucleotide is deemed capable of recruiting RNase H if it, when provided with a complementary target nucleic acid sequence, has an initial rate, as measured in pmol/l/min, of at least 5%, such as at least 10% or more than 20% of the of the initial rate determined when using a oligonucleotide having the same base sequence as the modified oligonucleotide being tested, but containing only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide, and using the methodology provided by Example 91 - 95 of WO01/23613 (hereby incorporated by reference).
  • recombinant human RNase Hl is available from Lubio Science GmbH, Lucerne, Switzerland.
  • the invention relates to a method of synthesis of alcohol protected allofuranose from alcohol protected glucofuranose comprising the steps of:
  • the alcohol groups in glucofuranose are protected at its postions 1, 2, 5 and 6, for example as depicted in the following formula:
  • PG represents a protecting group protecting the alcohol function.
  • protecting groups for alcohols are well knon in the art. Such protecting groups can for example be esters, ethers or acetals such as describedin Data from: J. Chem. Soc., Perkin Trans. 1, 1992, 3043-3048; Chem. Int. Ed., 1996, 35, 2056-2083 or J. Org. Chem., 1984, 49, 4674- 4682. Such examples are not exhaustive and other methods for protecting alcohols can be used. By the way of illustration, in the following examples benzyl ether (OBn) or mesylate (MsO) are examples of protected alcohol moieties.
  • OBn benzyl ether
  • MsO mesylate
  • the catalytic oxidation step is performed in the presence of a TEMPO catalyst
  • TEMPO catalyst can be selected from the group consisting of 2,2,6,6-Tetramethylpiperidin-l-yl)oxyl and (2, 2,6,6- tetramethylpiperidin- 1 -yl)oxidanyl.
  • the concentration of the oxidative catalysts is less than 1% of the reaction solution in volume.
  • the hypochlorite salt is a metal hypochlorite of the formula M + ClO .
  • NaClO has been used as a hypochlorite metal salt.
  • other metal salts can be formed such as KOC1, LIOC1, CaOCl, but in practice, sodium hypochlorite is the most commonly available.
  • the hypochlorite salt is selected from the group consisting of NaOCl, KOC1, LIOC1, CaOCl or a combination thereof
  • the solution of hypochlorite salt added to the reaction s can be between about 10% and about 15% of the reaction solution in volume. In an embodiment of the invention the solution of hypochlorite salt added to the reaction can be about 10% or 11% or 12% or 13% or 14% or 15% of the reaction solution in volume. In an embodiment of the invention the solution of hypochlorite salt added to the is about 12% of the solution in volume.
  • the catalytic oxidation is performed at a temperature of about l0°C or less.
  • the hypochlorite salt is removed. Removal of the hypochlorite salt can be peformed by conventional method know in the art, for example via a process of filtration and/or solvent exchange.
  • reaction solution further comprises a buffer such as sodium bicarbonate (NaHCCb).
  • a buffer such as sodium bicarbonate (NaHCCb).
  • reaction solution further comprises an organic solvent such as ethyl acetate (EtOAc).
  • organic solvent such as ethyl acetate (EtOAc).
  • the reduction step is performed with a reducing agent.
  • a reducing agent can be a solution of NaBH 4 .
  • TEMPO for example about 1% of the reation solution
  • reaction solution e.g. about 9 vol. equiv.
  • the invention relates to a method for the synthesis of bismesylate of formula (P-D-S-7): Chiral
  • said method comprising the steps of synthesizing allofuranose from glucosefuranose according to the method of the invention.
  • the method for the synthesis of bismesylate of formula (P-D-S-7) according to the invention can further comprise the steps of:
  • the compound of formula (b-D-S-S) can be prepared by reacting a compound of formula (b- ⁇ -8-4) with acetic acid in water: Chiral Chiral
  • the compound of formula (P-D-S-4) can be prepared by reacting a compound of formula (P-D-S-3) with tetrabutylammonium hydrogen sulfate (TBAHS) in an appropriate solvent, e.g. toluene and then adding benzylchloride.
