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WO1998021226A1 - Derive oligonucleotidique h-phosphonate et procede de production associe - Google Patents

Derive oligonucleotidique h-phosphonate et procede de production associe Download PDF

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Publication number
WO1998021226A1
WO1998021226A1 PCT/JP1997/004128 JP9704128W WO9821226A1 WO 1998021226 A1 WO1998021226 A1 WO 1998021226A1 JP 9704128 W JP9704128 W JP 9704128W WO 9821226 A1 WO9821226 A1 WO 9821226A1
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group
phosphonate
general formula
derivative
oligonucleotide
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PCT/JP1997/004128
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English (en)
Japanese (ja)
Inventor
Mitsuo Sekine
Takeshi Wada
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Chugai Seiyaku Kabushiki Kaisha
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Application filed by Chugai Seiyaku Kabushiki Kaisha filed Critical Chugai Seiyaku Kabushiki Kaisha
Priority to AU49648/97A priority Critical patent/AU4964897A/en
Publication of WO1998021226A1 publication Critical patent/WO1998021226A1/fr

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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/141—Esters of phosphorous acids
    • C07F9/1411—Esters of phosphorous acids with hydroxyalkyl compounds with further substituents on alkyl

Definitions

  • the present invention relates to an H-phosphonate oligonucleotide derivative and a method for producing the derivative.
  • the present invention relates to a novel H-phosphonucleotide nucleotide derivative and a method for producing the derivative. More specifically, the present invention relates to an H-phosphonate oligonucleotide derivative that is resistant to phosphodiesterase and has high uptake efficiency into cells. Background art
  • HSV simple herpes virus
  • BPV pipapi lipovirus
  • cmyconcogene ELWickstrom et al, In Vitro Cell Develop.Biol., 25,297 (1989)
  • BCL-2 oncogene JC Reed et al, Proc. Natl. Acad. Sci.
  • VSV vesicular stomatitis virus
  • PSMiller et al Biochimie, 67, 769 (1985)
  • N-ras oncogene expression control DMTidd, Anti-Cancer Drug Design, 3,117 (1988)
  • JGlobin expression control KR Blake, Biochemistry, 24, 6132 (1985)
  • chloramphenicol acetyl chloride transfer Regulation of the expression of Ise CJMarcus-Sekura, Nucleic Acid Res., 15, 5749 (1987) and the like have been reported.
  • the phosphorothioate-type DNA has a problem that the stability of the duplex is low, and it binds to proteins other than the target mA and causes non-selective inhibition.
  • Methylphosphonate-type DNA has high specificity for the target mMA, but must be used at a high concentration in order to exhibit effective biological activity due to low affinity. However, it has a problem that it cannot be administered to cells at a high concentration due to its low solubility.
  • An object of the present invention is to provide a novel H-phosphonate oligonucleotide derivative and a method for synthesizing the derivative.
  • H-phosphonate oligonucleotides are extremely unstable under basic conditions. For this reason, when removing the acyl-type protecting group at the base site and excising the oligomer from the solid support in the ordinary DNA synthesis, it is completely decomposed in the process of using ammonia water. Even in this case, if a protecting group is introduced into the 3 ′ and 5 ′ hydroxyl groups of the H-phosphonate oligonucleotide, it is relatively stable even under anhydrous basic conditions. However, when the protecting group is removed, the free hydroxyl group at the 3 'end of the H-phosphonate DNA is converted to an H-phosphonate diester adjacent to the 5' side via 2'-deoxyribose.
  • the present inventors have conducted intensive studies on a method for synthesizing H-phosphonate oligonucleotides in a high yield by suppressing the decomposition of H-phosphonate oligonucleotides under basic conditions in the synthesis process.
  • the present inventors introduce an alkoxyphosphonic acid having an alkylene group having an appropriate chain length at the 3 ′ end and the 5 ′ end of the oligomer, thereby obtaining oligomers.
  • the H-phosphone mononucleotide derivative of the present invention is represented by the following general formula (I).
