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WO1992001673A1 - Synthese et utilisations de ribonucleosides et de ribonucleotides marques par spin - Google Patents

Synthese et utilisations de ribonucleosides et de ribonucleotides marques par spin Download PDF

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Publication number
WO1992001673A1
WO1992001673A1 PCT/GB1991/001146 GB9101146W WO9201673A1 WO 1992001673 A1 WO1992001673 A1 WO 1992001673A1 GB 9101146 W GB9101146 W GB 9101146W WO 9201673 A1 WO9201673 A1 WO 9201673A1
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Prior art keywords
compound
group
formula
deuterium
hydrogen
Prior art date
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Ceased
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PCT/GB1991/001146
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English (en)
Inventor
David Rostron Trentham
John Edgar Thomas Corrie
Dario Renato Alessi
Ian Patrick Trayer
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Medical Research Council
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Medical Research Council
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Publication of WO1992001673A1 publication Critical patent/WO1992001673A1/fr
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Classifications

    • 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
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/92—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with a hetero atom directly attached to the ring nitrogen atom
    • C07D211/94—Oxygen atom, e.g. piperidine N-oxide
    • 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
    • C07H19/16—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
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids

Definitions

  • This invention relates to the preparation of spin labelled compounds from novel precursors and their use as probes. More specifically, the invention provides conformationally restricted spin labelled
  • ribonucleosides and ribonucleotides which are useful, for example, in protein orientation studies.
  • the present invention aims to provide such compounds.
  • R represents a CH 3 or CD 3 group
  • R 1 represents an alkyl group
  • R 2 represents an alkyl or aryl group; and N may be either an 14 N or 15 N atom,
  • R 1 may be any alkyl group and R 2 may be any alkyl or aryl group. It will be appreciated, however, that the presence of relatively small alkyl and aryl groups is usually to be preferred.
  • X represents hydrogen, a mono-, di- or
  • Y represents a purine or pyrimidine base, with the further proviso that when R represents CD3, the methylene hydrogen atoms of the six-membered
  • piperidine ring are hydrogen or deuterium.
  • either of X or Y may be radiolabel led.
  • group X as a phosphate ester derivative examples include the 3-thiotriphosphate, the
  • the spin labelled compound (2) incorporates the skeleton of the
  • the spin labelled nucleotide and nucleoside compounds of this invention may comprise either a purine or a pyrimidine base.
  • group Y can represent adenine, guanine, cytosine, uracil, thymine or 5-methylcytosine.
  • a particularly preferred spin labelled compound of the invention has the
  • Steps i), ii) and iii) involve relatively conventional chemistry, so that suitable reactants and process conditions will be readily appreciated by those skilled in the art.
  • the initial reduction of step i) may be performed, for example, in the presence of ascorbic acid.
  • the product is acylated in step ii), for example using acetic anhydride, and then converted in step iii) to a ketal.
  • step iv) the ketal is converted to an enol ether.
  • This latter product is the desired precursor compound (1) suitable for use in the synthesis of the spin labelled
  • Step (iv) proceeds with unexpected
  • Steps i) to iv) are illustrated in Example 1 below.
  • Z is a protecting group for the 5'-hydroxyl position and which may either equate to group X (as hereinbefore defined, except when X represents hydrogen) or be capable of conversion or remo v a l to leave a group 1 in that position;
  • R, R 1 , R 2 and Y are as previously defined, with the further proviso that when R represents CD 3 , the 2'- and 3'- hydroxyl hydrogen atoms in compound (8) may be deuterium in order that all four methylene hydrogen atoms of the six-membered piperidine ring of compound (9) are deuterium; vi) either a) where group 2 equates to group X, optionally further phosphorylating the compound (9) at that position, or b) treating the compound (9) such that Z is removed, and optionally phosphorylating, to leave a group X at that position; and
  • step vi) will depend upon the nature of group Z.
  • groups of the group Z when Z does not equate with X, include acyl (R 3 CO-, where R 3 may be alkyl, alkenyl, cycloalkyl or aryl), alkoxycarbonyl (R 3 OCO-, where R 3 is as defined above), allyl or substituted benzyl, such as 2-methoxybenzyl, o-nitrobenzyl or 1-(2-nitrophenyl)ethyl.