  • TSAHS tetrabutylammonium hydrogen sulfate
  • the compound of formula (P-D-S-3) can be prepared by reacting allofuranose with P2O5 (for example 545 g, 3.85 mol) in DMSO is to obtain a compound of formula (S- 2) and then reacting the compound of formula (S-2) with an aquous solution of NaBH 4 (for example 92 g, 2.43 mol) in water (for example 4 L). to obtain l,2:5,6-di-0-isopropyliden- a-D-allofuranose (P-D-S-3).
  • the compound of formula (P-D-S-6) can be prepared from compound of formula (b-D-S-S), by reacting with NaI0 4 (for example 757 g, 3.54 mol) in water and then with dioxane (for example 2.0L), formaldehyde and sodium hydroxide.
  • the invention relates to a method for the synthesis of an LNA-diol of formula 1 :
  • said method comprising the step of synthesizing allofuranose from glucosefuranose according to the method of the invention.
  • method for the synthesis of an LNA- diol further comprises the steps of preparing a bismesylate of formula (P-D-S-7) as described herein.
  • method for the synthesis of an LNA- diol further comprises the steps of:
  • the invention relates to a method for the synthesis of a LNA monomer, said method comprising the steps of synthesizing allofuranose from
  • the method for the synthesis of a LNA monomer further comprises the steps of preparing a bismesylate of formula (P-D-S-7) as described herein.
  • the method for the synthesis of a LNA monomer further comprises the steps of preparing a bismesylate of formula (1) as described herein.
  • LNA-G monomers are LNA monomers wherein the nucleobase is guanine.
  • LNA-G monomers have the following chemical formula:
  • PG is a protecting group protecting an alcohol or an amine group.
  • LNA-G monomers are stabilized under the form of their amidites, wich thereafter can conveniently be used for the synthesis of oligonucleotides.
  • LNA-A monomers are LNA monomers wherein the nucleobase is adenosine.
  • LNA-A monomers have the following chemical formula:
  • PG represents a protecting group for either an alcohol or an amine group.
  • LNA-A monomers are stabilized under the form of their amidites, wich thereafter can conveniently be used for the synthesis of oligonucleotides.
  • LNA-T monomers are LNA monomers wherein the nucleobase is thymine.
  • LNA-T monomers have the following chemical formula, wherein OPG is an protected alcohol and APG is an adenosine
  • nucleobase wherein the amine group is protected :
  • LNA-T monomers are stabilized under the form of their amidites, wich thereafter can conveniently be used for the synthesis of oligonucleotides.
  • LNA-C monomers are LNA monomers wherein the nucleobase is cytosine.
  • LNA-C monomers have the following chemical formula: Chiral
  • OPG is an alcohol protecting group and me-CPG is a methylcytosine nucleobase wherein the amine group is protected.
  • LNA-C monomers are stabilized under the form of their amidites, wich thereafter can conveniently be used for the synthesis of oligonucleotides.
  • a fifth object of the invention is an oligonucleotide prepared using LNA monomers as made according to the methods of the invention described herein.
  • the person skilled in the art will know how to prepare oligonucleotides from monomers according to methods known in the art.
  • the invention relates to a gapmer oligonucleotide,
  • the method of the invention can be used to prepare a gapmer oligonucleotide, pharmaceutically acceptable salt or conjugate as a medicament.
  • Said oligonucleotides can be useful in a method of modulating the expression of a target RNA in a cell comprising administering an oligonucleotide or gapmer oligonucleotide according to the invention to a cell expressing said target RNA so as to modulate the expression of said target RNA.
  • Said oligonucleotides can be useful in a method of inhibiting the expression of target RNA in a cell comprising administering an oligonucleotide or gapmer oligonucleotide according to the invention to a cell expressing said target RNA so as to inhibit the expression of said target RNA.
  • Said oligonucleotides can further be useful in an in vitro method of modulating or inhibiting a target RNA in a cell comprising administering an
  • oligonucleotide or gapmer oligonucleotide according to the invention to a cell expressing said target RNA, so as to modulate or inhibit said target RNA in said cell.
  • the target RNA can, for example be a mammalian mRNA, such as a pre-mRNA or mature mRNA, a human mRNA, a viral RNA or a non-coding RNA, such as a microRNA or a long non coding RNA.
  • the modulation is inhibition which may occur via target degradation (e.g . via recruitment of RNaseH, such as RNaseHl or RISC), or the inhibition may occur via an occupancy mediate mechanism which inhibits the normal biological function of the target RNA (e.g. mixmer or totalmer inhibition of microRNAs or long non coding RNAs).