  • R1 is a hydrogen atom, an alkyl group such as a methyl group, an ethyl group, a propyl group or a butyl group, an alkenyl group such as a vinyl group or an aryl group, a hydroxy group, a methoxy group, an ethoxy group, a propyloxy group, a butyloxy group, or a methoxyxetoxy group.
  • alkoxy group such as a group, an alkenyl group such as an aryloxy group, or an acyl group such as a methylcarbonyl group, an ethylcarbonyl group, a methoxycarbonyl group or an ethoxycarbonyl group, preferably a hydrogen atom, a hydroxy group, a methoxy group, or the like.
  • R 2 represents an optionally branched alkylene group having 1 to 10 carbon atoms, preferably 6 to 8 carbon atoms. Further, these alkylene groups may be via one or more oxygen atoms. For example,
  • H-phosphonate oligonucleotide derivative of the present invention can be synthesized, for example, by a method comprising the following steps.
  • R 3 represents a protecting group such as a trityl group, a monomethoxytrityl group, a dimethyloxytrityl group, a pixyl group, and the like;
  • R 5 represents a carrier such as aminopropyl CPG, long-chain alkylamino CPG, aminomethylpolystyrene, etc.
  • R 2 has the same meaning as described above.
  • R 6 represents the same protecting group as R 3 in formula (II);
  • a base monomer unit represented by The monomer unit is usually reacted in the presence of 1 to 5 equivalents of a dehydration condensing agent in pyridine or acetonitrile-pyridine (l: l, v / v) at room temperature for about 1 to 10 minutes.
  • the compound represented by the general formula (II) and the base monomer represented by the general formula (III) can be obtained by a method shown in Examples described later (see FIGS. 2 and 7). ).
  • a monomer is further bound to the monomer bound to the solid phase obtained in reaction step 1 to extend the nucleotide chain (see FIG. 5).
  • the protecting group R 6 of the compound represented by the general formula obtained in the reaction step 1 (IV) typically removed under conditions such as Torifuruoro acetate / CH 2 C1 2 to about, represented by the general formula (III)
  • the basic monomer units are reacted in the same manner as in the reaction step 1.
  • ⁇ , ⁇ ,, ⁇ 1 2 ,, ⁇ , ⁇ 1 6 and n are as defined above] can be obtained oligomer represented by.
  • an alkylene group is introduced into the 5 ′ end of the oligomer synthesized in Reaction Step 2 in the presence of a condensing agent (see FIG. 5).
  • R 7 is the same as R 3 in the general formula (II), or represents a protecting group or a functional group such as intercalation such as acridine,
  • R 8 may be different from each other, and may be an alkyl group such as a methyl group, an ethyl group, an isopropyl group or a t-butyl group, an aryl group such as a phenyl group, a methoxy group, an ethoxy group, or an isopropoxy group.
  • Alkoxy groups such as t-butoxy group, phenoxy group, aryloxy groups such as hexafluorophenoxy group, N, N-dimethylamino group, N, N-methylethylamino group, N, N-methylethylamino group And N, N-dialkylamino groups such as N, N-diphenylamino groups, N, N-diarylamino groups such as N, N-diphenylamino groups, pyrrolidinyl groups, biperidinyl groups, and cyclic amine residues such as monophonyl groups. Represents a virolidinyl group.
  • X 3 is a halogen atom such as a chlorine atom or a bromine atom
  • R represents a hydrogen atom, a halogen atom or N0 2.
  • R represents a hydrogen atom or a N0 2.
  • R represents a hydrogen atom or a nitrophenyl group.
  • Azolide groups such as
  • i represents a hydrogen atom or a phenyl group.
  • R represents a halogen atom which may be different from each other, CF 3 or NO 2.
  • R 3 is a lower alkyl group having 1 to 6 carbon atoms such as a methyl group and an ethyl group], for example,
  • the H-phosphonate oligonucleotide derivative synthesized in the solid phase in the reaction step 3 is separated from the solid phase under basic conditions, deprotected by hydrolysis, and the H-phosphonate oligonucleotide derivative is simply synthesized. This is the separation step (see Fig. 6).
