WO2004048376A1 - Nucleosides de napthylidine bicycliques - Google Patents

Nucleosides de napthylidine bicycliques Download PDF

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Publication number
WO2004048376A1
WO2004048376A1 PCT/JP2003/014985 JP0314985W WO2004048376A1 WO 2004048376 A1 WO2004048376 A1 WO 2004048376A1 JP 0314985 W JP0314985 W JP 0314985W WO 2004048376 A1 WO2004048376 A1 WO 2004048376A1
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mmol
group
compound
nucleoside
formula
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PCT/JP2003/014985
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English (en)
Japanese (ja)
Inventor
Sadao Hikishima
Noriaki Minakawa
Akira Matsuda
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Geneticlab Co., Ltd.
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Application filed by Geneticlab Co., Ltd. filed Critical Geneticlab Co., Ltd.
Priority to AU2003284668A priority Critical patent/AU2003284668A1/en
Publication of WO2004048376A1 publication Critical patent/WO2004048376A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • 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
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • 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

Definitions

  • the present invention relates to a novel nucleoside derivative and an oligonucleotide incorporating the nucleoside derivative.
  • DNA stores and transmits genetic information as a carrier.
  • DNA double-stranded structure mainly consists of 1) formation of hydrogen bond by two pairs of complementary base pairs (A: T, G: C), 2) scanning by adjacent base pairs due to stacking of these base pairs.
  • Interactions such as those found in DNA duplexes are also involved in molecular recognition between DNA and RNA and between nucleic acids and proteins, and are deeply involved in the regulation and expression of various functions in vivo.
  • advances in nucleic acid synthesis technology have made it possible to easily synthesize DNA and RNA having various sequences, and have brought dramatic advances in molecular biology.
  • nucleic acid drugs such as the antisense method, the antigene method, and the decoy method is being studied.
  • many properties have been reported so far when many base-modified nucleoside derivatives are chemically synthesized and incorporated into DNA chains.
  • Matteucci et al Synthesized various nucleosides as shown below and found that they stabilize a DNA duplex by base pairing with the guanine base of a complementary strand in a DNA strand (Lin Jones, RJ; Matteucci, MJ Am. Chem. Soc, 1995, 117, 3873 ⁇ 3874, Lin, KY; Matteucci, MJ Am. Chem. Soc, 1998, 120, 3531-3532).
  • the present inventors have previously synthesized four types of tricyclic imidazopyridopyrimidine nucleosides (Ira-NN, Im-OO, Im-NO, InrON) having four hydrogen bonding ability. (Kojima, NJ Ueno, Y .; Minakawa, NJ N. Matsuda, A "Nucleic Acis Symp. Ser., 1997, 37, 23-24).
  • An object of the present invention is to provide a novel nucleoside derivative for forming a base pair motif capable of further stabilizing the higher-order structure of a nucleic acid, and an oligonucleotide containing such a nucleoside analog. Disclosure of the invention
  • the present invention relates to nucleoside analogs having four hydrogen bonding functional groups that can participate in base pairing, comprising a compound of formula I:
  • the hydrogen-bonding functional group means a functional group and an atom that can participate in a hydrogen bond, for example, hydrogen, N of primary, secondary and tertiary amines or amides, aromatic N, carbonyl Or ⁇ ⁇ ⁇ ⁇ of carboxylic acid, H and ⁇ of water molecule, but are not limited thereto.
  • the present invention also provides an oligonucleotide comprising at least one of the above-described nucleoside analogs of the present invention.
  • the present invention also provides a compound of formula I I I:
  • R 2 is a protecting group for an amine group, and L is a leaving group
  • FIG. 1 shows a schematic diagram of a DNA duplex containing a tricyclic imidazopyridopyrimidine nucleoside.
  • FIG. 2 shows stabilization of a DNA double strand by the bicyclic naphthyridine nucleosides of the present invention.
  • FIG. 3 shows the thermal stability of the duplex of DNA containing the bicyclic naphthyridine nucleoside of the present invention and DNA containing the tricyclic imidazopyridopyrimidine nucleoside.
  • novel bicyclic naphthyridine nucleosides (Na-N, Na-OO, Na-NO, Na-ON) of the present invention are novel base pair motifs that can further stabilize the higher-order structure of nucleic acids. These compounds form four hydrogen bonds without distorting the DNA duplex like the base pairs between the tricyclic imidazopyridopyrimidine nucleosides, further stabilizing the DNA duplex. ( Figure 2).
  • the nucleoside analog of the present invention is a novel base pair motif that forms a base pair by four hydrogen bonds, and by incorporating this into a DNA, a very useful functional artificial nucleic acid can be created. Therefore, the present invention is useful for application to nucleic acid pharmaceuticals for controlling and stabilizing the higher-order structure of nucleic acids such as double helical structures. For example, by combining with the above-mentioned tricyclic nucleoside, it is possible to obtain a thermally stable DNA double strand, which is expected to be usable as a decoy molecule. I can wait. In addition, this naphthyridine base can be expected to form a base pair with a natural nucleic acid base, and is considered to be usable as an antisense molecule.
  • this compound is a fluorescent nucleoside, similar to the above tricyclic nucleoside. If these compounds show different responses depending on the nucleobases on the complementary strand side, they can be used for the detection of SNPs.
  • base-responsive fluorescent nucleosides such as BPP and BDA, have been reported by the group of Saito et al. (Okamoto, A .; Tainaka, K; Saito, I. 17th Symposium on Biological Function-Related Studies) , 90-91, Okamoto, A .; Tanaka, K; Fukuta, ⁇ ⁇ .; Saito, I. The 17th Symposium on Biofunctional Chemistry, 274-275).