  • target degradation e.g . via recruitment of RNaseH, such as RNaseHl or RISC
  • occupancy mediate mechanism which inhibits the normal biological function of the target RNA (e.g. mixmer or totalmer inhibition of microRNAs or long non coding RNAs).
  • Ethyl acetate (20 vol. equiv.) was added to a 20L reactor at 20 - 35°C.
  • l,2:5,6-Di-0- isopropylidene-a-D-glucofuranose S-l (1 wt. equiv.) was added and stirred into ethyl acetate.
  • a 10% Potassium bromide solution (0.3 wt. equiv.) was added into the reaction mass at 20 - 35°C and the reaction was stirred for 5 - 10 minutes at 20 - 35°C.
  • TEMPO 0.05 wt. equiv. was added into reaction mass at 20-35°C.
  • reaction was stirred for 15 - 20 minutes at 20-35°C and then cooled to 0 - l0°C.
  • a solution of aqueous basic Sodium hypochlorite (10-14%, 9 vol. equiv.) was then added to the reaction mixture slowly at 0 -
  • a solution of sodium bicarbonate was prepared using sodium bicarbonate (1 wt. equiv.) and water (5 vol. equiv.) at 20 - 35 °C and then cooled to below 20 °C and then NLT 10% of sodium hypochlorite below 20 °C. The mixture was stirred well and the solution kept at 5 - 10 °C. The content of the well stirred solution was added to the reaction.
  • the reaction was settled for 10 - 15 minutes and the upper layer (Ethyl acetate) was submitted to quality control for P-D-S-3 content by TLC.
  • the reaction mass was homogeneous biphasic (top layer was organic layer, bottom layer was aqueous layer).
  • Example 1.1 synthesis of allofuranose (P-D-S-3) from 100 g of glucosefuranose (SI) 1.2 Allofuranose was prepared according to the general synthesis of example 1 from 100 g of glucosefuranose. The yield was 70%.
  • Example 1.2 synthesis of allofuranose (P-D-S-3) from 500 g of glucosefuranose (SI)
  • LNA monomer and LNA amidite and LNA phosphoramidites are used interchangeably.
  • This synthesis of four LNA monomers comprises of 37 synthetic steps starting from commercial glucose and is outlined in scheme 1. The following describes the chemical manufacture of the four LNA monomers analytical methods for the detection of these. In this synthesis, LNA monomers are considered API starting material.
  • Example 2 monomer synthesis - Overview
  • the total synthesis of the four LNA monomers comprises of 37 synthetic steps starting from commercial glucose and is outlined in scheme 1.
  • the synthetic strategy is convergent where the first seven steps results in a modified sugar - the bismesylate (P-D-S-7) which is the common building block for all four monomers.
  • the pyrimidine synthesis is also convergent because the LNA-T diol (b- ⁇ -T-6) functions as starting material for the LNA T and the LNA MeC monomers.
  • the final steps from the diols to the amidites is the formation of the LNA monomeric building blocks that are compatible with the phosphoramidite approach for incorporated into oligonucleotides.
  • the synthesis of the LNA-A diol starts with removal of the 1 ,2-isopropylidine group from the the Bismesylate (P-D-S-7) followed by acetylation which yields the coupling sugar (b- D-S-8).
  • the coupling sugar is an anomeric mixture that is a sticky oil and therefore typically generated immediately before the following coupling step.
  • the coupling step is a Vorbruggen coupling between the coupling sugar and unprotected adenine.
  • SnCl 4 as Lewis catalyst results in the sole formation of b- ⁇ -A-2 without formation of the a-isomer and the N-6 sugar adduct.
  • the synthesis of the LNA-G diol starts with removal of the 1 ,2-isopropylidine group from the the Bismesylate (P-D-S-7) followed by acetylation which yields the coupling sugar (b- D-S-8).
  • the coupling sugar is an anomeric mixture that is a sticky oil and therefore typically generated immediately before the following coupling step.
  • the coupling step is a Vorbruggen coupling between the coupling sugar and 6-Chloroguanine.
  • TMSOTf as Lewis acid catalyst
  • 6-Chloroguanine results in the sole formation of P-D-G-2 without formation of the a-isomer and the N-7 isomer.
