WO2022123501A1 - Désoxydidéhydro-nucléosides protégés - Google Patents

Désoxydidéhydro-nucléosides protégés Download PDF

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WO2022123501A1
WO2022123501A1 PCT/IB2021/061540 IB2021061540W WO2022123501A1 WO 2022123501 A1 WO2022123501 A1 WO 2022123501A1 IB 2021061540 W IB2021061540 W IB 2021061540W WO 2022123501 A1 WO2022123501 A1 WO 2022123501A1
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compound
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butyldimethylsilyl
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alkyl
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Rinu SHRESTHA
Lawrence Daniel Harris
James Michael Wood
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Victoria Link Limited
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    • 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
    • C07H19/048—Pyridine radicals
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    • 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
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    • 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
    • C07H19/067—Pyrimidine radicals with ribosyl as the saccharide radical
    • 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
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • 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
    • C07H19/12—Triazine 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
    • C07H19/14—Pyrrolo-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
    • 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
    • C07H19/167—Purine radicals with ribosyl as the saccharide radical
    • 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
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids

Definitions

  • the invention relates generally to protected intermediates in the synthesis of nucleotide analogue drugs.
  • the invention relates to the synthesis of protected nucleoside compounds which can be used as intermediates in the synthesis of 3′-deoxy- 3′,4′-didehydro-nucleosides and related compounds.
  • BACKGROUND OF THE INVENTION The recent emergence of the COVID-19 pandemic has brought widespread attention to the impacts viruses can have on human life and healthcare systems. Viruses are implicated in the majority of epidemic and pandemic diseases, which reflects their high transmissibility and the paucity of effective antiviral therapies.
  • Viperin Virus Inhibitory Protein, Endoplasmic Reticulum-associated, Interferon- inducible
  • RSAD2 radio SAM domain-containing 2
  • Viperin inhibits replication of both RNA and DNA viruses by diverse mechanisms, including protein-protein interactions and modulation of immune-signalling, thereby contributing to the innate antiviral response.
  • the role of 3′-deoxy-3′,4′-didehydro-cytidine triphosphate (ddhCTP) in the antiviral activity of viperin is still not completely understood.
  • CTP cytidine triphosphate
  • Flaviviridae RdRps dengue virus, West Nile virus, Zika virus and hepatitis C virus
  • ddhCTP can inhibit NAD + - dependent enzymes, leading to downstream and/or upstream effects in metabolic/signalling pathways.
  • ddhCTP production does not affect the viability or growth rate of Vero and HEK293T cells.
  • ddhCTP and 3′-deoxy-3′,4′-didehydro-cytidine therefore represent a promising platform for the development of broad-spectrum antiviral agents.
  • ddhCTP, and related compounds can be challenging and improved synthetic routes are needed for the more efficient and cost-effective development of this important class of compounds.
  • the applicant has now developed a protecting group strategy to provide a convenient synthesis of important intermediate compounds useful for the synthesis of, not only ddhC and related compounds, but a broad range of 3’-deoxy-3’,4’-didehydroribonucleosides including compounds derived from both natural and non-natural nucleosides.
  • the invention seeks to address the need for improved methods of synthesising 3’- deoxy-3’,4’-didehydroribonucleoside based antiviral drugs by providing a novel class of compounds useful as intermediates in the synthetic process.
  • X1 is a silyl ether protecting group
  • Y is H, C 1-3 alkyl, C 2-3 alkenyl, or C 2-3 alkynyl
  • A is selected from: wherein: R 1 and R 2 are each H, OH, OX 2 where X 2 is a silyl protecting group, or a C 1-6 alkyl, C 1-12 acyl, C 1-6 alkoxymethyl, C 3 -C 12 cycloalkyl, alkyloxycarbonyl, aryl, aryloxy or aryloxymethyl group each of which is optionally substituted with one or more alkyl, alkenyl, alkyn
  • the compound is a compound of Formula (I). In other embodiments the compound is a compound of Formula (Ia).
  • X1 is an alkylsilyl group or an arylsilyl group.
  • the alkylsilyl group is trimethylsilyl (TMS), triethylsilyl (TES), t-butyldimethylsilyl (TBDMS), or triisopropylsilyl (TIPS).
  • the arylsilyl group may be t-butyldiphenylsilyl (TBDPS).
  • Y is H.
  • Y is C 1-3 alkyl, such as methyl.
  • A is: In some specific embodiments A is: In other embodiments of the invention A is:
  • R 1 or R 2 is benzoyl (Bz) optionally substituted with a methoxy group or a halo group, 9-fluorenylmethoxycarbonyl (Fmoc), t-butoxy carbonyl (Boc), benzyloxy carbonyl (Cbz), acetyl (Ac), trifluoroacetyl, benzyl (Bn) optionally substituted with a methoxy group or a halo group, triphenylmethyl (Tr), methoxytriphenylmethyl (MTr), dimethoxytriphenylmethyl (DMTr), p-toluenesulfonyl (Ts), isobutyryl, propionyl, or pivaloyl.
  • R 1 or R 2 taken together is phthalimide or benzylideneamine.
  • one of R 1 and R 2 is H and the other is OH or OX 2 .
  • X 2 is an alkylsilyl group or an arylsilyl group.
  • the alkylsilyl group is trimethylsilyl (TMS), triethylsilyl (TES), t-butyldimethylsilyl (TBDMS), or triisopropylsilyl (TIPS).
  • the arylsilyl group is t-butyldiphenylsilyl (TBDPS).
  • R 3 and R 4 are each H, methyl or trifluoromethyl.
  • R 5 is H or methyl.
  • R 6 is H, methyl, benzyloxy, 2- (phenyl)thioethyl, 2-(4-nitrophenyl)thioethyl, 2-(phenyl)sulfonylethyl, 2-(4- nitrophenyl)sulfonylethyl, 2-cyanoethyl, 2-(trimethylsilyl)ethyl, 2-(4-nitrophenyl)ethyl, or 2- (4-cyanophenyl)ethyl.
  • R 1 is H and R 2 is benzoyl.
  • the compound has the Formula (II) or the Formula (IIa): .
