WO1996006833A1 - Nouveaux analogues de nucleotides - Google Patents

Nouveaux analogues de nucleotides Download PDF

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Publication number
WO1996006833A1
WO1996006833A1 PCT/JP1995/001729 JP9501729W WO9606833A1 WO 1996006833 A1 WO1996006833 A1 WO 1996006833A1 JP 9501729 W JP9501729 W JP 9501729W WO 9606833 A1 WO9606833 A1 WO 9606833A1
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synthesis
solution
type
formula
added
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PCT/JP1995/001729
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English (en)
Japanese (ja)
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Takeshi Imanishi
Satoshi Obika
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Takeshi Imanishi
Satoshi Obika
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Priority to AU33545/95A priority Critical patent/AU3354595A/en
Publication of WO1996006833A1 publication Critical patent/WO1996006833A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring 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

Definitions

  • the present invention relates to novel nucleotide analogs, and more particularly, to nucleotide analogs suitable for antisense molecules.
  • Antisense oligonucleotides are one of the most promising fields in recent years as pharmaceuticals because they specifically regulate the expression of unwanted genes.
  • the antisense method is based on the concept of controlling the flow of so-called central bears from DNA to RNA to protein using an antisense oligonucleotide.
  • nucleic acid derivatives have been synthesized and studied. For example, phosphorothioate in which the oxygen atom on the phosphorus atom has been replaced with a zeo atom, methylphosphonate in which the methyl group has been replaced with a methyl group, and more recently, those in which the phosphorus atom has been replaced with a carbon atom and ribose is acyclic.
  • Backbone molecules have also been synthesized (F. Eckstein et al., Biochem..
  • the inventors of the present invention designed a nucleic acid derivative that would be useful in the antisense method, synthesized it, and confirmed its usefulness. Hereinafter, the present invention will be described.
  • nucleic acid derivatives for the antisense method was performed with the following three points in mind.
  • the nucleotide analog of the present invention can be represented by the following general formula.
  • X and Y are independently oxygen or sulfur
  • R is hydrogen, an alkyl group, or an acyl group
  • W may be hydrogen, an alkyl group, an acyl group or, when X is oxygen, a nucleotide, an oligonucleotide or a polynucleotide via a phosphate bond.
  • n is an integer of 1 to 50. However, when n is 2 or more, B is not limited to the same base.
  • B 2 are the same or different and are pyrimidine or purine nucleobase or a derivative thereof, X and Y are independently oxygen or sulfur, W is the same or different, hydrogen, It may be a nucleotide, oligonucleotide or polynucleotide via an alkyl group, an acyl group, or a phosphate bond.
  • m is an integer of 1 to 25. However, when m is 2 or more, and B 2 are not limited to the same base.
  • the pyrimidine or purine nucleobase is thymine, peracil, cytosine, adenine, guanine and derivatives thereof.
  • the nucleotide analog of the present invention can be synthesized as follows. For the sake of simplicity, a compound in which both X and Y in the above formula are oxygen will be described first.
  • optically active (s) -glycidol (compound 1) is selected as a raw material, and this is reacted with aqueous ammonia to form compound 2.
  • Mirror protection of the amino group with the tert-butoxycarbonyl (Boc) group gives compound 3 as a colorless oil.
  • the primary hydroxyl group is selectively protected with a t-tert-butyldifuryl-2-silylyl (TBDPS) group to obtain compound (silyl form) 4 as colorless crystals.
  • TDPS t-tert-butyldifuryl-2-silylyl
  • the secondary hydroxyl group is treated with dimethyl sulfoxide, acetic anhydride, Tylation gives compound 5. Further, it is reacted with a nucleobase ( ⁇ ,) activated with a trimethylsilyl (TMS) group, for example, thymine-2TMS, to obtain a monomer unit (R-form) 6.
  • TMS trimethylsilyl
  • the TBDPS group is deprotected to remove the silyl form 7 and the ⁇ 0c group to remove the ⁇ 0c group.
  • the desilyl form 7 of the monomer unit (R form) 6 synthesized above and the de-Boc form 8 are condensed using N, N'-carbonyldiimidazole to obtain a homodimer 13. This is further deprotected to obtain the desilyl form 14 and the Boc form 15.
  • homotetramer desilyl form 14 and the Boc form 15 are condensed in the same manner to obtain the homotetramer 16.
  • homotrimer 17 is synthesized by condensing monomer-unit desilylated form 7 and homodimer de-Boc form 15 in the same manner, or by condensing monomer unit de-Boc form 8 and homo-dimer desilylated form 14. it can.
  • a homo-oligomer [Chemical Formula 1]
  • S-form monomer unit
  • an S-form homo-oligomer can also be synthesized.
  • nucleoside 18 for example, a thymidine compound
  • DMT r dimethoxytrityl
  • N, N′-carbonyldiimidazole Condensed with the de-B0c-isomer (R-isomer) 8 to obtain dimerunit 19.
  • a type II oligonucleotide analog of the present invention can be obtained by a DNA synthesizer.
  • an R-form ( ⁇ ) oligonucleotide analog can be obtained by using a de-Boc-form (R-form) 8 instead of the de-Boc-form (S-form) 12. Purification and identification are as described in (3) above.