  • the synthesis of the LNA-T diol starts with removal of the 1 ,2-isopropylidine group from the the Bismesylate (P-D-S-7) followed by acetylation which yields the coupling sugar (b- D-S-8).
  • the coupling sugar is an anomeric mixture that is a sticky oil and therefore typically generated immediately before the following coupling step.
  • the coupling step is a Vorbruggen coupling between the coupling sugar and unprotected thymine.
  • TMSOTf as Lewis catalyst results in the sole formation of b- ⁇ -T-2 without formation of the a-isomer and the N-3 isomer.
  • the LNA-A diol (b- ⁇ -A-6) is protected with 4,4’-dimethoxytrityl at the 5’ position and with a transient trimethylsilyl group at the 3’ position followed by benzoylation at the exocyclic N-6 position.
  • Treatment with ammonium hydroxide removes the silyl group giving b- ⁇ -A-7.
  • Phosphitylation with 2-cyanoethyl N, N, N', A'-tctraisopropy l phosphord i- amidite gives the LNA-A monomer b- ⁇ -A-8.
  • the LNA-T diol (b- ⁇ -T-6) is protected with 4,4’-dimethoxytrityl at the 5’ position and with an acetyl group at the 3’ position giving b-0- 3.
  • the exocyclic carbonyl at C-4 is converted into a triazolate b- ⁇ - 4 via reaction with phosphoroxy chloride.
  • b- ⁇ - 4 is treated with concentrated aqueous ammonia resulting in the formation of the 5 -Me Cytosine nucleobase and the deprotection of the 3’ -OH (b- ⁇ - 6).
  • the LNA-G diol (b-D-G-T) is protected with dimethylformimine at the 2-N exocyclic amine and with 4,4’-dimethoxytrityl at the 5’ position giving b-D-G-S.
  • Phosphitylation with 2-cyanoethyl N, N, A'", A - 1 c t r a i s o p r o p y l p h o s p h o r d i a m i d i t c gives the LNA-G monomer b- ⁇ -0-9.
  • the LNA-T diol (b- ⁇ -T-6) is protected with 4,4’-dimethoxytrityl at the 5’ -OH giving b- D-T-7.
  • Phosphitylation with 2-cyanoethyl /V, /V, A'", A'"- 1 c t r a i s o p r o p y l p h o s p h o r d i a m i d i t c gives the LNA-T monomer b- ⁇ -T-8.
  • the mixture was cooled to 20°C and pH adjust to 7-8 with 1 N aq. NaOH solution.
  • a pH adjusting the pH to 7-8 may be helpful: if pH is below 7 impurity formation is observed. pH higher than 8, will form a sticky mass, which is difficult to filtrate.
  • the mixture was filtered and the salts were washed with 2 x 500 mL acetone.
  • the mixture was evaporated to a vol. of 5 L and 4 L DCM was added together with 5 L 1 N NaOH.
  • the mixture was stirred for 15 min and phases separated.
  • the aqueous phase was washed 2 times with 2 L DCM, and the combined organic phases were washed with 5 L brine and separated.
  • the mixture was evaporated to 2 L and added 2 L hexane.
  • the mixture was evaporated again to 2 L and again added 2 L hexane. This was repeated 2 times more.
  • the resulting 2 L mixture was left at 45°C under stirring. After 45 min the mixture was slowly cooled to 30°C and left for stirring at that temperature. After 30 min the mixture was slowly cooled to l0°C and left there for 60 min.
  • the product was filtered on a GF-P3 and washed with 1 L pre chilled (l0°C) hexane.
  • the crystals were dry in air-vented oven at 55 - 60°C.
  • DMSO 4.0 L
  • P2O5 545 g, 3.85 mol
  • DMSO 3.7 L
  • S-l Glucofuranose
  • DMSO 3.7 L
  • the concentrated MTBE-phase is added to a cold stirred solution of NaBEE (92 g, 2.43 mol) in water (4 L) over a period of 1 hour.
  • P-D-S-3 1000 g is dissolved in 5.0 L toluene and 2.0 L caustic lye is slowly added at 25- 30°C. 100 g TBAHS is added and the mixture is heated to 50°C. Then 608 g
  • the reaction is cooled to 25-30°C and stirring stopped. After 30 min the bottom phase is separated out.