  • the invention provides a process for the preparation of a compound of Formula (I) or Formula (Ia) comprising the steps: i. reacting a nucleoside of the Formula (III) or Formula (IIIa) with a silyl protecting reagent to protect the hydroxyl groups at the 2’ and 5’ positions with silyl protecting groups wherein: Y is H, C 1-3 alkyl, C 2-3 alkenyl, or C 2-3 alkynyl; A is selected from:
  • R 1 and R 2 are each H, OH, OX 2 where X 2 is a silyl protecting group, or a C 1-6 alkyl, C 1-12 acyl, C 1-6 alkoxymethyl, C 3 -C 12 cycloalkyl, alkyloxycarbonyl, aryl, aryloxy or aryloxymethyl group each of which is optionally substituted with one or more alkyl, alkenyl, alkynyl, alkoxy, aryl, aryloxy, amino, halo or hydroxy groups, provided R 1 and R 2 are not both H; or R 1 and R 2 taken together form a cyclic group optionally substituted with one or more alkyl, alkenyl, alkynyl, alkoxy, aryloxy, amino, halo or hydroxy groups; R 3 and R 4 are each H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, haloalkyl, or halogen;
  • R 1 or R 2 is benzoyl (Bz) optionally substituted with a methoxy group or a halo group, 9-fluorenylmethoxycarbonyl (Fmoc), t-butoxy carbonyl (Boc), benzyloxy carbonyl (Cbz), acetyl (Ac), trifluoroacetyl, benzyl (Bn) optionally substituted with a methoxy group or a halo group, triphenylmethyl (Tr), methoxytriphenylmethyl (MTr), dimethoxytriphenylmethyl (DMTr), p-toluenesulfonyl (Ts), isobutyryl, propionyl, or pivaloyl.
  • R 1 or R 2 taken together is phthalimide or benzylideneamine.
  • one of R 1 and R 2 is H and the other is OH or OX 2 .
  • X 2 is an alkylsilyl group or an arylsilyl group.
  • the alkylsilyl group is trimethylsilyl (TMS), triethylsilyl (TES), t-butyldimethylsilyl (TBDMS), or triisopropylsilyl (TIPS).
  • the arylsilyl group is t-butyldiphenylsilyl (TBDPS).
  • R 3 and R 4 are each H, methyl or trifluoromethyl.
  • R 5 is H or methyl.
  • R 6 is H, methyl, benzyloxy, 2- (phenyl)thioethyl, 2-(4-nitrophenyl)thioethyl, 2-(phenyl)sulfonylethyl, 2-(4- nitrophenyl)sulfonylethyl, 2-cyanoethyl, 2-(trimethylsilyl)ethyl, 2-(4-nitrophenyl)ethyl, or 2- (4-cyanophenyl)ethyl.
  • R 1 is H and R 2 is benzoyl.
  • A is:
  • the nucleoside of the Formula (III) or Formula (IIIa) is a 4-N-protected cytidine, for example 4-N-benzoylcytidine.
  • each silyl protecting group is an alkylsilyl group or an arylsilyl group.
  • the alkylsilyl group is trimethylsilyl (TMS), triethylsilyl (TES), t-butyldimethylsilyl (TBDMS), or triisopropylsilyl (TIPS).
  • the arylsilyl group is t-butyldiphenylsilyl (TBDPS).
  • TDPS t-butyldiphenylsilyl
  • the process is for the preparation of a compound of Formula (II) or Formula (IIa) comprising the steps: i. reacting a 4-N-benzoylcytidine with t-butyldimethylsilyl chloride to give 4-N- benzoyl-2′,5′-bis-O-(t-butyldimethylsilyl)cytidine; ii.
  • the process is for the preparation of a compound of Formula (II) where 4-N-benzoylcytidine in step i. is 4-N-benzoyl-D-cytidine.
  • the process is for the preparation of a compound of Formula (IIa) where 4-N-benzoylcytidine in step i. is 4-N-benzoyl-L-cytidine.
  • the invention provides the use of a compound of the invention in the preparation of a 3’-deoxy-3’,4’-didehydro nucleoside monophosphate, diphosphate, or triphosphate of the Formula (IV) or the Formula (IVa): wherein A, X 1 and Y are as defined above and Z is , or a salt thereof.
  • alkyl means any saturated hydrocarbon radical and is intended to include both straight- and branched-chain alkyl groups.
  • C 1 -C 6 alkyl means any saturated hydrocarbon radical having up to 6 carbon atoms.
  • alkenyl means any hydrocarbon radical having at least one double bond and is intended to include both straight- and branched-chain alkenyl groups.
  • alkenyl groups include, but are not limited to, ethenyl, n-propenyl, iso-propenyl, n-butenyl, iso-butenyl, sec-butenyl, n-pentenyl, 1,1-dimethylpropenyl, 1,2-dimethylpropenyl, , 1- ethylpropenyl, 2-ethylpropenyl, n-hexenyl and 1-methyl-2-ethylpropenyl.
  • alkynyl means any hydrocarbon radical having at least one carbon- carbon triple bond and is intended to include both straight- and branched-chain alkynyl groups.
  • alkynyl groups include, but are not limited to, ethynyl, n-propynyl and n-butynyl.
  • aryl means an aromatic radical having 1 to 18 carbon atoms and includes heteroaromatic radicals. Examples include monocyclic groups, as well as fused groups such as bicyclic groups and tricyclic groups.
  • Examples include, but are not limited to, phenyl, indenyl, 1-naphthyl, 2-naphthyl, azulenyl, heptalenyl, biphenyl, indacenyl, acenaphthyl, fluorenyl, phenalenyl, phenanthrenyl, anthracenyl, cyclopentacyclooctenyl, and benzocyclooctenyl, pyridyl, pyrrolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazolyl, tetrazolyl, and imidazolyl.
  • alkoxy means an OR group, where R is alkyl, alkenyl or alkynyl as defined above.
  • aryloxy means an OR group, where R is aryl as defined above.
  • alkylene means a diradical corresponding to an alkyl group and is intended to include straight chain alkyl groups. Examples of alkylene groups include, but are not limited to, methylene and ethylene.
  • protecting group means a group that selectively protects an organic functional group, temporarily masking the chemistry of that functional group and allowing other sites in the molecule to be manipulated without affecting the functional group. Suitable protecting groups are known to those skilled in the art and are described, for example, in Protective Groups in Organic Synthesis (3 rd Ed.), T. W. Greene and P. G. M.
  • protecting groups include, but are not limited to, O-benzyl, O-benzhydryl, O-trityl, O-t-butyldimethylsilyl, O-t-butyldiphenylsilyl, O-4-methylbenzyl, O-acetyl, O-chloroacetyl, O-methoxyacetyl, O-benzoyl, O-4- bromobenzoyl, O-4-methylbenzoyl, O-fluorenylmethoxycarbonyl and O-levulinoyl.
  • sil ether protecting group is a protecting group such as O-t- butyldimethylsilyl and O-t-butyldiphenylsilyl.
  • nucleoside means a compound that consists of a purine or pyrimidine base combined with deoxyribose or ribose and is found especially in DNA or RNA, and includes adenosine, cytidine, uridine and guanosine.