  • a total of eight types of fluorescent nucleosides can be obtained in combination with the above-mentioned tricyclic nucleoside, so that it can be used as a more general-purpose fluorescent probe for SNPs detection or hybridization detection.
  • the nucleoside derivative of the present invention can be synthesized as follows. Naphthyridine nucleosides, which are the target compounds, are C-nucleosides. After constructing the base moiety and the sugar moiety, respectively, they are synthesized by performing 'C-glycosylation using a palladium catalyst.
  • the sugar moiety is protected with a TBS group at the sugar moiety of thymidine according to the method described in the literature, and then treated with HMDS and ammonium sulfate to obtain a glycal form 2 (Coleman, RS; Madams, MLJ Org. Chem., 1998, 63, 5700-5703). Then, it is treated with TBAF, and the TBS group at the 5-position is selectively deprotected to give compound 3 (Scheme 1).
  • the base moiety is reacted with 2,6-diaminopyrimidine and malic acid in sulfuric acid according to the method described in the literature, and neutralized with aqueous ammonia to obtain naphthyridine derivative 4 (Newkome, G.R; Garbis, SJ; Majestic, V. K; Fronczek, FR; Chiari, GJ Org. Chem., 1981, 46, 833-839). Thereafter, the compound 4 is treated with an equivalent amount of NIS, and the amino group is protected with a dimethylamidine group to give the 6-position 5 (Scheme 2).
  • the compound 6 is obtained by the Heck reaction of the glycal derivative 3 and the naphthyridine derivative 5 and then treated with TBAF to obtain a 3′-position ketone 7. Next, this is reduced, and after deprotection of the dimethylamidine group, bicyclic naphthyridine nucleoside (Na-NO) can be synthesized (Scheme 3) (Zhang, HC; Daves, GD, Jr. J. Org. Chem., 1992, 57, 4690-4696).
  • the present invention also provides a compound of the formula II I I:
  • R 2 is a protecting group for an amine group and L is a leaving group
  • a natural nucleoside for example, 3′-OH of thymidine
  • Ri protecting group
  • the nucleoside analogs of the present invention can be converted to amidites by methods known in the art and incorporated into oligonucleosides. Specifically, the obtained Na-NO form was protected with a dibutylamidine group at the amino group of the base to give compound 9, and then the 5'-7K acid group was subjected to dimethoxytrityl iridyl according to a conventional method. Subsequently, the hydroxyl group at the 3'-position is converted to an amidite 11 by phosphoramidation (Scheme 4). The obtained amidite body 11 can be introduced into a DNA oligomer according to a solid phase phosphoramidite method.
  • the present invention provides an oligonucleotide comprising the above-described bicyclic naphthyridine nucleoside.
  • the thus obtained oligonucleotides of the present invention can be used as diagnostic, therapeutic and research reagents as antisense oligonucleotides, lipozymes, primers, abutamas, antigenes, probes and the like.
  • the oligonucleotides of the invention are from about 6 to about 100 nucleotides in length.
  • the oligonucleotide is from about 12 to about 20 nucleotides in length.
  • Oligonucleotides may include modified sugars, such as those having a substituent at the 2 'position, and nucleic acid bases other than adenine, guanine, cytosine, thymine, peracil, such as hypoxanthine, 5-alkylcytosine, It may contain 5-alkylperacyl, 5-haloperacyl, 6-azapyrimidine, 6-alkylpyrimidine and the like. Further, it may contain an internucleoside bond other than the phosphodiester, for example, a phosphorothioate bond.
  • thymidine (9.7 g, 40.0 mmol) was dissolved in DMF (150 mL), and imidazole (13.6 g, 200.0 mmoL) and TBSC1 (15.0 g, 100.0 bandol) were added, followed by stirring at room temperature for 2 hours. did.
  • the solvent was distilled off, and the residue was dissolved in ethyl acetate (600 mL), washed twice with water (200 mL) and once with brine (200 mL), and dried over anhydrous sodium sulfate. The solvent is distilled off, and the residue is dissolved in a small amount of ethyl acetate.
  • the resulting amidite 11 is introduced into a DNA oligomer according to the solid phase phosphoramidite method, and the DNA oligomer incorporating the tricyclic imidazopyridopyrimidine nucleoside Im-ON is used as a complementary strand to form a DNA double strand
  • the thermal stability was evaluated. As a result, even when one base pair of Im-ON: Na-NO base pair is introduced, the DNA duplex is extremely stable, unlike the base pair of tricyclic imidazopyridopyrimidines, and G: C It was found to be 8.6 degrees more stable than the base pair (Fig. 3A).
  • the Tm value was 95.7 ° C, which revealed that the DNA duplex was stabilized at 26.7 ° C. This is stabilization of 8.9 degrees per base pair, and the DNA double strand was able to be stabilized when either one or three base pairs were introduced (Fig. 3B).
  • the novel bicyclic naphthyridine nucleoside functions as a complementary base to the tricyclic imidazopyridopyrimidine, forms four hydrogen bonds without causing distortion in the DNA duplex, and forms the DNA double strand. It has become clear that this will further stabilize.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
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  • Molecular Biology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
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  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
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  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne un analogue de nucléoside qui comporte quatre groupes fonctionnels à liaison hydrogène, capable de participer à une formation de paire de bases et ayant une structure sélectionnée dans la série des groupes ci-après. L'invention concerne également un oligonucléotide renfermant l'analogue considéré. Ledit analogue est utile pour la formation d'un motif de paire de bases capable de stabiliser plus avant la structure d'ordre supérieur propre à un acide nucléique.
PCT/JP2003/014985 2002-11-26 2003-11-25 Nucleosides de napthylidine bicycliques WO2004048376A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003284668A AU2003284668A1 (en) 2002-11-26 2003-11-25 Bicyclic naphthylidine nucleosides

