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WO1990003370A1 - DERIVES DE PYRAZOLO[3,4-d]PYRIMIDINE - Google Patents

DERIVES DE PYRAZOLO[3,4-d]PYRIMIDINE Download PDF

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Publication number
WO1990003370A1
WO1990003370A1 PCT/US1989/004184 US8904184W WO9003370A1 WO 1990003370 A1 WO1990003370 A1 WO 1990003370A1 US 8904184 W US8904184 W US 8904184W WO 9003370 A1 WO9003370 A1 WO 9003370A1
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Prior art keywords
hydrogen
independently
group
sugar moiety
pyrazolo
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PCT/US1989/004184
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English (en)
Inventor
Charles R. Petrie
Rich B. Meyer
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Microprobe Corporation
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Publication of WO1990003370A1 publication Critical patent/WO1990003370A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • 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
    • C07H19/06—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
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • This invention relates to derivatives of pyrazolo- [3,4-d]pyrimidine and to the use of these compounds for the preparation of oligonucleotides.
  • Oligonucleotides are useful as diagnostic 0 probes for the detection of "target" DNA or RNA sequences.
  • probes were made up of sequences of nucleic acid containing purine, pyrimidine or 7-deazapurine nucleotide bases (U.S. Patent 4,711,955).
  • the method for attaching chemical moieties 5 to these bases has been via an acetoxy- mercuration reaction, which introduces covalently bound mercury atoms into the 5-position of the pyrimidine ring, the C-8 position of the purine ring or the C-7 position of a 7-deazapurine ring (Dale et al., Proc. Natl. Acad. Q Sci. USA 7jD:2238 (1973); Dale et al., Biochemistry
  • oligonucleotide probes The sugar component of oligonucleotide probes has been, until the present, composed of nucleic acid containing ribose or deoxyribose or, in one case, Q natural 0-arabinose (patent publication EP 227,459).
  • a novel class of nucleotide base the 3,4-disubst ituted and 3, ,6-trisubstituted pyrazolo[3,4-d]pyrimidines, has now been found which offers several advantages over the prior art.
  • the de 5 novo chemical synthesis of the pyrazolopyrimidine and the resulting nucleotide allows for the incorporation of a wide range of functional groups in a variety of different positions on the nucleotide base and for the use of different sugar moieties.
  • adenine, guanine and hypoxanthine analogs are obtained from a single nucleoside precursor. Additionally, the synthesis does not require the use of toxic heavy metals or expensive catalysts.
  • This invention is directed to novel 3,4-disubsti- tuted and 3 ,4,6-trisubstituted pyrazolo[3,4-d]pyrimidines and to the use of these compounds in the preparation of oligonucleotides.
  • the invention is also directed to nucleosides and mono- and oligonucleotides comprising at least one of these pyrazolopyrimidines, and to the use of the resulting novel oligonucleotides for diagnostic purposes.
  • pyrazolopyrimidines of the present invention are of the following formula (I) :
  • • l is hydrogen, or a sugar moiety optionally substituted at its 3• or its 5' position with monophosphate, diphosphate, triphosphate, or a reactive group suitable for nucleotide bond formation; provided that when R 3 is hydrogen, then R*-_ cannot be hydrogen;
  • R 3 is hydrogen or the group -w-(X) n -A; each of and X is independently a chemical linker arm;
  • A is an intercalator, an electrophilic crosslinker, a photoactivatable crosslinker, or a reporter group; each of R 4 and Rg is independently H, OH, SR,
  • R is H or C 1 personally 6 alkyl; n is zero or one; and t is zero to twelve.
  • the invention also provides novel nucleosides and nucleotides comprising at least one of the above pyrazolo- pyrimidines.
  • Nucleotides of this invention and oligo- and polynucleotides into which the nucleotides have been incorporated may be used as probes.
  • the novel oligo- and polynucleotides are useful in the identification, isolation, localization and/or detection of complementary nucleic acid sequences of interest in cell-free or cellular systems. Therefore, the invention further provides a method for identifying target nucleic acid sequences, which method comprises utilizing an oligo- or polynucleotide probe comprising at least one of a labeled pyrazolo[3,4-d]pyrimi- dine of the present invention.
  • This invention provides novel substituted pyrazolo[3,4-d]pyrimidines which are used as the nucleotide base in preparing nucleosides and nucleotides, rather than the natural purine or pyrimidine bases or the deazapurine analogs.
  • the synthesis of 3,4-disubstituted and 3,4,6-tri- substituted pyrazolo[3,4-d]pyrimidine nucleosides and their use as reagents for incorporation into nucleic acids either enzymatically or via chemical synthesis offers several advantages over current procedures.