  • One advantage of the process of the invention is that it may be applied generally to a range of compounds, by commencing from the appropriate nucleoside analogue, which may be available from a commercial supplier, and choosing appropriate protection for the nucleobase substituent.
  • the process affords direct access to 2’-silyl protected 3’-deoxy- 3’,4’-didehydroribo compounds by a very efficient synthetic route.
  • This route is advantageous because it requires a low number of high-yielding chemical steps to obtain the 2’-silyl protected 3’-deoxy-3’,4’-didehydroribo compounds.
  • the 2’-silylated intermediates in this process generally have good solubility in organic solvents which allows for simple practical handling.
  • the enantiomer of compound 5A namely compound ent-5A (Scheme 2)
  • ent-1 also sourced from Biosynth Carbosynth ®
  • Monophosphorylation of alcohol 5A was first attempted by the Yoshikawa method (POCl3 in trimethylphosphate, Tetrahedron Letters 1967, 8 (50), 5065–5068).
  • Phosphoramidite chemistry was instead employed to access the monophosphate (Tetrahedron Lett. 1997, 38, 7407) delivering difluorenylmethyl phosphate 12 (Scheme 3).
  • Both the fluorenylmethyl and N-benzoyl groups could be removed from phosphate ester 12 using ammonia in methanol to deliver free phosphate 11. Phosphorylation of the 5′-alcohol was also achieved without protection of the 4-N group. Removal of the 4-N-benzoyl group with ammonia in methanol afforded alcohol 13, which then underwent selective reaction with phosphoramidite to give phosphate ester 14. Compound 13 could be converted to the alternatively protected 4-N-DMTr-protected intermediate 15 by transient protection of the 5’-alcohol via chlorotrimethylsilane and reaction with 4,4′-dimethoxytrityl chloride.
  • tert- butyl phosphate esters were cleaved by treatment with triethylamine and chlorotrimethylsilane in hot acetonitrile (Tetrahedron Letters 1991, 32 (3), 395–398) to give phosphate 11 without affecting the 2′-O-TBDMS ether.
  • 2′-O-TBDMS-protected phosphate 11 provided access to the phosphorylated forms of ddhC; ddhCMP (16), ddhCDP (18) and ddhCTP (20) (Scheme 4).
  • the TBDMS group of phosphate 11 underwent traceless deprotection under mild acidic conditions to give ddhCMP (16) quantitatively.
  • ddhCMP (16) was isolated as its sodium salt following ion exchange.
  • ddhCDP (18) and ddhCTP (20) were both prepared from monophosphate 11 using the method reported by Hoard and Ott (J. Am. Chem. Soc. 1965, 87 (8), 1785–1788).
  • Adoption of this strategy over direct di- or triphosphorylation of alcohol 5A was motivated by the scalability of the reaction (J. Org. Chem. 1990, 55 (6), 1834–1841), as well as the simplicity of the reagent system, which would streamline purification.
  • Activation of monophosphate 11 as the imidazolidate was performed using CDI, the excess of which was quenched using water.
  • ddhCDP (18) was also readily accessed from monophosphate 11 by activation with CDI, treatment with orthophosphate, and Dowex-mediated TBDMS cleavage.
  • the same TBDMS-protecting group strategy was applied to the synthesis of ddhUMP (33) and ddhUTP (32) (Scheme 5).
  • ddhUMP (31) was accessible from alcohol 25 via fluorenylmethyl phosphate 29. Cleavage of the phosphate esters using triethylamine in acetonitrile afforded free phosphate 30 as the triethylamine salt. Traceless deprotection of the TBDMS group was achieved using Dowex resin (H form) as an acid catalyst in water, affording ddhUMP (31) quantitatively.
  • ddhUTP (33) was accessed from TBDMS-protected monophosphate 30 by preparation of the imidazolidate using CDI, treatment with pyrophosphate tributylammonium salt to give triphosphate 32, then TBDMS cleavage using Dowex resin (H form) in water.
  • Appel reaction of alcohol 38 to afford iodide 39 can be achieved with either a triphenylphosphine-iodine-imidazole reagent system, or triphenoxy methylphosphonium iodide.
  • ddhGTP (44) was accessed from alcohol 41 via fluorenylmethyl phosphate ester 42.
  • the 5-fluorouridine analogue 56 (Scheme 9) was prepared from known compound 4- N-benzoyl-5-fluorocytidine (52) (Nucleic Acids Research, 2009, 37(22), 7728-7740) by a similar method to that which furnished the 2’-O-TBDMS-N-4-Benzoyl-ddhC intermediate 5A. Selective 2’,5’-bis-silyl ether protection followed by iodination afforded compound 54. However, in this instance, upon treatment of iodide 54 with TFA/water, uridine compound 55 was unexpectedly obtained, where the 5-fluoro-cytosine base moiety underwent hydrolysis to the 5-fluoro-uracil base moiety. Compound 55 could be converted to 2’-O- TBDMS-protected 5-fluoro-ddhU compound 56 by DABCO-mediated elimination.
  • the synthetic route utilising a 2′-O-TBDMS protected ddhC derivative enables the facile synthesis of biologically-relevant phosphates of ddhC.
  • the silyl group protection strategy also has the benefit of providing a lipophilic handle that enables reverse phase flash chromatographic purification of highly charged compounds that would otherwise require more intensive purification methods. Further, this route enables the TBDMS protecting group to be removed without trace under mild conditions to provide the deprotected targets in good purity. Taken together, these properties have facilitated a robust and scalable synthesis of ddhCTP (20), and similar compounds, providing useful quantities of antiviral metabolites and their prodrugs for biological studies.
  • the synthetic methodology is applicable not only to 3’- deoxy-3’,4’-didehydrocytidines, but also to 3’-deoxy-3’,4’-didehydrouridines and 3’-deoxy- 3’,4’-didehydroguanidines.
  • the methodology may be used for the synthesis of 3’-deoxy- 3’,4’-didehydroribonucleosides in general.
  • the chemical synthesis route developed by the applicant has led to a novel class of compounds useful as intermediates for the synthesis of a wide range of 3’-deoxy-3’,4’-didehydroribonucleoside based antiviral drugs.
  • Example 2 1-(2′,5′-Bis-O-(tert-butyldimethylsilyl)-3′-iodo- ⁇ -D-threo- pentofuranosyl)-4-N-benzoylcytosine (3A) A flask was charged with methyltriphenoxyphosphonium iodide (6.41 g, 11.3 mmol) under argon, followed by 2′,5′-bis-O-(tert-butyldimethylsilyl)-4-N-benzoylcytidine (2A) (4.35 g, 7.55 mmol).