  • the compound represented by the general formula (VI) obtained in the reaction step 3 was converted into ⁇ , ⁇ -bistrimethylsilyl trifluoroacetamide-provylamine-acetonitrinole (1: 2: 2, ⁇ / ⁇ / ⁇ ) or the like, usually at room temperature for 30 minutes to 1 hour, then the solid phase carrier is removed by filtration, the filtrate is evaporated under reduced pressure, and then acetonitrile-water (1 : 1, ⁇ / ⁇ ) or dioxane-water (1: 1, ⁇ / ⁇ ) or THF-water (1: 1 ⁇ / ⁇ ) at room temperature for 5 minutes to give the present invention.
  • the ⁇ -phosphonate oligonucleotide derivative represented by the general formula (I) can be obtained.
  • the ⁇ -phosphonate oligonucleotide derivative of the present invention obtained by the above reaction steps has the following features.
  • the ⁇ -phosphonate oligonucleotide derivative of the present invention has less steric hindrance at the phosphate site than conventional ones, and when used as an antisense, easily forms a double strand with the target gene, and has a phosphorothioate type.
  • DNA and methylphosphonate DNA Further, it is more advantageous than natural DNA. Also, since it is a neutral molecule, it does not generate electrostatic repulsion with the negative charge of phosphate of the complementary strand when forming a double strand, so it can form a more stable duplex than natural DNA. It is advantageous.
  • the phosphate moiety of DNA does not have a negative charge
  • the double strand formed with RNA is unlikely to be a substrate for RNaseH, an enzyme that recognizes the negative charge of DNA and degrades A.
  • the selectivity of the antisense effect on the target gene is advantageous over phosphorothioate-type DNA.
  • it since it has higher water solubility than methylphosphonate-type DNA, it can be administered to cells at a high concentration.
  • the H-phosphonate oligonucleotide derivative of the present invention is particularly useful as an antisense, for example, a messenger RNA produced upon propagation of a virus such as HIV, HCV, HCMV, HSV, HTLV, etc.
  • Messenger RNAs encoding proteins essential for the survival of the virus for example, HIV TAR, U5, rev, rtatj (O. Zelphati, et al., Antisense Res. Dev., 3, 323 (1993), B. Bordier, et al., Nucleic Acids Res., 20,5999 (1992)), HCV "5 to UTR" (M.
  • HSV "IE110", “UL30", “UL37”, “UL37j (A. Peyman, et al., Biol. Chem. Hoppe Seyler, 376, 195 (1995), JASmith, et al., J. Virol, 69, 1925 (1995)), as a virus growth inhibitor having a nucleotide sequence complementary to the "tax" of HTLV-1, etc., or acute and chronic leukemia, liver cancer, breast cancer, colorectal cancer, etc.
  • Messenger RNA specifically generated during the growth of tumor cells usually messenger MA encoding a protein essential for the survival of the target tumor cells, such as AML-1 in acute leukemia (C.
  • This antisense comprises, as an active ingredient, usually an oligonucleotide derivative represented by the general formula (I) having a length of about 4 to 30, preferably about 10 to 20.
  • an oligonucleotide derivative represented by the general formula (I) having a length of about 4 to 30, preferably about 10 to 20.
  • the H-phosphonate oligonucleotide derivative of the present invention does not require the use of a protective group particularly in the base and phosphate groups due to its synthetic chemistry, the synthesis reaction process is shortened and is suitable for mass synthesis. .
  • FIG. 1 is a diagram showing the behavior of an H-phosphonate nucleotide under basic conditions.
  • FIG. 2 is a diagram showing a method for synthesizing a monomer as a unit of an H-phosphonate oligonucleotide.
  • FIG. 3 is a diagram showing a method for synthesizing a 5 ′ terminal phosphonate.
  • FIG. 4 is a diagram showing a method for introducing a linker into a solid phase.
  • FIG. 5 is a diagram showing a step of solid-phase synthesis of an H-phosphonate oligonucleotide.
  • FIG. 6 is a view showing a step of removing a protecting group from a solid-phase synthesized H-phosphonate oligonucleotide.
  • FIG. 7 is a diagram showing a method for synthesizing a monomer that is a unit of an H-phosphonate oligonucleotide.
  • FIG. 8 is a diagram showing a method for synthesizing 5, terminal phosphonate.