  • the organic phase is isolated, and the aqueous phase washed with 1 L toluene and separated out.
  • the combined organic phases are washed with 1 L demineralised water for 30 min, allowed to separate, and the bottom phase is unloaded.
  • the organic phase is evaporated to an oil, and 4.0 L hexane is added.
  • the mixture is cooled to 0°C and allowed to stir for 2h.
  • the product is filtered on a GF-P3 and washed with 2 L pre chilled hexane.
  • the reaction is cooled to 10 °C and pH is adjusted to 8 - 8.5 using 50 % sodium hydroxide (approx.: 5000 mL).
  • the mixture is filtered on a GF-P3, and the filtrate is extracted with 3 x 3 L DCM.
  • the combined organic phases are concentrated to afford colorless oil.
  • Heptane (1.5 L) is added and the mixture allowed to cool down to room temperature.
  • the first crop of crystals are isolated on a GF-P3, and the filter cake is washed with MTBE/ heptane (1 :3) (1.5 L, 20°C).
  • the filtrate is evaporated, and MTBE (500 mL) and heptane (200 mL) is added to the residue.
  • the hot solution (45-50°C) is transferred to a flask and allowed to reach RT.
  • the secound crop of crystals are isolated on a GF-P3.
  • the material is left to dry in a vacuum oven over night to give 750 g (yield: 75 %) white solid.
  • P-D-S-7 1000 g is suspended in Acetic acid (3.0 L) to give an off-white suspension.
  • Sulfuric acid (5.7 mL) is dissolved in 100 mL Acetic acid and added to the suspension.
  • Acetic anhydride (364 mL) is added drop wise over a period of 1 hour at 20°C. The stirred reaction is left at 25°C until LC-MS analysis shows full conversion to P-D-S-8 (typical 5- 16 h).
  • the organic phase is concentrated under reduced pressure using a rotary evaporator to get approximately 1200 g clear oil.
  • P-D-S-8 1000 g is dissolved in anh. acetonitrile (2 L) under nitrogen atmosphere to give a colorless solution. Adenine (291 g) is added and the mixture is stirred at 25°C. SnCl 4 (1123 g) is added dropwise over a period of 30 min. at max. 45°C. The stirred reaction is left at 40°C until LC-MS analysis shows full conversion to b- ⁇ -A-2 (typical 2 h).
  • reaction mixture is cooled to 0°C and pH adjusted to 4.5 - 5.0 using 14 M NaOH (aq).
  • Celite added (450 g) to the mixture, filtered on a GF-P3, and the filter cake is washed with EtOAc (2 x 1 L).
  • the combined organic phases are washed with 1 M K2HPO4 (1 x 3 L).
  • the organic phase is concentrated under reduced pressure using a rotary evaporator to orange oil.
  • the reaction mixture is concentrated under reduced pressure using a rotary evaporator until 2000 mL is collected.
  • the mixture is added 2.5 L demineralized water, filtered on a GF-P3, and the filter cake is washed with demineralized water (3 x 1.5 L).
  • P-D-A-3 1000 g are dissolved in acetonitrile (3 L) and coevaporated 2 times with acetonitrile (2 x 3 L), 2 times with toluene (2 x 2 L) and finally dissolved in DMF (9.0 L).
  • NaOBz 805 g is added and the mixture is stirred at l05°C until LC-MS analysis shows full conversion to b- ⁇ -A-4 (typical 2 h).
  • the white suspension is cooled to 25°C and added 20 L demineralized water.
  • the mixture is cooled to 5°C and filtered on a GF-P3.
  • the filter cake is washed with demineralized water (1 x 9 L). The wet filter cake is used without further purification in next step.
  • P-D-A-4 1000 g is dissolved in THF (6.0 L) to give a yellow solution.
  • Water 5.0 L
  • Lithium hydroxide hydrate 115 g is added and the mixture is stirred at 25 °C until LC-MS analysis shows full conversion to b-D-A-S (typical 1 h).
  • the reaction mixture is concentrated under reduced pressure using a rotary evaporator until 4000 mL is collected.
  • the mixture is added 4.5 L demineralalized water, cooled to l0°C, filtered on a GF-P3, and the filter cake is washed with demineralized water (1 x 3.0 L).