  • the crude oil was purified by flash column chromatography (silica gel, 10–30% EtOAc-Hex) to afford the title compound (3.38 g, 65%) as an off-white solid.
  • the worked-up crude reaction mixture can be recrystallised from methanol at between 40-60 °C.
  • Example 3 1-(2′-O-(tert-Butyldimethylsilyl)-3′-iodo- ⁇ -D-threo-pentofuranosyl)-4- N-benzoylcytosine (4A) To a solution of 1-(2′,5′-bis-O-(tert-butyldimethylsilyl)-3′-iodo- ⁇ -D-threo- pentofuranosyl)-4-N-benzoylcytosine (3A) (5.64 g, 8.23 mmol) in THF (24 mL) at 0 °C was added a mixture of TFA-H 2 O (1:1, 7.2 mL) dropwise over 3 min.
  • the reaction mixture was warmed to room temperature and stirred for 3.5 h.
  • the reaction was neutralised by addition of sat aq NaHCO 3 (50 mL), then extracted with EtOAc (3 ⁇ 50 mL).
  • the combined organic layers were washed with sat aq NaHCO 3 (20 mL), brine (20 mL), dried over anhydrous MgSO 4 , filtered and concentrated in vacuo.
  • the crude oil was purified by flash column chromatography (120 g silica gel, 10%–100% EtOAc-Hex) afforded the title compound (4.17 g, 89% yield) as a colourless solid.
  • the worked-up crude reaction mixture can be recrystallised by suspending and stirring in 5% EtOAc in petroleum ether (60-80) (at a volume equivalent to around 2 mL/g of the starting material 3A used) followed by addition of CHCl 3 (at a volume equivalent to around 0.4-0.7 mL/g of starting material 3A) at 40-50 °C.
  • Example 7 1-(2′-O-(Triisopropylsilyl)-3′-iodo- ⁇ -D-threo-pentofuranosyl)-4-N- benzoylcytosine (4B) To a solution of 1-(2′,5′-bis-O-(triisopropylsilyl)-3′-iodo- ⁇ -D-threo-pentofuranosyl)- 4-N-benzoylcytosine (3B) (200 mg, 0.260 mmol) in THF (12 mL) at 0 °C was added a mixture of TFA-H 2 O (1:1, 6 mL) dropwise over 3 min.
  • the reaction mixture was warmed to room temperature and stirred for 10 h.
  • the reaction was neutralised by addition of sat aq NaHCO 3 (50 mL), then extracted with EtOAc (3 ⁇ 50 mL).
  • the combined organic layers were washed with sat aq NaHCO 3 (20 mL), brine (20 mL), dried over anhydrous MgSO 4 , filtered and concentrated in vacuo.
  • the crude oil was purified by flash column chromatography (silica gel, 10–50% EtOAc-Hex) to afford the title compound (146 mg, 92% yield) as a colourless solid.
  • Example 8 4-N-Benzoyl-2′-O-(triisopropylsilyl)-3′-deoxy-3′,4′-didehydrocytidine (5B) To a suspension of 1-(2′-O-(triisopropylsilyl)-3′-iodo- ⁇ -D-threo-pentofuranosyl)-4-N- benzoylcytosine (4B) (108 mg, 0.176 mmol) in PhMe (3 mL) was added DABCO (70 mg, 0.59 mmol) and the resulting suspension was heated to 75 °C and stirred for 5 h.
  • DABCO 70 mg, 0.59 mmol
  • Example 9 2′,5′-Bis-O-tert-butyldiphenylsilyl-4-N-benzoylcytidine (2C) To a suspension of 4-N-benzoylcytidine (1) (1.50 g, 4.32 mmol) in pyridine (8.6 mL) was added TBDPSCl (3.2 mL, 11.9 mmol) and the reaction mixture was stirred at room temperature for 18 h, then heated to 50 °C and stirred for another 24 h.
  • Example 10 1-(2′,5′-Bis-O-(tert-butyldiphenylsilyl)-3′-iodo- ⁇ -D-threo- pentofuranosyl)-4-N-benzoylcytosine (3C) 2′,5′-Bis-O-tert-butyldiphenylsilyl-4-N-benzoylcytidine (2C) (540 mg, 0.655 mmol) and methyltriphenoxyphosphonium iodide (581 mg, 1.03 mmol) were dissolved in DMF (4.5 mL) and treated with pyridine (0.11 mL, 1.4 mmol).
  • Example 11 1-(2′-O-(tert-Butyldiphenylsilyl)-3′-iodo- ⁇ -D-threo-pentofuranosyl)- 4-N-benzoylcytosine (4C)
  • 1-(2′,5′-bis-O-(tert-butyldiphenylsilyl)-3′-iodo- ⁇ -D-threo- pentofuranosyl)-4-N-benzoylcytosine (166 mg, 0.178 mmol) in MeCN (0.9 mL) was added TfOH-SiO 2 (2 mmol/g TfOH on silica gel, 150 mg, 0.30 mmol) prepared according to Carbohyd.
  • Example 12 1-(2′-O-(tert-Butyldiphenylsilyl)-3′-deoxy-3′,4′-didehydro-4-N- benzoylcytosine (5C) To a solution of 1-(2′-O-(tert-butyldiphenylsilyl)-3′-iodo- ⁇ -D-threo-pentofuranosyl)- 4-N-benzoylcytosine (4C) (39 mg, 0.056 mmol) in PhMe (1.1 mL) was added DABCO (23 mg, 0.20 mmol) and the reaction mixture was heated to 70 °C for 5 h, then 80 °C for 1 h.
  • 5C 1-(2′-O-(tert-butyldiphenylsilyl)-3′-iodo- ⁇ -D-threo-pentofuranosyl)- 4-N-benzoylcytosine (4C) (39 mg, 0.056 mmol
  • Example 13 4-N-Benzoyl-2’,3’-O-methoxymethylidenecytidine (6)
  • 4-N-Benzoylcytidine (1) (10.7 g, 30.2 mmol) was suspended in dry DMF (100 mL) and trimethyl orthoformate (130 mL) before PTSA (250 mg, 1.3 mmol) was added. The mixture was stirred for 24 h at r.t. After the reaction had consumed all starting material an excess of Amberlyst® A-21 resin was added and the mixture stirred for 15 mins before filtering. Triethylamine (5 mL) was added to the filtrate before concentrating to dryness using high vacuum at 50 °C.