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JP2002-342980 2002-11-26
JP2002342980A JP2004175708A (ja) 2002-11-26 2002-11-26 二環性ナフチリジンヌクレオシド

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006094347A1 (fr) * 2005-03-08 2006-09-14 Biota Scientific Management Pty Ltd. Nucleosides bicycliques et nucleotides servant d’agents therapeutiques
US7666851B1 (en) * 2006-05-18 2010-02-23 Steven Albert Benner Three ring fused analogs of isoguanosine

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000511160A (ja) * 1996-05-15 2000-08-29 リサーチ コーポレイション テクノロジーズ インコーポレイテッド 多環芳香族基が結合した新規ヌクレオシドアナログ、その合成方法および使用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000511160A (ja) * 1996-05-15 2000-08-29 リサーチ コーポレイション テクノロジーズ インコーポレイテッド 多環芳香族基が結合した新規ヌクレオシドアナログ、その合成方法および使用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006094347A1 (fr) * 2005-03-08 2006-09-14 Biota Scientific Management Pty Ltd. Nucleosides bicycliques et nucleotides servant d’agents therapeutiques
US7666851B1 (en) * 2006-05-18 2010-02-23 Steven Albert Benner Three ring fused analogs of isoguanosine

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JP2004175708A (ja) 2004-06-24

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