  • the method for synthesizing the oligonucleotide analogue of the present invention has been described, among the compounds represented by the above formulas (Chemical Formula 1 and Chemical Formula 2), the thio derivative in which X and / or Y in the formula is sulfur is Use 3'-thionucleoside or 5'-mononucleoside or an analog thereof in place of natural nucleoside, or use N, N'-thiocarbodildimidazole in place of N, N'-carbodildimidazole
  • a thio derivative in which X or Y is sulfur can be synthesized.
  • by performing both at the same time it is possible to obtain a derivative in which both X and Y are sulfur. Wear.
  • type I and type II heterodimeric derivatives can be produced by the following synthesis method.
  • the starting material is 5'-monomethoxytrityl-3'-deokin-1 3'-thiothymidine (R. Crosstick, JS Vyle, J. Chem. Soc., Cheo. Commun., 993 (1988)) which is known in the literature.
  • N, N'-thiocarbonyldiimidazole W. Walter, KD Bode, Angew. Chem., 79.285 (1967)
  • N, N'-carbonyldiimidazole de-Boc form of monomer unit
  • FIG. 1 shows the time-dependent changes in ultraviolet absorption (260 nm) when various oligonucleotides of the present invention were digested with exonuclease.
  • FIG. 2 is an electrophoretogram showing changes over time when the oligonucleotide of the present invention and a natural oligonucleotide are degraded by exonuclease.
  • FIG. 3 is a graph showing that the antisense oligonucleotide of the present invention (IL16RAS1 to 4-IR) in Example 6 suppresses the expression of soluble IL-16R.
  • an antisense molecule having the nucleotide analog of the present invention introduced at a required position in a required number (length) can be synthesized. Entire nucleotide analog It is preferable that nucleoside units have a length of 10 to 30.
  • Such antisense molecules are not easily degraded not only for exonucleases but also for endonucleases, and can exist in vivo for a long time after administration to the living body. For example, it forms a duplex with the sense strand RNA to inhibit the formation (translation) of pathogenic biological components (proteins) or forms a triplex with the duplex DNA to form the mR Inhibits transcription to NA. It may also inhibit the growth of infected viruses.
  • antisense molecules using the nucleotide analogs of the present invention have utility as pharmaceuticals for treating diseases by inhibiting the function of genes such as antitumor agents and antiviral agents. Expected.
  • the antisense molecule using the nucleotide analog of the present invention can be formulated into a preparation for parenteral administration by blending a conventional auxiliary agent such as a buffer and Z or a stabilizer.
  • a conventional pharmaceutical carrier can be blended and formulated into a descendant, cream-solution, salve or the like.
  • Aqueous ammonia (440 ml) was added to 68 mmo 1), and the mixture was stirred for 17 hours under ice cooling. After distilling off ammonia water at a low temperature, the mixture was diluted with anhydrous ethanol (120 ml), dried over anhydrous calcium carbonate, and the solvent was distilled off. Triethylamine (9.4 ml, 1 eq.) And dibutyl butyrate carbonate (15.5 ml, 1 eQ.) Were added to the obtained DMF solution (100 ml) of the supernatant, and the mixture was added at room temperature. ⁇ The mixture was stirred.
  • Compound 6c was obtained according to the same method and conditions as in 2-a.
  • Trifluoroacetic acid (0.5 ml) was added dropwise to a methylene chloride solution (5.5 ml) of monomer unit (R form) 6a (372 mg, 0.65 mmo 1, 1.1 eq. :) under ice-cooling and room temperature. After stirring for 120 minutes at room temperature, the solvent was distilled off, and the de-Boc form 8a obtained was used in the crude state for condensation.
  • the desilylated 7a (20 Omg. 0.6 lmmo 1.1.0 eq.) was dehydrated by azeotropic distillation with anhydrous pyridine three times to obtain an anhydrous pyridine solution (5.5 ml).
  • N, N'-carbonyldiimidazole (198 mg.
  • Trifluoroacetic acid (0.11 ml) was added dropwise to a solution of homodimer (R-isomer) 13a (119 mg, 0.145 mmo 1, 1.1 eq. :) in methylene chloride (1.0 ml) under ice-cooling, and the solution was added at room temperature for 120 minutes. After stirring for 1 minute, the solvent was distilled off, and the homodimer-derived de-B0c form 15a was used for condensation without purification.
  • N, N'-force Luponyldiimidazole (44 mg. 2.0 eq.) was added, and the mixture was stirred at room temperature under a nitrogen stream for 150 minutes.
  • Water (301, 12 eq.) was added, and the mixture was stirred for 30 minutes to decompose excess N, ⁇ '-carbonyldiimidazole, and then the solvent was distilled off. Dehydration was performed by azeotropic distillation with anhydrous pyridine three times to obtain an anhydrous pyridine solution (0.6 ml).
  • the following heterodimer unit was synthesized according to the method and conditions described in (1) above.
  • Anhydrous DMF solution (2m1) of bis (412trotropinyl) carbonate (38Omg, 1.3mmo1) was added to triethylamine (0.10m1.0.69mmo1) and 5'-0- (4,4'dimethoxytrityl).
  • An anhydrous DMF solution (2 ml) of 1 N-benzoylol 2'-doxycytidine (40 Omg, 0.63 mmo1) (compound 18c) was added dropwise at room temperature and stirred for 5 hours. After the solvent was distilled off, the residue was diluted with methylene chloride, and washed twice with a 0.01 N aqueous sodium hydroxide solution, twice with water, and once with a saturated saline solution.
  • the target 5′-GCAGCCTCCTTCCCATG-Cc-A-3 ′ (11.3 nmo 1) was obtained in the same manner as in 6-a.