  • Toluene 250 mL was added to the crude and the suspension was concentrated again. Toluene was added and concentrated twice more before the crude was suspended in ethyl acetate and brought to 70 °C for ca. 30 mins. After cooling to 15 °C the solution was allowed to stir for 20 mins before the first crop of white precipitate was collected by filtration, and washing with cold ethyl acetate. On standing (24-40 h), more product precipitated from the mother-liquor as a white solid and was collected by filtration, washing with cold ethyl acetate. The overall yield for the three crops isolated was 89% (10.4 g). The white, solid product was a mixture of epimers at the orthoformate stereocentre in the proportion ca.
  • Example 14 4-N-Benzoyl-3’-deoxy-3’,4’-didehydrocytidine-5’-aldehyde (7)
  • 4-N-benzoyl-2’,3’-O-methoxymethylidenecytidine (6) (3.30 g, 8.47 mmol) was converted to the crude then semi-purified by column chromatography by dry loading and eluting with a gradient from 5-10% MeOH in CHCl 3 .
  • Example 15 4-N-Benzoyl-2’-O-tert-butyldimethylsilyl-3’-deoxy-3’,4’- didehydrocytidine-5’-aldehyde (8) 4-N-Benzoyl-3’-deoxy-3’,4’-didehydrocytidine-5’-aldehyde (7) (400 mg, 1.22 mmol) and imidazole (170 mg, 2.44 mmol) were dissolved in dry DMF (10 mL) before TBDMSCl (250 mg, 1.61 mmol) was added at r.t. The mixture was stirred for 3 h before more TBDMSCl (80 mg, 0.51 mmol) was added.
  • Example 16 4-N-Benzoyl-2’-O-tert-butyldimethylsilyl-3’-deoxy-3’,4’- didehydrocytidine
  • 5A 4-N-Benzoyl-2’-O-tert-butyldimethylsilyl-3’-deoxy-3’,4’-didehydrocytidine-5’- aldehyde (8) (400 mg, 0.91 mmol) was dissolved in methanol (10 mL) and cooled to 0 °C before sodium borohydride (35 mg, 0.91 mmol) was added. After 10 mins, and all starting material had been consumed, acetone (0.2 mL) was added.
  • Example 19 1-(2′-O-(tert-Butyldimethylsilyl)-3′-iodo- ⁇ -L-threo-pentofuranosyl)- 4-N-benzoylcytosine (ent-4A) Analogous to the reaction of 3A to give 4A, compound ent-3A afforded ent-4A (1.12 g, 55% over two steps). [ ⁇ ]D 20 -32.86 (1.05, CHCl 3 ); 1 H and 13 C NMR spectroscopic data were identical to those for the enantiomer.
  • Example 21 Di-tert-butyl-4-N-benzoyl-2′-O-(tert-butyldimethylsilyl)-3′-deoxy- 3′,4′-didehydrocytidine-5′-phosphate (9) To a solution of 4-N-benzoyl-2′-O-(tert-butyldimethylsilyl)-3′-deoxy-3′,4′- didehydrocytidine (5A) (1.41 g, 3.18 mmol) in anhydrous MeCN (16 mL) at room temperature was added 1H-tetrazole (0.45 M in MeCN, 21.0 mL, 9.45 mmol), followed by di-tert-butyl N,N-diisopropylphosphoramidite (1.60 mL, 5.07 mmol) dropwise.
  • reaction mixture was stirred for 1 h, then cooled to 0 °C, and tert-butyl hydroperoxide (70 w/w% in H 2 O, 1.10 mL, 7.95 mmol) was added dropwise.
  • the reaction mixture was allowed to warm to room temperature slowly over 2 h, then quenched by addition of sat aq NaHCO 3 (25 mL).
  • the reaction mixture was diluted with water (50 mL) and the aqueous layer extracted with EtOAc (3 ⁇ 50 mL). The combined organic layers were washed with brine (30 mL), dried over anhydrous MgSO 4 , filtered and concentrated in vacuo.
  • Example 22 Di-tert-butyl-2′-O-(tert-butyldimethylsilyl)-3′-deoxy-3′,4′- didehydrocytidine-5′-phosphate (10) Di-tert-butyl-4-N-benzoyl-2′-O-(tert-butyldimethylsilyl)-3′-deoxy-3′,4′- didehydrocytidine-5′-phosphate (9) (220 mg, 0.346 mmol) was dissolved in a solution of ammonia (7 M in MeOH, 3.5 mL, 25.0 mmol) and the resulting solution was stirred at room temperature for 18 h, then concentrated in vacuo.
  • ammonia 7 M in MeOH, 3.5 mL, 25.0 mmol
  • Example 23 2′-O-(tert-Butyldimethylsilyl)-3′-deoxy-3′,4′-didehydrocytidine-5′- phosphate triethylammonium salt (11)
  • MeCN MeCN
  • Et 3 N 1.9 mL, 14 mmol
  • TMSCl 1.3 mL, 10 mmol
  • reaction mixture was then placed in a pre-heated heating block at 75 °C and stirred at this temperature for 2 h.
  • the reaction mixture was allowed to cool to 40 °C, then volatiles were removed from the reaction mixture in vacuo, while still under an inert atmosphere.
  • the slurry thus obtained was co-evaporated twice with MeCN to remove any residual volatile impurities, then purified by flash chromatography (12 g C18 column, 10%– 100% MeOH/H 2 O with 0.5% Et 3 N) to afford the title compound (131 mg, 76%) as a colourless solid.
  • reaction mixture was stirred for 1 h, then cooled to 0 °C, and tert-butyl hydroperoxide (70 w/w% in H 2 O, 1.10 mL, 7.95 mmol) was added dropwise.
  • the reaction mixture was allowed to warm to room temperature and stirred for 90 min, then quenched by addition of 10% aq Na 2 S 2 O 3 (5 mL) and sat aq NaHCO 3 (100 mL).
  • the reaction mixture was extracted with EtOAc (3 ⁇ 50 mL) and the combined organic layers were washed with brine (30 mL), dried over anhydrous MgSO 4 , filtered and concentrated in vacuo.
  • Example 25 2′-O-(tert-Butyldimethylsilyl)-3′-deoxy-3′,4′-didehydrocytidine-5′- phosphate triethylammonium salt (11) Difluorenylmethyl-4-N-benzoyl-2′-O-(tert-butyldimethylsilyl)-3′-deoxy-3′,4′- didehydrocytidine-5′-phosphate (12) (2.20 g, 2.50 mmol) was dissolved in a 7 M solution of anhydrous NH3 in MeOH (25 mL) and stirred at room temperature for 18 h.
  • Example 27 Difluorenylmethyl-2′-O-(tert-butyldimethylsilyl)-3′-deoxy-3′,4′- didehydrocytidine-5′-phosphate (14) To a suspension of 2′-O-(tert-butyldimethylsilyl)-3′-deoxy-3′,4′-didehydrocytidine (13) (100 mg, 0.295 mmol) in MeCN (1.5 mL) at room temperature was added a solution of 1H-tetrazole (0.45 M in MeCN, 1.6 mL, 0.72 mmol), followed by a solution of difluorenyl N,N-diisopropylphosphoramidite (1.0 M in benzene, 0.37 mL, 0.37 mmol) dropwise.