  • FIG. 9 is a diagram showing a method for introducing a linker into a solid phase used in the present invention.
  • This mixture was diluted with MeOH (5 mL), and 1 M of triethylammonium hydrogen carbonate (10 mL) was added. The mixture was washed 3 times with Et 2 0 (10 mL x 3 ), and back extracted the organic phase with 1 M of Bok Li ethyl ammonium Niu arm hydrogencarbonate carbonate (Triethyla ⁇ onium hydrogencarbonate).
  • This aqueous layer (The aqueous layer) and the washings (washings) were mixed and extracted three times with CHC-MeOH (2: 1, v / v, 10 mL x 3). The aqueous phase CHC1 3 - MeOH (2: 1 , v / v) several times were back extracted with.
  • 1,6-Hexanediol (0.104 g, 1 olol) was dried by repeated co-dehydration using anhydrous pyridine, and finally dissolved in anhydrous pyridine (10 mL). Dimethoxytrityl chloride (0.373 g, 1.1 ol) was added to the solution, and the mixture was stirred at room temperature for 3 hours. The mixture was diluted with CHC1 3, washed three times with 5% NaHCO 3, and back extracted the aqueous phase with CHC1 3.
  • the aqueous phase and washings (washings) were combined, extracted 3 times with CHC1 3, was back extracted the organic phase with CHC1 3.
  • the residue was passed through a silica gel column (30 g silica gel). By applying a gradient of methanol (0-4) was subjected to chromatography using a CHC1 3 containing 0.5% Torichiruamin. The fractions containing "3" were combined, concentrated and dried.
  • filters were performed (10 ⁇ X 50 ⁇ ) manually with each cycle of chain extension are detritylation (detritylati on) (CH 2 C1 1% TFA in 2; 15 seconds), washed (CH 2 C1 2, then pyridine), (0.1 M monomers in one pyridine ( "2a”, “2b”, “2c") cup-ring and 0.5 M of BDPP; 2 min), washing (pyridine), tubing (0.1 M triethylammonium methylphosphonate, 0.5 M BDPP in pyridine; 2 min), washing (pyridine, then CH 2 Cl 2 ).
  • trimethylammonium 6- (dimethoxytrityloxy) hexylphosphonate (triethyla onium 6- (dime thoxytrityloxy) hexyl phosphonate) (3) is converted into monomer units (“2a”, “2b”). ”And“ 2c ”) (Fig. 5).
  • the average yield per cycle was estimated to be 96-99% by DMTr analysis. After chain extension, it was removed by treating 15 seconds DMTr group in 1% TFA in CH 2 C1 in 2, CH 2 C1 2, then washed with CHaCN.
  • CAGT tetranucleotide H-phosphonate
  • tetranucleotide H-phosphoneone is obtained from l ⁇ mol of 6- (dirmethoxytrityloxy) hexyl oxalate bound to LCM-CPG. (31.0 A2 60 units) was obtained at a yield of 84 °.
  • Example 2 Chemical synthesis of H-phosphonate oligodeoxyribonucleotide Chemical synthesis of H-phosphonate oligodeoxyribonucleotide was performed under different reaction conditions from Example 1.
  • the mixture was diluted with pyridine (5 mL) and 1 M triethylammonium hydrogen canolebonate (1 O mL) was added.
  • the mixture was washed 5 times with Et 2 0, the organic phase 1 M Toryechiru Anmoniumuhi Dorogen force Honoré non non of - back-extracted with preparative (triethylammonium hydrogencarbonate).
  • the aqueous phase and washings were mixed and extracted three times with CHC1 3, was back extracted the aqueous phase with CHC1 3.
  • the organic phase and washings were combined, dried through Na 2 S0 4, filtered and reduced pressure to dryness and concentrated.
  • the residue was passed through a silica gel column (30 g silica gel).
  • the CHC1 3 containing 4% Torichiruamin 27 was used for chromatography.
  • the fractions containing "2a” were combined, concentrated and dried.
  • the product was dissolved in CHC1 3 (10 mL), and lavage with 1 M tri E chill ammonium Niu arm hydro Genkanorebone Bok of (Triethyla thigh onium hydrogenca bonate) (10 mL) , to remove the sheet Rikageru.