  • Z may be a group that is retained in the final product, such as a phosphate (as in
  • step vi) has the effect of converting group Z to group X.
  • protecting group Z when protecting group Z is removed by alkaline hydrolysis (i.e. is acyl or alkoxycarbonyl), isolation of compound (9a), where X is hydrogen, is still possible because the protecting group is more susceptible to alkaline hydrolysis than the N-acyloxy group that is removed in step vii).
  • step v) comprises reacting a precursor compound (1), where R, R 1 and R 2 are each CH 3 , with adenosine 5'-monophosphate (AMP) to produce a compound of the formula:-
  • step vi) comprises pyrophosphorylating the product of step v) to produce a compound of the formula:-
  • step vii) comprises deacetylation and air
  • steps vi) and vii) the benzoyl and acetyl groups are sequentially removed and the resulting N-hydroxy compound oxidized in air to produce a compound of the formula (14):-
  • a range of spin labelled compounds for use as probes, or as a part of such probes, can be derived from the precursor compound (1) of the present invention.
  • the spin label is rigidly attached to nucleotide or nucleoside sugar residues and thereby, for example, rigidly oriented on proteins.
  • This tight coupling of the spin label avoids the previously mentioned disadvantage of mobile nucleotide labels and thus opens up the possibility of new and improved orientation or structure studies of certain proteins.
  • the use of such probes should enable betterinvestigation of how muscle cross-bridges move during contraction and relaxation, or how DNA interacts with DNA-binding proteins that control gene expression.
  • the spin label precursor compounds (1) could be used to produce other types of labelled probes based on, for example, the common ribonucleoside 5' di- and 5'-triphosphates, caged ATP compounds (i.e. photo-labile derivatives of ATP from which ATP can be readily regenerated), pyridine nucleotides (e.g. NAD + , NADH), ribonucleotide analogues and ol igonucleotides.
  • the spin label precursor compounds (1) could be used to produce other types of labelled probes based on, for example, the common ribonucleoside 5' di- and 5'-triphosphates, caged ATP compounds (i.e. photo-labile derivatives of ATP from which ATP can be readily regenerated), pyridine nucleotides (e.g. NAD + , NADH), ribonucleotide analogues and ol igonucleotides.
  • Example 1 relates to the preparation of a spin label precursor compound
  • Example 2 relates to the introduction of that molecule into AMP and further elaboration to produce a labelled compound suitable for use as a probe.
  • Example 3 relates to the
  • N-Acetoxy-2,2,6,6-tetramethyl-4-piperidone (6), 4-Oxo-2,2,6,6-tetramethyl piperidin-1-oxyl (20g, 188 mmol) was melted by gentle warming and treated with a solution of L-ascorbic acid (37.6g, 190 mmol) in water (320 ml). The solution was stirred vigorously at ambient temperature for 5 min, during which its colour changed rapidly from dark red to pale yellow. It was then diluted with saturated aqueous NaHCO 3 (800 ml) in a 5 litre conical flask and cooled in ice.
  • AMP monohydrate (free acid form) (2.2g, 6 mmol) was suspended in dry dimethylformamide (50 ml) and the solvent was removed under vacuum ( ⁇ 1 mm Hg) at a bath temperature of 35°C. The procedure was repeated a further three times in order to remove traces of water from the nucleotide, and at the end of the final cycle the residue was thoroughly pumped under vacuum to ensure complete removal of the dimethylformamide.
  • toluenesulphonic acid monohydrate (5.7g; 30 mmol) was similarly dried by repeated vacuum evaporation from anhydrous acetonitrile (4 ⁇ 50 ml), then dissolved in anhydrous acetonitrile (440 ml) together with the enol ether (1) (20g, 88 mmol).
  • This solution was adde rapidly to the dried nucleotide and the resulting suspension was stirre for 7 days at room temperature.
  • the undissolved fraction of the AMP was removed by filtration, the filtrate was diluted with 10 mM triethylammonium bicarbonate (TEAB) buffer, pH 7.4 (2 1) and the mixture was extracted with petroleum ether (3 ⁇ 400 ml).
  • TEAB triethylammonium bicarbonate
  • the AMP spiroketal (10) (120 umol) was pyrophosphorylated by known procedures 2 ' 3 and purified by ion-exchange chromatography on DEAE-cellulose as described above, using a column of void volume 150 ml, a linear gradient formed from 10 and 350 mM TEAB (each 600 ml) and a flow rate of 50 ml/h. Fractions were analysed by reverse-phase h.p.l.c. (Conditions as above). The retention time for the ATP spiroketal (11) was 1.83 min). Fractions containing pure product were combined and freed from buffer salts as above to afford the ATP spiroketal acetate (11) as its

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Saccharide Compounds (AREA)