  • 1H-tetrazole 0.45 M in MeCN, 1.6 mL, 0.72 mmol
  • reaction mixture was stirred for 1 h, then cooled to 0 °C, and tert-butyl hydroperoxide (70 w/w% in H 2 O, 85 ⁇ L, 0.61 mmol) was added dropwise.
  • the reaction mixture was allowed to warm to room temperature and stirred for 90 min, then quenched by addition of 10% aq Na 2 S 2 O 3 (1 mL) and sat aq NaHCO 3 (10 mL).
  • the reaction mixture was extracted with EtOAc (3 ⁇ 10 mL) and the combined organic layers were washed with brine (5 mL), dried over anhydrous MgSO 4 , filtered and concentrated in vacuo.
  • Example 29 2′-O-tert-Butyldimethylsilyl-3′,4′-didehydro-3′-deoxy-4-N- (4,4′-dimethoxytrityl)-cytidine (15) To a solution of 2′-O-tert-butyldimethylsilyl-3′,4′-didehydro-3′-deoxycytidine (13) (100 mg, 0.295 mmol) in pyridine (1.5 mL) at room temperature was added chlorotrimethylsilane (80 ⁇ L, 0.6 mmol).
  • Example 30 3′-Deoxy-3′,4′-didehydrocytidine-5′-phosphate sodium salt (16) 2′-O-(tert-Butyldimethylsilyl)-3′-deoxy-3′,4′-didehydrocytidine-5′-phosphate triethylammonium salt (11) (115 mg, 0.221 mmol) was dissolved in AcOH-DI H 2 O (1:1, 4.4 mL) and the resulting solution was stirred at room temperature for 18 h, then concentrated in vacuo at or below 40 °C. The residue obtained was co-evaporated with DI H 2 O three times to remove residual AcOH, affording the triethylammonium phosphate as a colourless solid.
  • the reaction mixture was stirred for 1 h, then loaded onto Dowex ® 50WX8 Et 3 NH form and eluted with MeCN to remove imidazole from the crude mixture. Fractions containing the intermediate imidazolidate were combined and concentrated in vacuo to dryness.
  • the crude imidazolidate was dissolved in MeCN (1.0 mL), to which solution was added tributylammonium phosphate (1 M in MeCN, 0.96 mL, 0.96 mmol). The reaction mixture was stirred at room temperature for 24 h, then concentrated in vacuo.
  • reaction mixture was stirred for 1 h, then bis(tributylammonium) pyrophosphate (530 mg, 0.966 mmol) was added and the reaction mixture stirred for a further 48 h, after which the reaction was complete by TLC analysis (silica gel, 6:1:3 i-PrOH-H 2 O-28% aq NH 4 OH).
  • the reaction solvent was removed in vacuo, then the crude triphosphate was dissolved in DI H 2 O and converted to its triethylammonium salt by passage through an ion exchange column (Dowex ® 50WX8 Et 3 NH form) eluting with DI H 2 O. Product-containing fractions were combined, then concentrated in vacuo.
  • Example 34 3′-Deoxy-3′,4′-didehydrocytidine-5′-triphosphate tris(triethylammonium) salt (20) To a solution of 2′-O-(tert-butyldimethylsilyl)-3′-deoxy-3′,4′-didehydrocytidine-5′- triphosphate tris(triethylammonium) salt (19) (97 mg, 0.110 mmol) in deionized water (10 mL) was added Dowex® 50WX8 hydrogen form (216 mg), which had previously been washed with MeOH and deionized water.
  • Example 36 1-(2′,5′-Bis-O-(tert-butyldimethylsilyl)-3′-iodo- ⁇ -D-threo- pentofuranosyl)-uracil (23)
  • triphenylphosphine 113 mg, 0.422 mmol
  • imidazole 58 mg, 0.843 mmol
  • I2 108 mg, 0.426 mmol
  • THF 3.0 mL
  • 2′,5′-bis-O-(tert-butyldimethylsilyl)uridine 2′,5′-bis-O-(tert-butyldimethylsilyl)uridine (22) (100 mg, 0.212 mmol) in THF (2.1 mL).
  • reaction mixture was heated to 60 °C and stirred for 4 h, then cooled to room temperature.
  • the reaction mixture was diluted with EtOAc (15 mL), then silica gel (750 mg) was added to form a slurry.
  • the slurry was concentrated in vacuo to give a free- flowing solid mixture was purification by dry load; flash column chromatography (25 g silica gel, 5–50% EtOAc-Hex) afforded the title compound (99.8 mg, 81% yield) as a colourless solid.
  • Example 37 1-(2′-O-(tert-Butyldimethylsilyl)-3′-iodo- ⁇ -D-threo-pentofuranosyl)- uracil (24) To a solution of 1-(2′,5′-bis-O-(tert-butyldimethylsilyl)-3′-iodo- ⁇ -D-threo- pentofuranosyl)-uracil (23) (131 mg, 0.225 mmol) in THF (0.5 mL) at 0 °C was added a mixture of TFA-H 2 O (1:1, 0.14 mL) dropwise over 3 min.
  • the reaction mixture was warmed to room temperature and stirred for 3.5 h.
  • the reaction was neutralised by addition of sat aq NaHCO 3 (5 mL), then extracted with EtOAc (3 ⁇ 5 mL).
  • the combined organic layers were washed with sat aq NaHCO 3 (5 mL), brine (5 mL), dried over anhydrous MgSO 4 , filtered and concentrated in vacuo.
  • the crude oil was purified by flash column chromatography (12 g silica gel, 15%–80% EtOAc-Hex) to afford the title compound (90 mg, 85% yield) as a colourless solid.
  • reaction mixture was then cooled to 0 °C and quenched by addition of sodium methoxide (25% w/w in MeOH, 0.21 mL, 0.92 mmol) and PhMe (5 mL).
  • the reaction mixture was concentrated in vacuo and the resultant oil purified by flash column chromatography (120 g silica gel, 3%–20% MeOH-CH 2 Cl 2 ) to afford the title compound (3.18 g, 54% yield) as a colourless gum.
  • the product was an approximately 1:1 mixture of diastereomers at the orthoformate stereocentre, designated as A and B.
  • Example 40 3′-Deoxy-3′,4′-didehydro-uridine-5′-aldehyde (27)
  • the title compound was prepared according to a procedure adapted from Petrová et al. (Tetrahedron Letters, 2010, 51, 6874-6876).