  • the white crystals are dried in vacuum oven (typical 16 h at 50°C, 10-50 mbar).
  • the warm reaction mixture is filtered on celite at approx. 60°C.
  • the filter is washed with a preheated (70°C) mixture of ethanol/ water (3000 mL ethanol/ 1000 mL water).
  • the combined filtrates are concentrated under reduced pressure using a rotary evaporator until approx. 4 L solution is left.
  • 2-propanol (3.5 L) is added, and evaporated until 5.0 L are collected. Then more 2-propanol (3.5 L) is added and the mixture is cooled to 0°C, filtered on a GF-P3, and the filter cake is washed with cold 2-propanol (1 x 2.0 L at 0°C).
  • the white crystals are dried in oven (typical 16 h at 40°C, airflow).
  • P-D-A-6 1000 g is dissolved in anh. pyridine (30 L) under nitrogen atmosphere and DMTrCl (2014 g) is added. The deep red reaction mixture is stirred at room temperature until TLC analysis (10 % MeOH in DCM) shows full conversion (typical 3 h). Reaction cooled to 0°C and TMSC1 (2064 g) is added over a period of 1 h. Benzoylchloride (2670 g) is added at 0°C, and the reaction mixture is allowed to warm up to 20 - 25°C and left overnight.
  • the beige suspension is cooled to 0°C and MeOH (5.0 L) is added drop wise at max. l0°C (exothermic, jacket set to minus l0°C).
  • MeOH 5.0 L
  • a mixture of 25% NH3 aq (8300 mL) and purified water (3.0 L) is added to the mixture (exothermic) at max. 20°C.
  • the reaction mixture is stirred at room temperature until TLC analysis (10 % MeOH in DCM) shows full conversion to TM (typical 5 h).
  • the reaction is added water (5.0 L) and toluene (20.0 L).
  • the phases are separated and the organic phase is concentrated under reduced pressure using a rotary evaporator (water bath 60°C).
  • PN2 2-Cyanoethyl-N, N, N' , N' ,-tetraisopropylphosphorodiamidite
  • reaction mixture is applied directly on a silica gel column (10 kg) packed in
  • CEbCkiAcOEt (9:1 , v/v) + 0.1% Et 3 N followed by flash chromatography in QUCUTHF (88: 12, v/v) + 0.1% EtsN.
  • P-D-T-6 1000 g is dissolved in anh. pyridine (10 L) under nitrogen atmosphere and DMTrCl (1505 g) is added. The deep red reaction mixture is stirred at 28°C until LC-MS analysis shows full conversion to the DMT -intermediate. Acetic anhydride (1.0 L) is added over a period of 30 min. at max. 25°C. The stirred reaction is left at 25°C until LC- MS analysis shows full conversion to P-D-C-3 (typical 16 h).
  • the reaction mixture is concentrated under reduced pressure using a rotary evaporator to get approximately 7 kg red oil.
  • the oil is dissolved in 2.2 L CELCN at 70°C and slowly added to 14 L stirred EtOAc at 20°C.
  • the resulting mixture is filtered (the removed solid is probably pyridine chloride), and the mother liquor concentrated under reduced pressure using a rotary evaporator to get approximately 5 kg red oil.
  • the 5 kg red oil is dissolved in toluene (6.0 L) and washed with sat. NaHCCh aq (2 x 3.0 L), water (1 x 3.0 L) and brine (1 x 3.0 L).
  • the organic phase is concentrated under reduced pressure using a rotary evaporator to approximately 2800 g red oil.
  • the crude oil is dissolved in 2 L toluene + 20 mL TEA. 8000 g Silica gel 60 is washed with toluene + 1% TEA.
  • Eluent 1 10 % EtOAc in toluene + 0.1 % TEA until all DMT impurities are out (typical 6 column volumes). Shown with LC-MS. Eluent 2: EtOAc + 0.1 % TEA to eluate the b-D-C 3 out (typical 8 column volumes).
  • the product containing fractions are pooled and concentrated under reduced pressure using a rotary evaporator.
  • the water phase is washed with EtOAc (4.0 L) and the combined organic phases are washed with water (8.0 L) and brine (4.0 L).