  • the de novo chemical synthesis of the nucleotide allows for the incorporation of a wide range of functional groups (e.g., NH 2 , SH, OH, halogen, COOH, CN, CONH 2 ) and the use of different sugar moieties.
  • adenine, guanine, and hypoxanthine analogs are obtained from a single nucleoside precursor. And, the synthesis does not require the use of toxic heavy metals or expensive catalysts.
  • pyrazolopyrimidines of the present invention are of the following formula (I) :
  • Rl is hydrogen, or a sugar moiety optionally substituted at its 3* or its 5* position with monophosphate, diphosphate, triphosphate, or a reactive group suitable for nucleotide bond formation; provided that when 3 is hydrogen, then R ⁇ cannot be hydrogen;
  • R 3 is hydrogen or the group - -(X) n -A; each of W and X is independently a chemical linker arm;
  • A is an intercalator, an electrophilic crosslinker or a reporter group; each of R4 and is independently H, OH, SR,
  • NH 2 or NH (C H 2 ) t NH 2' R is H or C ⁇ .galkyl; n is zero or one; and t is zero to twelve.
  • the sugar moiety is selected from those useful as a component of a nucleotide. Such a moiety may be selected from, for example, ribose, deoxyribose, glucose, arabinose, xylose and lyxose. The sugar moiety is preferably ribose, deoxyribose or arabinose and embraces either anomer, ⁇ or ⁇ .
  • a reactive group suitable for internucleotide bond formation is one which is useful during chain extension in the synthesis of an oligonucleotide.
  • Reactive groups particularly useful in the present invention are those containing phosphorus.
  • Phosphorus-containing groups suitable for internucleotide bond formation are preferably alkyl phosphorchloridites or alkylphosphora idites.
  • activated phosphate diesters may- be employed for this purpose.
  • a chemical linker arm ( alone or together with X) serves to make the functional group (A) more able to readily interact with antibodies, detector proteins, or chemical reagents, for example.
  • the linkage holds the functional group away from the base when the base is paired with another within the double-stranded complex.
  • Linker arms may include alkylene groups of 1 to 12 carbon atoms, alkenylene groups of 2 to 12 carbon atoms and 1 or 2 olefinic bonds, alkynylene groups of 2 to 12 carbon atoms and 1 or 2 acetylenic bonds, or such groups substituted at a terminal point with nucleophilic groups such as oxy, thio, amino or chemically blocked derivatives thereof (e.g., trifluoroacetamido) .
  • Such functionalities including aliphatic or aromatic amines, exhibit nucleophilic properties and are capable of serving as a point of attachment of the functional group (A) .
  • the linker arm moiety ( alone or together with X) is preferably of at least three atoms and more preferably of at least five atoms.
  • the terminal nucleophilic group is preferably amino or chemically blocked derivatives thereof (e.g., trifluoroacetamido) .
  • Intercalators are planar aromatic bi-, tri- or polycyclic molecules whose dimensions are roughly the same as those of a purine-pyrimidine pair and which can insert themselves between two adjacent base pairs in a double stranded helix of nucleic acid.
  • Intercalators have been used to cause frameshift mutations in DNA and RNA. It has also recently been shown that when an intercalator is covalently bound via a linker arm ("tethered") to the end of a deoxyoligonucleotide, it increases the binding affinity of the oligonucleotide for its target sequence, resulting in strongly enhanced stability of the complementary sequence complex. At least some of the tethered intercalators also protect the oligonucleotide against exonucleases, but not against endonucleases. See, Sun et al.. Nucleic Acids Res. 15:6149-6158 (1987); Le Doan et al. , Nucleic Acids Res. 15:7749-7760 (1987) .
  • tetherable intercalating agents examples include oxazolopyridocarbazole, acridine orange, proflavine, acriflavine and derivatives of proflavine and acridine such as 3-azido-6-(3-bromopropylamino)acridine, 3-amino-6-(3-bromopentylamino)acridine, and
  • Oligonucleotides capable of crosslinking to the complementary sequence of target nucleic acids are valuable in chemotherapy because they increase the efficiency of inhibition of mRNA translation or gene expression control by covalent attachment of the oligonucleotide to the target sequence. This can be accomplished by crosslinking agents being covalently attached to the oligonucleotide, which can then be chemically activated to form crosslinkages which can then induce chain breaks in the target complementary sequence, thus inducing irreversible damage in the sequence.
  • electrophilic crosslinking moieties include alpha-halocarbonyl compounds, 2-chloroethylamines and epoxides.
  • oligonucleotides comprising at least one nucleotide base moiety of the invention are utilized as a probe in nucleic acid assays
  • a label is attached to detect the presence of hybrid polynucleotides.