  • 2′,3′-Di-O-methoxymethylidene-uridine (26) (3.18 g, 11.1 mmol)
  • 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (4.50 g, 22.2 mmol) were placed under argon, then suspended in DMF (22 mL).
  • Example 41 2′-O-(tert-Butyldimethylsilyl)-3′-deoxy-3′,4′-didehydrouridine-5′- aldehyde (28)
  • Et 3 N 4.0 mL, 29 mmol
  • TBDMSCl TBDMSCl
  • reaction mixture was stirred for 1 h, then cooled to 0 °C and treated with tBuOOH (70% in H 2 O, 0.59 mL, 4.23 mmol).
  • the reaction was allowed to warm to room temperature and stirred for 90 min, then diluted with sat aq NaHCO 3 (50 mL) and EtOAc (25 mL).
  • the aqueous phase was extracted with EtOAc (2 ⁇ 25 ml), then the combined organic layers were washed with brine (20 mL), dried over anhydrous MgSO 4 , filtered and concentrated in vacuo.
  • reaction mixture was stirred for 40 min, then bis(tributylammonium) pyrophosphate (790 mg, 1.44 mmol) and Bu 3 N (0.14 mL, 0.58 mmol) were added and the reaction mixture stirred for a further 72 h, after which the reaction was complete by TLC analysis (silica gel, 6:1:3 i-PrOH-H 2 O-28% aq NH 4 OH).
  • the reaction solvent was removed in vacuo, then the crude residue was purified by flash chromatography on C18 silica gel (40–80% solvent B-buffer, where buffer A is 20 mM Bu 3 N and 30 mM AcOH in H 2 O, solvent B is 15 mM Bu 3 N in MeOH).
  • the triphosphate was converted to its triethylammonium salt by passage through an ion exchange column (Dowex ® 50WX8 Et 3 NH form) eluting with DI H 2 O, then lyophilized to afford the title compound (143 mg, 50% yield) as a colourless solid.
  • Example 48 2′,5′-Bis-O-(tert-butyldimethylsilyl)-3′-deoxy-3′,4′-didehydrouridine (34) To a solution of iodide 23 (250 mg, 0.429 mmol) in PhMe (3 mL) was added DABCO (150 mg, 1.27 mmol), then the reaction mixture was heated to 80 °C and stirred for 18 h. The reaction mixture was cooled down to room temperature and washed with 1 M aq Na 2 S 2 O 3 solution. The organic layer was then dried over anhydrous MgSO 4 , filtered and concentrated in vacuo.
  • DABCO 150 mg, 1.27 mmol
  • Example 51 2′,5′-Bis-O-(tert-butyldimethylsilyl)-2-N-isobutyryl-guanosine (38) 2-N-Isobutyryl-guanosine (37) (4.04 g, 11.2 mmol) and TBDMSCl (4.15 g, 27.3 mmol) were dissolved in DMF (65 mL), then treated with imidazole (3.02 g, 44.0 mmol) at room temperature. The reaction mixture was stirred for 3 d, then partitioned between water (100 mL) and EtOAc (150 mL).
  • Example 52 9-(2′,5′-Bis-O-(tert-butyldimethylsilyl)-3′-iodo- ⁇ - D -threo- pentofuranosyl)-2-N-isobutyrylguanine (39)
  • PPh3 94 mg, 0.35 mmol
  • I2 90 mg, 0.35 mmol
  • imidazole 47 mg, 0.68 mmol
  • THF 2.5 mL
  • 2′,5′- bis-O-(tert-butyldimethylsilyl)-2-N-isobutyryl-guanosine (100 mg, 0.17 mmol) in THF (1.7 mL).
  • the reaction mixture was heated to 60 °C and stirred for 5 h, then cooled to room temperature.
  • the crude reaction mixture was decanted from precipitated triphenylphosphine oxide, then concentrated in vacuo.
  • the crude product was purified by flash column chromatography (silica gel, 0%–10% acetone-CH 2 Cl 2 ) to afford the title compound (70 mg, 59% yield) as a colourless solid.
  • the reaction mixture was allowed to warm to room temperature slowly, then stirred for 3 h.
  • the reaction was then diluted with EtOAc (10 mL) and neutralised by addition of sat aq NaHCO 3 (2 mL).
  • the biphasic mixture was separated, then the organic layer was washed with water, brine, dried over anhydrous MgSO 4 , filtered and concentrated in vacuo.
  • the crude product obtained was purified by flash column chromatography (silica gel, 5% MeOH-EtOAc) to afford the title compound (96 mg, 84% yield) as a colourless solid.
  • reaction mixture was stirred for 1 h, then cooled to 0 °C, and tert-butyl hydroperoxide (70 w/w% in H 2 O, 0.38 mL, 2.7 mmol) was added dropwise.
  • the reaction mixture was allowed to warm to room temperature and stirred for 3 h, then quenched by addition of 10% aq Na 2 S 2 O 3 (2 mL) and sat aq NaHCO 3 (50 mL).
  • the reaction mixture was extracted with EtOAc (3 ⁇ 30 mL) and the combined organic layers were washed with brine (30 mL), dried over anhydrous MgSO 4 , filtered and concentrated in vacuo.
  • Example 56 2′-O-(tert-Butyldimethylsilyl)-3′-deoxy-3′,4′-didehydroguanosine-5′- phosphate (43)
  • Difluorenylmethyl-2′-O-(tert-butyldimethylsilyl)-3′-deoxy-3′,4′-didehydro-2-N- isobutyryl-guanosine-5′-phosphate (42) (420 mg, 0.474 mmol) was dissolved in ammonia (7 M in MeOH, 5 mL) and stirred at room temperature for 20 h.
  • Example 57 3′-Deoxy-3′,4′-didehydroguanosine-5′-triphosphate triethylammonium salt (44) 2′-O-(tert-Butyldimethylsilyl)-3′-deoxy-3′,4′-didehydroguanidine-5′-phosphate triethylammonium salt (43) (172 mg, 0.307 mmol) and CDI (265 mg, 1.55 mmol) were dissolved in MeCN (3.0 mL) and DMF (1.5 mL) under argon. The reaction mixture was stirred at room temperature for 2 h, then excess CDI was quenched by addition of H 2 O (50 ⁇ L, 3 mmol).
  • reaction mixture was stirred for 1 h, then passed through a Dowex ® 50WX8 Et 3 NH-form column to remove imidazole, eluting with MeCN.
  • the eluted phosphorimidazolidate intermediate was concentrated in vacuo, brought under argon, redissolved in DMF (3.0 mL) and treated with bis(tributylammonium) pyrophosphate (500 mg, 0.911 mmol).