  • the organic phase is concentrated under reduced pressure using rotary evaporator until yellow crispy foam is obtained.
  • the mixture is allowed to separate, and the organic phase is concentrated under reduced pressure using rotary evaporator until white crispy foam is obtained.
  • the aqueous phase is washed with toluene (3.0 L) and the combined toluene phases are washed with water (5.0 L) and brine (5.0 L) and separated.
  • the organic phase is concentrated under reduced pressure using rotary evaporator until yellow oil is obtained.
  • Eluent 2 EtOAc + 0.1 % TEA to eluate the b-D-C 7 (typical 6 column volumes).
  • the product containing fractions are pooled and concentrated under reduced pressure using a rotary evaporator.
  • Typical yield 75 - 85 % white crispy foam.
  • PN2 2-Cyanoethyl-N, N, N' , N' ,-tetraisopropylphosphorodiamidite
  • PN2 (650 mL) is added to a solution of 1 M DCI (168 g in 1000 mL DCM). This mixture is added to a solution of P-D-C-7 (1000 g) dissolved in anh. DCM (5000 mL) under nitrogen atmosphere at l6°C - l8°C. The mixture was stirred at 24°C until TLC shows full conversion to P-D-C-8.
  • the filtered DCM fractions are pooled and concentrated under reduced pressure. 2 L MeCN is added and this mixture is concentrated. 5 L MeCN is added and the mixture is cooled to 0°C. The mixture is filtered on a GF-P3 to isolate the white crystals.
  • P-D-S-8 1000 g is dissolved in anh. acetonitrile (8.4 L) under nitrogen atmosphere to give a colorless solution. 6-Chloroguanine (382 g) is added and the mixture is stirred at 25°C. BSA (805 g) is added dropwise over a period of 15 min. at 50°C. The reaction is heated to 85 - 90°C and stirred until the mixture is clear brown (typical 2 h).
  • the reaction mixture is cooled to lO°C and tap water is added (2.8 L). pH adjusted to 8 using 3M NaOH aq (approx. 1.5 L). The resulting solution is extracted with EtOAc (3.0 L + 2 x 1.5L). The combined organic phases are washed with 1 M K2HPO4 aq (2 x 3 L) and with 25 % sat. NaCl in tap water (2 x 1.8 L). The organic phase is concentrated under reduced pressure using a rotary evaporator to give brown oil.
  • P-D-G-3 1000 g is dissolved in DCM (7.0 L) and cooled to minus 5°C followed by addition of benzyl alcohol (292 g). Potassium t-butoxide (243 g) is added in portions over a period of 3 hours. Temp. 0 +/- 5°C. The reaction is stirred at minus 5°C until LC-MS analysis shows full conversion to P-D-G-4 (typical 1 h after completed addition).
  • the reaction mixture is added water (7 L) and stirred for 10 min.
  • the phases are separated and the aqueous phase is extracted once with DCM (1.5 L).
  • the combined organic phases are washed with brine (5 L) and separated.
  • Silica 100-200 is added to the mixture and stirred for 1 h. Filtered on a GF-P3 and the filter cake is washed with DCM.
  • This combined organic phases is concentrated (50°C, 50 mbar).
  • Toluene (2.0 L) is added to the residue.
  • the suspension is concentrated on rotary evaporator (50°C, 50 mbar).
  • Toluene (3.0 L) is added to the residue again and stirred for 2-3 h.
  • the product is isolated by filtration.
  • the filter cake is washed with toluene (500 mL).
  • P-D-G-4 1000 g is dissolved in DMSO (molsive dry, 5.0 L) under N2-atm. NaOBz (364 g) is added to the reaction mixture. The unclear red-brown reaction mixture is heated to l00°C until LC-MS analysis shows full conversion to b-D-G-S (typical 2 h).
  • the reaction is cooled to 35°C, and poured into cold (0°C) demineralised water (20 L) and stirring is continued night over.
  • the suspension is filtered on a GF-P3 (very slow!) and the off-white filter cake is suspended in 4 L water (still on the filter) to remove DMSO.
  • the wet product b-D-G-S is used in the next step without drying!
  • the reaction is concentrated under reduced pressure (60°C, 100 mbar) until 3.0 L is distilled off. Then 6 L demineralised water is added to the rubber like matrix and the mixture is stirred at 25°C night over.