  • Such labels act as reporter groups and act as means for detecting duplex formation between the target nucleotides and their complementary oligonucleotide probes.
  • a reporter group as used herein is a group which has a physical or chemical characteristic which can be measured or detected. Detectability may be provided by such characteristics as color change, luminescence, fluorescence, or radioactivity; or it may be provided by the ability of the reporter group to serve as a ligand recognition site.
  • Probes may be labeled by any one of several methods typically used in the art. A common method of detection is the use of autoradiography with 3 H, 125 I, 35 S, 1 C, or 32 P labeled probes or the like. Other reporter groups include ligands which bind to antibodies labeled with fluorophores , chemiluminescent agents, and enzymes. Alternatively, probes can be conjugated directly with labels such as fluorophores, chemiluminescent agents, enzymes and enzyme substrates. Alternatively, the same components may be indirectly bonded through a ligand-antiligand complex, such as antibodies reactive with a ligand conjugated with label. The choice of label depends on sensitivity required, ease of conjugation with the probe, stability requirements, and available instrumentation.
  • Radioactive probes are typically made using commercially available nucleotides containing the desired radioactive isotope.
  • the radioactive nucleotides can be incorporated into probes, for example, by using DNA synthesizers, by nick-translation, by tailing of radioactive bases to the 3• end of probes with terminal transferase, by 8 copying M13 plasmids having specific inserts with the Klenow fragment of DNA polymerase in the presence of radioactive dNTP's, or by transcribing RNA from templates using RNA polymerase in the presence of radioactive rNTP's.
  • Non-radioactive probes can be labeled directly with a signal (e.g., fluorophore, chemiluminescent agent or enzyme) or labeled indirectly by conjugation with a ligand.
  • a ligand molecule is covalently bound to the probe. This ligand then binds to a receptor molecule which is either inherently detectable or covalently bound to a detectable signal, such as an enzyme or photoreactive compound.
  • Ligands and antiligands may be varied widely. Where a ligand has a natural "antiligand", namely ligands such as biotin, thyroxine, and cortisol, it can be used in conjunction with its labeled, naturally occurring antiligand.
  • any haptenic or antigenic compound can be used in combination with a suitably labeled antibody.
  • a preferred labeling method utilizes biotin-labeled analogs of oligonucleotides, as disclosed in P. Langer et al., Proc. Natl. Acad. Sci. USA 78.:6633-6637 (1981), which is incorporated herein by reference.
  • Enzymes of interest as reporter groups will primarily be hydrolases, particularly phosphatases, esterases, ureases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
  • Chemiluminescers include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
  • hybridization conditions are not critical and will vary in accordance with the investigators preferences and needs.
  • Various hybridization solutions may be employed, comprising from about 20 to 60% volume, preferably 30%, of a polar organic solvent.
  • a common hybridization solution employs about 30-60% v/v formamide, about 0.5 to 1M sodium chloride, about 0.05 to 0.1M buffers, such as sodium citrate, Tris HC1, PIPES or HEPES, about 0.05 to 0.5% detergent, such as sodium dodecylsulfate, and between 1-10 mM EDTA, 0.01 to 5% ficoll (about 300-500 kilodaltons) , 0.1 to 5% polyvinylpyrrolidone (about 250-500 kdal) , and 0.01 to 10% bovine serum albumin.
  • unlabelled carrier nucleic acids from about 0.1 to 5 mg/ml, e. ⁇ .. partially fragmented calf thymus or salmon sperm, DNA, and/or partially fragmented yeast RNA and optionally from about 0.5 to 2% wt./vol. glycine.
  • Other additives may also be included, such as volume exclusion agents which include a variety of polar water-soluble or swellable agents, such as anionic polyacrylate or polymethylacrylate, and charged saccharidic polymers, such as dextran sulfate.
  • hybridization technique is not essential to the invention.
  • Hybridization techniques are generally described in Nucleic Acid Hybridization, A Practical Approach, Ed. Hames, B.D. and Higgins, S.J., IRL Press, 1985; Gall and Pardue (1969), Proc. Natl. Acad. Sci., U.S.A., 63:378-383, and John, Burnsteil and Jones (1969) Nature, 223:582-587.
  • improvements are made in hybridization techniques, they can readily be applied.
  • the amount of labelled probe which is present in the hybridization solution may vary widely. Generally, substantial excesses of probe over the stoichio etric amount of the target nucleic acid will be employed to enhance the rate of binding of the probe to the target DNA.
  • degrees of stringency of hybridization can be employed. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for the formation of a stable duplex.