  • Literature-known 3'-0-acetylthymidine 22a (0.93.1.2 Ommo 1, 1.0 eq) 5 ) was azeotropically dehydrated with anhydrous pyridine three times and dehydrated with anhydrous pyridine solution (15m1). did. N, ⁇ '-carbonyldiimidazole (1.04 g, 2.0 e Q.) was added, and the mixture was stirred at room temperature under a nitrogen stream for 150 minutes. Water (0.70 ml, 12 eq.) was added and the mixture was stirred for 30 minutes to decompose excess N, N'-carbonyldiimidazole, and then the solvent was distilled off.
  • Dehydration was performed by azeotropic distillation with anhydrous pyridine three times to obtain an anhydrous pyridine solution (15 ml). To this was added 12a of anhydrous pyridine solution (15 ml) and 4-dimethylaminopyridine (4-DMAP) (0.39 g, 0.18 mmo 1, 0.5 eq.), And the mixture was added to a stream of nitrogen. The mixture was stirred at room temperature for 2 days. After the solvent was distilled off, the residue was diluted with ethyl acetate, washed four times with water and twice with a saturated saline solution. The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off.
  • 4-DMAP 4-dimethylaminopyridine
  • II-S type amidite 27 a (30 Omg, 0.30 mm o 1) was acetonitrile Using a natural solution of natural nucleoside as a lysate solution (2.5 ml), a II-S oligomer was synthesized on a 0.2 ⁇ mo 1 scale using a DNA synthesizer (5 ′ — DMTr: ON). Ammonia treatment was performed at 70 ° C for 3 hours, and the excised solution was purified using a simple reversed-phase column (Mi 11 ipore, 01igo-Pak TM SP), and further purified by reversed-phase HPLC. (Column: TOSOH, TSKgel ODS-120T (3.2 mm ID x l.
  • Example 9 Measurement of molar extinction coefficient £ of II-I type S heterodimer unit
  • antisense strand The hybridizing ability of the antisense strand was examined by measuring the Tm of annealed oligosaccharide (antisense strand), which is various antisense molecules synthesized in Examples 6 and 8, and the sense strand. .
  • a sample solution having a final concentration of 100 mM NaCl, 10 mM sodium phosphate buffer (pH 7.2), 4 antisense strands and 4 M sense strands was bathed in boiling water and cooled to room temperature over 10 hours.
  • a nitrogen stream was passed through the cell chamber of the UV spectrophotometer to prevent condensation, and the sample solution was slowly cooled to 0 ° C.
  • a cell with a lid was used, and a drop of mineral oil was dropped on the surface of the sample solution. The sequences of the antisense strand and the sense strand used for the measurement are shown below.
  • nucleosides are represented by capital letters such as T, C, A and G
  • analogs in the present invention are represented by small letters such as t, c, a and g. .
  • the I-R type hetero-oligomer measures the effect of the mismatch of the heterodimer unit (T t) on the analog (t), and this value is calculated as the II-S type hetero-dimer unit II-S ( Since the effect of mismatch on the natural nucleoside (T) of t T) is shown, it cannot be compared directly, but is shown as reference data.
  • Experimental example 2 Measurement of enzyme resistance (1)
  • FIG. 1 shows the change over time in the relative absorbance at 260 nm.
  • the heterodimer introduced into the oligonucleotide has resistance to enzymatic degradation, and the oligomer of the present invention satisfies the properties required for an antisense molecule.
  • the non-natural oligomer is terminated at the introduced heterodimer by enzymatic degradation compared to the natural oligomer (T11).
  • the heterodimer introduced into the oligonucleotide has resistance to enzymatic degradation, and the oligomer of the present invention satisfies the properties required for an antisense molecule.
  • Example 4 Inhibitory effect of human soluble IL-6R (sIL-6R)
  • pBSF2R.236 (Science, 241, 825-828 (1988)) was digested with SphI, and the 1205 bp IL-16Rc DNA fragment was inserted into mp18 (Amersham).
  • sIL-6RcDNA was prepared by preparing a synthetic oligomer of 5'-ATTATTCTAGAGAGCTTCT-3 'and using an in vitro mutagenesis system (manufactured by Amersham). As a result, the stop codon was at position 345 of the amino acid sequence.
  • the dhfr-l cDNA was inserted into the PvuII site of plasmid pECE (Cell, 45, 721-735 (1986)) to prepare plasmid pECEdhfr.
  • the HindII-SalI fragment of sIL-6R was inserted into plasmid pECEdhfr to prepare a soluble IL-16R expression vector brasmid pECEdhfr344.
  • pECEdhfr344 was introduced into dhfr-CHO cell DXB-11 (Pro. Natl. Acad. Sci. U.S.A .. 77, 4216-4220 (1980)) by the calcium phosphate method and amplified by MTX. Finally, 200 nMMTX-resistant sIL-16R-producing CHO cells (CHO. SR344) were produced (J. Biochem .. 108. 673-676 (1990)). Normal culture of the cells was performed in an IMDM medium (Gibeo) containing 5% FCS (Xavier Investments) and 200n MMTX.
  • SR344 cells are detached from the culture dish with trypsin-EDTA (manufactured by Gibco), washed with culture medium, and further washed with serum-free medium non-serum (Nippon Zenyaku Kogyo) and serum-free medium non-serum containing 200 nMMTX. Suspended in water. To a 96-well culture plate, add 2 M IL-16R antisense oligomer 100 c1 to CHO. SR344 cell suspension 100 1 (5 ⁇ 10 4 cells / ml), 37 ° C, 5% C 0 were cultured in Inkyubeta one 2 under.
  • the amount of soluble IL-16R at the time of the culture was determined using a sandwich ELISA using mouse anti-IL-16R monoclonal antibody (MT18) (Japanese Patent Laid-Open No. 2-288898) and Egret anti-IL-16R polyclonal antibody. It was measured by the method.
  • MT18 mouse anti-IL-16R monoclonal antibody
  • Egret anti-IL-16R polyclonal antibody It was measured by the method.
  • a sense oligomer derivative for the antisense oligomer IL-6RAS4-IR was used as a control.
  • the sense oligomer (IL-6RSE1) can be synthesized in the same manner as in Example 6 without using a Cc heterodimer.