Abstract

Composés correspondant aux formules: (1) dans laquelle R représente un groupe CH3 ou CD3; R1 représente un groupe alkyle; R2 représente un groupe alkyle ou aryle; et N peut représenter soit un atome 14N soit un atome 15N, à condition que, quand R représente CD3, les atomes de méthylène et d'hydrogène de vinyle du noyau à six éléments, soient deutérium; et (2) dans laquelle R et N sont comme définis ci-dessus, X représente hydrogène ou un groupe mono-, di- ou triphosphate, ou un dérivé d'ester de phosphate; et Y représente une base de purine ou de pyrimidine, à condition que, quand R représente CD3, les atomes d'hydrogène de méthylène du noyau de pipéridine à six éléments, soient hydrogène ou deutérium. L'invention décrit également le procédé de préparation de ces composés. Les composés de nucléosides et de nucléotides marqués par spin, trouvent leur utilisation comme sondes s'appliquant, par exemple, à des études de la structure et de l'orientation des protéines.
PCT/GB1991/001146 1990-07-17 1991-07-11 Synthese et utilisations de ribonucleosides et de ribonucleotides marques par spin Ceased WO1992001673A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9015684.5 1990-07-17
GB909015684A GB9015684D0 (en) 1990-07-17 1990-07-17 Synthesis and uses of spin labelled ribonucleosides and ribonucleotides

Publications (1)

Publication Number Publication Date
WO1992001673A1 true WO1992001673A1 (fr) 1992-02-06

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PCT/GB1991/001146 Ceased WO1992001673A1 (fr) 1990-07-17 1991-07-11 Synthese et utilisations de ribonucleosides et de ribonucleotides marques par spin

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AU (1) AU8193991A (fr)
GB (1) GB9015684D0 (fr)
WO (1) WO1992001673A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7018985B1 (en) 2000-08-21 2006-03-28 Inspire Pharmaceuticals, Inc. Composition and method for inhibiting platelet aggregation
US7132408B2 (en) 2000-08-21 2006-11-07 Inspire Pharmaceuticals, Inc. Composition and method for inhibiting platelet aggregation
US7452870B2 (en) 2000-08-21 2008-11-18 Inspire Pharmaceuticals, Inc. Drug-eluting stents coated with P2Y12 receptor antagonist compound
US11566038B2 (en) 2020-11-11 2023-01-31 deutraMed Solutions Ltd. Deuterium-stabilised ribonucleic acid (RNA) molecules displaying increased resistance to thermal and enzymatic hydrolysis, aqueous compositions comprising stabilised RNA molecules and methods for making same

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2235103A1 (en) * 1973-06-29 1975-01-24 Commissariat Energie Atomique Marked nitroxide derivatives of saccharides - by reaction of acid nitroxide with halogenated saccharides
EP0133674A1 (fr) * 1983-08-03 1985-03-06 Schering Aktiengesellschaft Composés nitroxylés, procédé pour leur préparation et agents diagnostiques les contenant

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2235103A1 (en) * 1973-06-29 1975-01-24 Commissariat Energie Atomique Marked nitroxide derivatives of saccharides - by reaction of acid nitroxide with halogenated saccharides
EP0133674A1 (fr) * 1983-08-03 1985-03-06 Schering Aktiengesellschaft Composés nitroxylés, procédé pour leur préparation et agents diagnostiques les contenant

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7018985B1 (en) 2000-08-21 2006-03-28 Inspire Pharmaceuticals, Inc. Composition and method for inhibiting platelet aggregation
US7101860B2 (en) 2000-08-21 2006-09-05 Inspire Pharmaceuticals, Inc. Composition and method for inhibiting platelet aggregation
US7132408B2 (en) 2000-08-21 2006-11-07 Inspire Pharmaceuticals, Inc. Composition and method for inhibiting platelet aggregation
US7452870B2 (en) 2000-08-21 2008-11-18 Inspire Pharmaceuticals, Inc. Drug-eluting stents coated with P2Y12 receptor antagonist compound
US7618949B2 (en) 2000-08-21 2009-11-17 Inspire Pharmaceuticals, Inc. Drug-eluting stents coated with P2Y12 receptor antagonist compound
US11566038B2 (en) 2020-11-11 2023-01-31 deutraMed Solutions Ltd. Deuterium-stabilised ribonucleic acid (RNA) molecules displaying increased resistance to thermal and enzymatic hydrolysis, aqueous compositions comprising stabilised RNA molecules and methods for making same
US11780869B2 (en) 2020-11-11 2023-10-10 deutraMed Solutions Ltd. Deuterium-stabilised ribonucleic acid (RNA) molecules displaying increased resistance to thermal and enzymatic hydrolysis, aqueous compositions comprising stabilised RNA molecules and methods for making same

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GB9015684D0 (en) 1990-09-05
AU8193991A (en) 1992-02-18

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