  • the degree of stringency can be controlled by temperature, ionic strength, the inclusion of polar organic solvents, and the like. For example, temperatures employed will normally be in the range of about 20 to 80 C, usually 25 to 75 C. For probes of 15- 50 nucleotides in 50% formamide, the optimal temperature range can vary from 22-65"C. With routine exper ⁇ imentation, one can define conditions which permit satisfactory hybridization at room temperature.
  • the stringency of hybridization is also conveniently varied by changing the ionic strength and polarity of the reactant solution through manipulation of the concentration of formamide within the range of 20% to 50%.
  • the glass, plastic, or filter support to which the probe-target hybrid is attached is introduced into a wash solution typically containing similar reagents (e.g., sodium chloride, buffers, organic solvents and detergent) , as provided in the hybridization solution.
  • reagents e.g., sodium chloride, buffers, organic solvents and detergent
  • the time period for which the support is maintained in the wash solutions may vary from minutes to several hours or more.
  • Either the hybridization or the wash medium can be stringent. After appropriate stringent washing, the correct hybridization complex may now be detected in accordance with the nature of the label.
  • the probe may be conjugated directly with the label.
  • the label is radioactive
  • the support surface with associated hybridization complex substrate is exposed to X-ray film.
  • the label is fluorescent
  • the sample is detected by first irradiating it with light of a particular wavelength. The sample absorbs this light and then emits light of a different wavelength which is picked up by a detector (Physical Biochemistry, Freifelder, D. , W.H. Freeman & Co., 1982, pp. 537-542).
  • the label is an enzyme
  • the sample is detected by incubation an appropriate substrate for the enzyme.
  • the signal generated may be a colored precipitate, a colored or fluorescent soluble material, or photons generated by bioluminescence or chemi-luminescence.
  • the preferred label for dipstick assays generates a colored precipitate to indicate a positive reading. For example, alkaline phosphatase will dephosphorylate indoxyl phosphate which then will participate in a reduction reaction to convert tetrazolium salts to highly colored and insoluble formazans.
  • Detection of a hybridization complex may require the binding of a signal generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal.
  • the binding of the signal generation complex is also readily amenable to accelerations by exposure to ultrasonic energy.
  • the label may also allow indirect detection of the hybridization complex.
  • the label is a hapten or antigen
  • the sample can be detected by using antibodies.
  • a signal is generated by attaching fluorescent or enzyme molecules to the antibodies or in some cases, by attachment to a radioactive label.
  • malononitrile (III) is treated with acyl halide (II) in the presence of a base to yield acylmalononitrile (IV) , which is subsequently methylated with dimethyl sulfate or diazomethane, for example, to give the substituted methoxymethylenemalononitrile (V) .
  • This compound is then reacted with hydrazine hydrate in boiling alcohol to give the
  • the carboxamide (VII) may alternatively be prepared by treating cyanoacetamide (XII) with acid halide (II) to give the acylcyanoacetamide (XIII) , which is then methylated and the resulting methoxy compound (XIV) is reacted with hydrazine hydrate. 13
  • Syntheses of pyrazolo[3,4-d]pyrimidines are accomplished from the two pyrazole intermediates, VI and VII.
  • 3,4-disubstituted pyrazolo[3,4-d]pyrimidines (VIII and X) are obtained by treating the corresponding VI and VII with boiling formamide.
  • VI may be treated with dialkoxymethyl ester of a carboxylic acid, at room temperature or above room temperature, and then with ammonia to give VIII
  • VII may be treated with dialkoxymethyl ester of a carboxylic acid (without subsequent ammonia treatment) , at room temperature or above room temperature, to give X.
  • VI and VII may be treated with an alkyl xanthate salt such as potassium ethyl xanthate and with alkyl halide such as methyl iodide, at a temperature above room temperature, followed by oxidation by a peroxide such as m-chloroperbenzoic acid (MCPBA) and subsequent treatment with ammonia to give IX and XI, respectively, where Rg is NH 2 . 14
  • the compounds of formula I may be recovered from the reaction mixture in which they are formed by established procedures.
  • the sugar may be either added to the 1-position of the pyrazole VI or VII prior to further treatment or added to the 1-position of the pyrazolo[3,4-d]pyrimidine VIII, IX, X or XI.
  • the pyrazole or pyrazolopyrimidine is treated with sodium hydride and then with the glycosyl halide of the blocked sugar.
  • Oligonucleotides of the present invention comprise at least one and up to all of their nucleotides from the substituted pyrazolo[3,4-d]pyrimidines of formula I.
  • oligonucleotides To prepare oligonucleotides, protective groups are introduced onto the nucleosides of formula I and the nucleosides are activated for use in the synthesis of oligonucleotides.