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Abstract

Molécules antisens de formule générale (I), où les B1 peuvent être identiques ou différents et représenter une base d'acide nucléique du type pyrimidinique ou purinique ou l'un de ses dérivés; X et Y représentent chacun indépendamment oxygène ou soufre; R représente hydrogène, alkyle ou acyle, w représente hydrogène, alkyle ou acyle ou, lorsque X représente oxygène, W peut être un nucléotide, un oligonucléotide ou un polynucléotide fixé par l'intermédiaire une liaison phosphate; et n représente un entier compris entre 1 et 50 sous réserve que lorsque n est égal à 2 ou plus, les B1 soient différents l'un de l'autre; ou de formules générales (II) et (III) où B2 est identique à B1, m est identique à n et W comporte un nucléotide, etc. fixé par une liaison phosphate.
PCT/JP1995/001729 1994-08-31 1995-08-31 Nouveaux analogues de nucleotides WO1996006833A1 (fr)

Priority Applications (1)

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AU33545/95A AU3354595A (en) 1994-08-31 1995-08-31 Novel nucleotide analogs

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JP20734394 1994-08-31

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03236396A (ja) * 1990-02-10 1991-10-22 Tosoh Corp 修飾オリゴデオキシヌクレオチド及びそれを含むｄｎａ

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03236396A (ja) * 1990-02-10 1991-10-22 Tosoh Corp 修飾オリゴデオキシヌクレオチド及びそれを含むｄｎａ

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