  • the reaction mixture was stirred at room temperature for 48 h, then concentrated in vacuo.
  • Example 58 2′,5′-Bis-O-(tert-butyldimethylsilyl)-5-azacytidine (46)
  • TBDMSCl 4.81 g, 31.0 mmol
  • the reaction mixture was stirred for 48 h, then concentrated in vacuo.
  • the oil obtained was triturated with EtOAc (100 mL), then filtered through Celite ® and the filtrate was washed sequentially with sat. aq. NaHCO 3 (25 mL), water (25 mL) and brine (25 mL).
  • Example 59 2′,5′-Bis-O-(tert-butyldimethylsilyl)-4-N-(4,4′-dimethoxytrityl)-5- azacytidine (47) 2′,5′-Bis-O-(tert-butyldimethylsilyl)-5-azacytidine (46) (1.55 g, 3.28 mmol), 4,4′- dimethoxytrityl chloride (1.29 g, 3.62 mmol) and AgNO3 (613 mg, 3.60 mmol) were placed under argon, then dissolved in CH 2 Cl 2 (33 mL).
  • the resulting solution was treated with sym- collidine (0.90 mL, 6.81 mmol), upon which a precipitate formed and the reaction mixture turned orange.
  • the reaction mixture was stirred at room temperature for 90 min, after which the colour of the mixture had changed to yellow-brown.
  • the reaction mixture was filtered through a pad of Celite ® , washing with CH 2 Cl 2 (2 ⁇ 10 mL), then the filtrate was concentrated in vacuo.
  • the residue obtained was dissolved in EtOAc (125 mL) and washed sequentially with 10% aq. CuSO 4 (3 ⁇ 30 mL), 5% aq.
  • the reaction mixture was heated to 60 °C and stirred for 18 h.
  • the reaction mixture was then cooled to room temperature, diluted with EtOAc-Hex (2:1, 75 mL), and filtered through a pad of Celite ® .
  • the filtrate was concentrated in vacuo, then the resulting residue was purified by flash column chromatography on silica gel (25 ⁇ 40% EtOAc-Hex) to afford the title compound (2.32 g, 93% yield) as a colourless foam.
  • the product existed as an approximately 2:1 mixture of rotamers, designated as A (major) and B (minor), respectively.
  • Example 62 2′-O-tert-Butyldimethylsilyl-3′-deoxy-3′,4′-didehydro-5- azacytosine (50) 1-(2′-O-(tert-Butyldimethylsilyl)-3′-iodo- ⁇ -D-xylofuranosyl)-5-azacytosine (498mg, 1.06 ⁇ mmol) and DABCO (420 mg, 3.71mg) were suspended in PhMe (21 ⁇ mL) and heated at 75 ⁇ °C for 18 ⁇ h.
  • the reaction mixture was cooled to room temperature and concentrated in vacuo, then the solid residue was adsorbed onto silica gel by concentration from a solution in CH 2 Cl 2 -MeOH.
  • the silica dry load thus obtained was subjected to flash column chromatography on silica gel (7 ⁇ 20% solvent B-EtOAc, solvent B: 20% conc NH 4 OH in MeOH) to afford the title compound (224mg, 62% yield) as a colourless solid.
  • Example 63 3′-Deoxy-3′,4′-didehydro-5-azacytosine (51) To a solution of 2′-O-(tert-butyldimethylsilyl-3′-deoxy-3′,4′-didehydro-5-azacytosine (50) (51 mg, 0.15 mmol) in MeOH (1.5 mL) was added NH 4 F (29 mg, 0.76 mmol) and the reaction mixture was heated to 60 °C for 1 h. The reaction was then charged with additional NH 4 F (10 g, 0.27 mmol) and stirred at 60 °C for 1 h further.
  • Example 64 4-N-Benzoyl-2′,5′-bis-O-(tert-butyldimethylsilyl)-5-fluorocytidine (53)
  • 4-N-benzoyl-5-fluorocytidine (52) (3.31 g, 9.06 mmol) in pyridine (20 mL) at room temperature
  • TBDMSCl 4.22 g, 27.2 mmol
  • the reaction was stirred for 72 h after which the volatiles were removed in vacuo.
  • the crude white solid was dissolved in EtOAc and washed with water (2 ⁇ 300 mL), then brine (250 mL) and was dried (MgSO 4 ), then concentrated in vacuo.
  • Example 65 1-(2′,5′-Bis-O-(tert-butyldimethylsilyl)-3′-iodo- ⁇ --threo- pentofuranosyl)-4-N-benzoyl-5-fluorocytosine (54)
  • 4-N-benzoyl-2′,5′-bis-O-(tert-butyldimethylsilyl)-5- fluorocytidine (1.60 g, 2.70 mmol) and triphenoxymethylphosphonium iodide (2.3 g, 4.1 mmol) was added DMF (11 mL) followed by pyridine (0.44 mL, 5.4 mmol).
  • Example 66 1-(2′-O-(tert-Butyldimethylsilyl)-3′-iodo- ⁇ -D-threo-pentofuranosyl)- 5-fluorouracil (55)
  • 1-(2′,5′-bis-O-(tert-butyldimethylsilyl)-3′-iodo- ⁇ -D-threo- pentofuranosyl)-4-N-benzoyl-5-fluorocytosine (930 mg, 1.32 mmol) in THF (2.8 mL) at 0 °C was added TFA-H 2 O (1:1, 0.8 mL) dropwise.
  • Example 67 2′-O-(tert-Butyldimethylsilyl)-3′-deoxy-3′,4′-didehydro-5- fluorouridine (56)
  • 1-(2′-O-(tert-Butyldimethylsilyl)-3′-iodo- ⁇ -D-threo-pentofuranosyl)-5-fluorouracil (55) (246 mg, 0.506 mmol) was dissolved in dry PhMe (8.5 mL) then heated to 75 °C and treated with DABCO (170 mg, 0.506 mmol). The reaction mixture was stirred at 75 °C for 4 h, then cooled to room temperature and concentrated in vacuo.

Abstract

L'invention concerne des composés représentés par la formule (I) ou la formule (Ia) qui sont des intermédiaires utiles dans la synthèse de médicaments antiviraux potentiels tels que le 3'-désoxy-3',4'-didéhydro-cytidine triphosphate (ddhCTP).
PCT/IB2021/061540 2020-12-10 2021-12-10 Désoxydidéhydro-nucléosides protégés WO2022123501A1 (fr)

Applications Claiming Priority (4)

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AU2020904583A AU2020904583A0 (en) 2020-12-10 Protected deoxydidehydro-nucleosides
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