  • the conversion to protected, activated forms follows the procedures as described for 2--deoxynucleosides in detail in several reviews. See, Sonveaux, Bioorganic Chemistry l ⁇ :274-325 (1986); Jones, in Oligonucleotide Synthesis, a Practical Approach , M.J. Gait, Ed., IRL Press, p. 23-34 (1984).
  • the activated nucleotides are incorporated into oligonucleotides in a manner analogous to that for DNA and RNA nucleotides, in that the correct nucleotides will be sequentially linked to form a chain of nucleotides which is complementary to a sequence of nucleotides in target DNA or RNA.
  • the nucleotides may be incorporated either enzymatically or via chemical synthesis.
  • the activated nucleotides may substitute for an adenine using the nick translation procedure, as described by Langer et al., Proc. Natl. Acad. Sci. USA 2 :6633-6637 (1981), the disclosure of which is incorporated herein by reference.
  • the activated nucleotides may be used directly on an automated DNA synthesizer according to the procedures and instructions of the particular synthesizer employed.
  • the oligonucleotides may be prepared on the synthesizer using the standard commercial phosphoramidite or H-phosphonate chemistries.
  • An oligonucleotide probe according to the invention includes at least one labeled substituted pyrazolo[3,4-d]pyrimidine nucleotide moiety of formula I.
  • the amount of labeled probe present in the hybridization solution may vary widely, depending upon the nature of the label, the amount of the labeled probe that can reasonably bind to the cellular target nucleic acid, and the precise stringency of the hybridization medium and/or wash medium. Generally, substantial probe excesses over the stoichiometric amount of the target will be employed to enhance the rate of binding of the probe to the target nucleic acids.
  • the invention is also directed to a method for identifying target nucleic acid sequences, which method comprises utilizing an oligonucleotide probe including at least one labeled substituted pyrazolo[3,4-d]pyrimidine nucleotide moiety of formula I.
  • the method comprises the steps of: 16
  • An assay for identifying target nucleic acid sequences utilizing an oligonucleotide probe including at least one labeled substituted pyrazolo[3,4-d]pyrimidine nucleotide moiety of formula I and comprising the above method is contemplated for carrying out the invention.
  • Such an assay may be provided in kit form.
  • a typical kit will include a probe reagent component comprising an oligonucletide including at least one labeled pyrazolo[3,4-d]pyrimidine, the oligonucleotide having a sequence complementary to that of the target nucleic acids; a denaturation reagent for converting double-stranded nucleic acid to single-stranded nucleic acid; and a hybridization reaction mixture.
  • the kit can also include a signal-generating system, such as an enzyme for example, and a substrate for the system.
  • 6-Aminocaproic acid (26 g, 0.2 mmole) was dissolved in dichloromethane (200 mL) by the addition of triethylamine (100 L) . Trityl chloride (120 g, 0.45 mmole) was added and the solution stirred for 36h. The resulting solution was extracted with IN HC1 and the organic layer evaporated to dryness. The residue was suspended in 2-propanol/lN NaOH (300 mL/100 mL) and refluxed for 3h. The solution was evaporated to a thick syrup and added to dichloromethane (500 mL) . Water was added and acidified. The phases were separated, the organic layer dried over sodium sulfate, and evaporated to dryness. The residue was suspended in hot 2-propanol, cooled, and filtered to give 43.5 g (58%) of 6-(tritylamino)caproic acid, useful as an intermediate compound.
  • Example 4 (3.5 g, 8 mmole) was treated with sodium hydride and stirred for 30 min at 0-4*C.
  • EXAMPLE 6 l-(2-Deoxy- -£-e ⁇ _ythropentofuranosyl)-3-[5-(tritylamin- o)pentyl]pyrazolo[3,4-d]pyrimidin-4-amine.
  • EXAMPLE 7 l-(2-Deoxy- -D-erythro-pentofuranosyl)-3-[5-(tritylami ⁇ no) ⁇ entyl]pyrazolo[3,4-d]pyrimidin-4-amine 5•-monophosphate.
  • Example 8 The monophosphate of Example 8 (80 mg, ca. 0.1 mmole) was dissolved in DMF with the addition of triethylamine (14 ⁇ L) . Carbonyldiimidazole (81 mg, 0.5 mmole) was added and the solution stirred at RT for 18 hr. The solution was treated with methanol (40 ⁇ L) , and after stirring for 30 min tributylammonium pyrophosphate (0.5 g in 0.5 mL DMF) was added. After stirring for 24 hr another aliquot of tributylammonium pyrophosphate was added and the solution stirred overnight. The reaction mixture was evaporated to dryness and chromatographed following the procedure in Example 8.
  • the triphosphate of Example 9 was incorporated into pHPV-16 using the nick tanslation protocol of Langer et al.