  • the warm reaction mixture is filtered through a cellulose pad at approx. 60°C.
  • the filter is washed with a preheated (70°C) mixture of ethanol/ water (3000 mL ethanol/ 1000 mL water).
  • the combined filtrates are concentrated under reduced pressure using a rotary evaporator until approx. 4 L solution is left.
  • 2-propanol (3.5 L) is added, and evaporated until 5.0 L are collected. Then more 2-propanol (3.5 L) is added and the mixture is cooled to 0°C, filtered on a GF-P3, and the filter cake is washed with cold 2-propanol (1 x 2.0 L at 0°C).
  • P-D-G-7 1000 g, 3.4 mol is suspended in pyridine (10 L).
  • Dimethylformamide dimethyl acetal (907 g, 9.1 mol) is added and the resulting suspension is stirred at approx. 25°C until a clear solution is obtained (app. 2 1 ⁇ 2 h) and then further 90 min after which in- process analysis shows full conversion of the starting material into the intermediate.
  • Tap water 60 mL, 3.4 mol
  • the reaction mixture is stirred 30 min at 25°C followed by evaporation to dryness under reduced pressure.
  • the reaction mixture is drained into jerry cans and aqueous NaHCCb (6%, 30 L) and dichloromethane (10 L) is transferred to the reactor.
  • the reaction mixture is transferred to the reactor at 20-25°C over 20 min. followed by dichloromethane (3.3 L).
  • the resulting emulsion is stirred for 5 min and the phases separated.
  • the aqueous phase is extracted with dichloromethane (10 L).
  • the combined organic phases are washed with aqueous NaCl (25%, 7 L).
  • the organic phase is concentrated under reduced pressure using a jacket temperature of 60°C. When approx. 20 L of distillate has been collected the resulting mixture is subjected to a polish filtration.
  • the centrifugation can be substituted with a filtration but this can be very slow.
  • PN2 2-Cyanoethyl-N, N, N' , N' ,-tetraisopropylphosphorodiamidite
  • Methyl fc/t-butyl ether (1.0 L) was added over 35 min. The resulting suspension was stirred at ca. 20°C for 90 min. after which it was filtered. The filter cake was washed with a mixture of methyl tert- butyl ether and toluene (1 :1, v/v, 1.55 L) followed by wash with methyl tert- butyl ether (700 mL). The wet cake was dried in vacuum.
  • the pH is adjusted to 6.1 with AcOH (app. 720 mL).
  • the phases are separated and the aq.- phase is extracted with 1.6 L DCM.
  • the combined organic phases are washed with 4.5 L H2O.
  • the organic phase is concentrated in vacuo (50°C, 40 mbar).

Abstract

La présente invention concerne la fabrication d'allofuranose à partir de glucofuranose tel que défini dans la description et dans la revendication. L'allofuranose est un intermédiaire dans la fabrication d'oligonucléotides qui peuvent être utilisés en tant que médicament.
PCT/EP2019/063044 2018-05-25 2019-05-21 Nouveau procédé de fabrication d'allofuranose à partir de glucofuranose WO2019224172A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021075538A1 (fr) * 2019-10-18 2021-04-22 第一三共株式会社 Procédé de fabrication de phosphoramidite bicyclique
CN113045612A (zh) * 2021-03-17 2021-06-29 常州市白云生物科技有限公司 3-O-苄基-4-C-羟甲基-1,2-O-异亚丙基-α-D-呋喃核糖的合成方法
WO2022124410A1 (fr) * 2020-12-11 2022-06-16 ヤマサ醤油株式会社 Cristaux d'amidite de nucléoside réticulé de type cytosine et leur procédé de production

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021075538A1 (fr) * 2019-10-18 2021-04-22 第一三共株式会社 Procédé de fabrication de phosphoramidite bicyclique
WO2022124410A1 (fr) * 2020-12-11 2022-06-16 ヤマサ醤油株式会社 Cristaux d'amidite de nucléoside réticulé de type cytosine et leur procédé de production
CN113045612A (zh) * 2021-03-17 2021-06-29 常州市白云生物科技有限公司 3-O-苄基-4-C-羟甲基-1,2-O-异亚丙基-α-D-呋喃核糖的合成方法

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