  • the probe prepared with the triphosphate of Example 9 was compared with probe prepared using commercially available bio-11-dUTP
  • Bio-12-dAPPTP (comprising the triphosphate of Example 9) and nucleotide mix A.
  • Nucleic acid was isolated by ethanol precipitation and hybridized to pHPV-16 slotted onto 5 nitrocellulose.
  • the hybridized biotinylated probe was visualized by a streptavidin - alkaline phosphatase conjugate with BCIP/NBT substrate.
  • Probe prepared using either biotinylated nucleotide gave identical signals.
  • the probes were also tested in an in situ o format on cervical smears and showed no qualitative differences in signal and background.
  • the carboxamide from Example 11 is reacted with potassium ethyl xanthate and ethanol at an elevated temperature to give the potassium salt of 4-hydroxypyrazolo- [3,4-d]pyrimidine-6-thiol. This salt is then reacted with iodomethane to give 4-hydroxy-6-methylthio-3-[(5-trityl- amino)pentyl]pyrazolo[3,4-d]pyrimidine.
  • Example 12 Following the procedure of Example 5, the pyrazolopyrimidine of Example 12 is treated with sodium hydride and reacted with l-chloro-l,2-dideoxy-3,5-di-o- toluoylribofuranose. The resulting compound is reacted with MCPBA and with methanolic ammonia, and the toluoyl protecting groups are removed to give the product.
  • EXAMPLE 14 l-(2-Deoxy-0-JD-erythro-pentofuranosyl)-4-hydroxy-3-[5- (6-biotinamido)hexamidopentyl]pyrazolo[3,4-d]pyrimidin- -6- amine 5'-monophosphate.
  • EXAMPLE 15 l-(2-Deoxy-/9-D-erythropentofuranosyl)-4-hydroxy-3-[5-(- 6-biotinamido)hexamidopentyl]pyrazolo[3,4-d]pyrimidin- 6-amine 5•-triphosphate.
  • Example 14 Following the procedure of Example 9, the 5'-mono- phosphate of Example 14 is treated with carbonyldii idazole and then reacted with tributylammonium pyrophosphate to give the corresponding 5•-triphosphate.
  • EXAMPLE 16 l-(2-Deoxy-9-D-erythropentofuranosyl)-3-[5-(tritylamin- o)pentyl]pyrazolo[3,4-d]pyrimidine-4-benzoylamine.
  • Example 16 Following the procedure of Example 8, the benzoyl- amine of Example 16 is treated with palladium hydroxide on carbon and then with trifluoroacetic anhydride to give l-(2-deoxy- -D-erythropentofuranosyl) -3-[5-(trifluoroacetamido)pentyl]pyrazolo[3,4-d] pyrimidine-4-benzoylamine.
  • Example 17 The compound of Example 17 is reacted with di ethoxytrityl chloride and pyridine to give the corresponding 5*-dimethoxytrityl compound. This compound is then reacted with cyanoethyl chloro-N,N-diisopropylphosphor- amidite (according to the method of Sinha et al.. Nucleic Acids Res. 12:4539 (1984)) to give the 3'-o-activated nucleoside.
  • compositions of the present invention are novel and are useful for diagnostic purposes.

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Abstract

L'invention concerne de nouvelles pyrazolo[3,4-d]pyrimidines, 3,4-bisubstituées et 3,4,6-trisubstituées ainsi que l'utilisation de ces composés dans la préparation d'oligonucléotides. L'invention concerne également des nucléosides ainsi que des mono- et oligonucléotides comprenant au moins une de ces pyrazolopyrimidines, et l'utilisation des nouveaux oligonucléotides obtenus à des fins de diagnostic. Plus particulièrement, les pyrazolopyrimidines de cette invention ont la formule (I), dans laquelle R1 représente hydrogène, ou une fraction de sucre facultativement remplacée en position 3' ou 5' par monophosphate, diphosphate, triphosphate, ou un groupe réactif adéquat pour la formation d'une liaison de nucléotide, à condition que lorsque R3 représente hydrogène, alors R1 ne peut pas être de l'hydrogène; R3 représente hydrogène ou le groupe -W-(X)n-A; W et X représentent chacun indépendamment un bras de liaison chimique; A représente un élément intercalaire, un agent de réticulation électrophile ou un groupe rapporteur; R4 et R6 représentent chacun indépendamment H, OH, SR, NH2, ou NH(CH2)tNH2, R représente H ou alkyle comportant 1 à 6 atomes de carbone; n représente zéro ou un; et t représente zéro ou douze. Les nouveaux oligo- et polynucléotides sont utiles pour l'identification, l'isolation, la localisation et/ou la détection de séquences d'acide nucléique complémentaires d'intérêt dans des systèmes sans cellule ou cellulaires. Par conséquent l'invention concerne également un procédé d'identification de séquences d'acide nucléique cible, ledit procédé consistant à utiliser une sonde d'oligo- ou polynucléotides comprenant au moins une des pyrazolo[3,4-d]pyrimidines marquées de l'invention.
PCT/US1989/004184 1988-09-28 1989-09-26 DERIVES DE PYRAZOLO[3,4-d]PYRIMIDINE WO1990003370A1 (fr)

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US6312925B1 (en) 1997-05-08 2001-11-06 Epoch Pharmaceuticals, Inc. Methods and compositions to facilitate D-loop formation by oligonucleotides
US6312894B1 (en) 1995-04-03 2001-11-06 Epoch Pharmaceuticals, Inc. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
WO2002012263A1 (fr) * 2000-08-03 2002-02-14 Roche Diagnostics Gmbh Composes de fixation d'acides nucleiques contenant des analogues pyrazolo[3,4-d]pyrimidine de purine 2,6-diamine et leurs utilisations
US6844433B2 (en) 1998-07-31 2005-01-18 Affymetrix, Inc. Nucleic acid labeling compounds
US6864059B2 (en) 1996-01-23 2005-03-08 Affymetrix, Inc. Biotin containing C-glycoside nucleic acid labeling compounds
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US7763624B2 (en) 2005-08-22 2010-07-27 Amgen Inc. Substituted pyrazolo[3,4-d]pyrimidines as ACK-1 and LCK inhibitors
US7794945B2 (en) 1995-04-03 2010-09-14 Elitech Holding B.V. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
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US6136601A (en) * 1991-08-21 2000-10-24 Epoch Pharmaceuticals, Inc. Targeted mutagenesis in living cells using modified oligonucleotides
US5594121A (en) * 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
US6498241B1 (en) 1991-12-09 2002-12-24 Roche Diagnostics Gmbh 2-deoxy-isoguanosines isosteric analogues and isoguanosine derivatives as well as their synthesis
WO1993012130A1 (fr) * 1991-12-09 1993-06-24 Boehringer Mannheim Gmbh 2'-desoxy-isoguanosines, analogues isosteres et derives d'isoguanosine, ainsi que leur utilisation
US6147199A (en) * 1991-12-09 2000-11-14 Boehringer Mannheim 2-deoxy-isoguanosines isoteric analogues and isoguanosine containing oligonucleotides
WO1994024144A2 (fr) * 1993-04-19 1994-10-27 Gilead Sciences, Inc. Formation a helice triple et double a l'aide d'oligomeres contenant des purines modifiees
WO1994024144A3 (fr) * 1993-04-19 1995-03-16 Gilead Sciences Inc Formation a helice triple et double a l'aide d'oligomeres contenant des purines modifiees
AU691814B2 (en) * 1993-06-18 1998-05-28 Nycomed Imaging As Factor VII-derived peptides
US6312894B1 (en) 1995-04-03 2001-11-06 Epoch Pharmaceuticals, Inc. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
US7556923B1 (en) 1995-04-03 2009-07-07 Epoch Biosciences, Inc. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
US7794945B2 (en) 1995-04-03 2010-09-14 Elitech Holding B.V. Hybridization and mismatch discrimination using oligonucleotides conjugated to minor groove binders
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US5935830A (en) * 1995-06-07 1999-08-10 Epoch Pharmaceuticals, Inc. Targeted mutagenesis in living cells using modified oligonucleotides
US7291463B2 (en) 1996-01-23 2007-11-06 Affymetrix, Inc. Nucleic acid labeling compounds
US7423143B2 (en) 1996-01-23 2008-09-09 Affymetrix. Inc. Nucleic acid labeling compounds
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US6965020B2 (en) 1996-01-23 2005-11-15 Affymetrix, Inc. Nucleic acid labeling compounds
US6864059B2 (en) 1996-01-23 2005-03-08 Affymetrix, Inc. Biotin containing C-glycoside nucleic acid labeling compounds
US6143877A (en) * 1997-04-30 2000-11-07 Epoch Pharmaceuticals, Inc. Oligonucleotides including pyrazolo[3,4-D]pyrimidine bases, bound in double stranded nucleic acids
US6312925B1 (en) 1997-05-08 2001-11-06 Epoch Pharmaceuticals, Inc. Methods and compositions to facilitate D-loop formation by oligonucleotides
US6949367B1 (en) 1998-04-03 2005-09-27 Epoch Pharmaceuticals, Inc. Modified oligonucleotides for mismatch discrimination
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JP2002510507A (ja) * 1998-04-03 2002-04-09 エポック・バイオサイエンシーズ・インコーポレイテッド ハイブリダイゼーション及び不一致識別のための、ピラゾロ［３，４−ｄ］ピリミジン含有オリゴヌクレオチド
WO1999051775A1 (fr) * 1998-04-03 1999-10-14 Epoch Pharmaceuticals, Inc. Oligonucleotides contenant des pyrazolo [3,4-d] pyrimidines et servant a l'hybridation et a la discrimination du defaut de complementarite
US7715989B2 (en) 1998-04-03 2010-05-11 Elitech Holding B.V. Systems and methods for predicting oligonucleotide melting temperature (TmS)
US7751982B2 (en) 1998-04-03 2010-07-06 Elitech Holding B.V. TM leveling methods
WO1999067378A1 (fr) * 1998-06-19 1999-12-29 Mcgill University CONSTRUCTIONS OLIGONUCLEOTIDES ANTISENSE A BASE DE β-ARABINOFURANOSE ET DE SES ANALOGUES
US7179905B2 (en) 1998-07-31 2007-02-20 Affymetrix, Inc. Nucleic acid labeling compounds
US7491818B2 (en) 1998-07-31 2009-02-17 Affymetrix, Inc. Nucleic acid labeling compounds
US6844433B2 (en) 1998-07-31 2005-01-18 Affymetrix, Inc. Nucleic acid labeling compounds
US6072046A (en) * 1998-08-26 2000-06-06 Epoch Pharmaceuticals, Inc. Diaziridinyl-aryl and bis-[di(chloroethyl)amino]-aryl oligonucleotide conjugates and reagents for making the same
US7205105B2 (en) 1999-12-08 2007-04-17 Epoch Biosciences, Inc. Real-time linear detection probes: sensitive 5′-minor groove binder-containing probes for PCR analysis
US7485442B2 (en) 1999-12-08 2009-02-03 Epoch Biosciences, Inc. Real-time linear detection probes: sensitive 5'-minor groove binder-containing probes for PCR analysis
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JP2003525292A (ja) * 2000-03-01 2003-08-26 エポック・バイオサイエンシーズ・インコーポレイテッド ミスマッチ識別のための修飾オリゴヌクレオチド
EP1944310A3 (fr) * 2000-03-01 2008-08-06 Epoch Biosciences, Inc. Oligonucléotides modifiées pour discrimination de désadaptation
WO2001064958A3 (fr) * 2000-03-01 2002-03-28 Epoch Bioscienecs Inc Oligonucleotides modifies permettant de discriminer des mesappariements
WO2001064958A2 (fr) * 2000-03-01 2001-09-07 Epoch Bioscienecs, Inc. Oligonucleotides modifies permettant de discriminer des mesappariements
US7238795B2 (en) 2000-08-03 2007-07-03 Roche Molecular Systems, Inc. Nucleic acid binding compounds containing pyrazolo[3,4-d]pyrimidine analogues of purin-2,6-diamine and their uses
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US8076072B2 (en) 2001-03-12 2011-12-13 Affymetrix, Inc. Nucleic acid labeling compounds
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US7468243B2 (en) 2001-03-12 2008-12-23 Affymetrix, Inc. 2-aminopyrimidin-4-one nucleic acid labeling compounds
US8497064B2 (en) 2001-03-12 2013-07-30 Affymetrix, Inc. Nucleic acid labelling compounds
US7718374B2 (en) 2003-10-02 2010-05-18 Elitech Holding B.V. Single nucleotide polymorphism analysis of highly polymorphic target sequences
US7348146B2 (en) 2003-10-02 2008-03-25 Epoch Biosciences, Inc. Single nucleotide polymorphism analysis of highly polymorphic target sequences
US7759126B2 (en) 2003-10-28 2010-07-20 Elitech Holding B.V. Real-time linear detection probes: sensitive 5′-minor groove binder-containing probes for amplification (or PCR) analysis
US7763624B2 (en) 2005-08-22 2010-07-27 Amgen Inc. Substituted pyrazolo[3,4-d]pyrimidines as ACK-1 and LCK inhibitors
EP2659000A1 (fr) * 2010-12-30 2013-11-06 Ventana Medical Systems, Inc. Dépôt optimisé de chromogènes utilisant des analogues de la pyrimidine
EP2659000B1 (fr) * 2010-12-30 2016-08-10 Ventana Medical Systems, Inc. Dépôt optimisé de chromogènes utilisant des analogues de la pyrimidine
US9435795B2 (en) 2010-12-30 2016-09-06 Ventana Medical Systems, Inc. Enhanced deposition of chromogens utilizing pyrimidine analogs
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