  • the aqueous phase was back extracted with CHC1 3, mixing an organic phase and washings (washings), dried through Na 2 S0 4, filtered under reduced pressure, concentrated to dryness, "2a” (0.5 89 g, 82%) as a colorless foam.
  • Method B ( Figure 7): A solution of phosphonic acid (0.082 g, 10 ml, dried by repeated co-dehydration with anhydrous pyridine) in anhydrous pyridine (10 mL) was added to bis (2-oxo). -(3-oxazolidinyl) phosphinic chloride (bis (2-OXO-3-oxazolidinyl) phosphinic chloride) (1.40 g, 5.5 t ol) was added. The reaction mixture was stirred at room temperature for 20 minutes. To this mixture was added 5'-0-dimethyloxytrityldeoxyadenosine (la) (la) (0.554 g, 1 t ol).
  • 5′-0-dimethoxytrityldeoxycytidine (5, -0-dimethoxytrityldeoxycytidine) (lc) (0.570 g, (0,708 g, 94%) as a colorless foam.
  • 1,5-Pentanediol (0.104 g, 1 ol) was dried by repeated co-dehydration using anhydrous pyridine, and finally dissolved in anhydrous pyridine (10 mL). To this solution was added dimethoxytrityl chloride (0.373 g, 1.1 ol) and the mixture was stirred at room temperature for 3 hours. The mixture was diluted with CHC1 3, washed three times with 5% NaHCO 3, and back extracted the aqueous phase with CHC1 3. That 30 Combine the organic phase and the washings, dry over Na 2 SO 4 , filter, concentrate and dry to dryness, and dry 5- (dimethoxytrityloxyl) pentanol. Was obtained as a crude mixture.
  • 1,5-Pentanediol (0.104 g, 1 mol) was dried by repeated co-dehydration using anhydrous pyridine, and finally dissolved in anhydrous pyridine (10 mL). To this solution was added dimethoxytrityl chloride (0.373 g, 1.1 mmol), and the mixture was stirred at room temperature for 3 hours. The mixture was diluted with CHC1 3, washed three times with 5% NaHCO 3, and back extracted the aqueous phase with CHCl.
  • Tetranucleotide H-phosphonate (1 mol) bound to LCAA-CPG via oxalyl linker is converted to 1 M N, 0-bis (dioxane) in dioxane.
  • the reaction was carried out at room temperature for 2 hours with trimethylsilyl) acetoamide (N, 0-bis (trimethyl hylsilyl) acetamide) and then for 10 minutes with gaseous formaldehyde. After washing with dioxane, the mixture was treated in n-PrNHr "ethanol (1: 4, v / v) at room temperature for 30 minutes.
  • the CPG gel was removed by filtration, and the filtrate was concentrated under reduced pressure. Purification of the product by reverse phase HPLC
  • Tetranucleotide H-phosphonate (1 mol) bound to LCAA-CPG via oxalyl linker is converted to 1 M N, 0-bis (trimethylsilyl) acetamide-Me 2
  • the reaction was performed with S ⁇ BH 3 (5: 2, v / v) at room temperature for 1 hour. After washing with methanol, it was treated in n-PrNH 3 -methanol (1: 4, v / v) at room temperature for 30 minutes. The CPG gel was removed by filtration, and the filtrate was concentrated under reduced pressure.
  • the crude product was purified by reverse phase HPLC to obtain tetranucleotide boranophosphonate.
  • the H-phosphonate oligonucleotide derivative of the present invention has properties such as easy formation of a double strand with a target gene, resistance to phosphodiesterase, and high efficiency of cell uptake.
  • the H-phosphonate oligonucleotide derivative of the present invention is expected to be used particularly as an antisense nucleic acid.

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Abstract

L'invention concerne un nouveau dérivé oligonucléotidique H-phosphonate, un procédé de synthèse de ce dérivé, consistant à synthétiser, à l'aide d'un processus en phase solide, un oligomère possédant un acide alcoxyphosphonique présentant un groupe alkylène à longueur de chaîne moyenne au niveau de chaque extrémité 3' et 5', afin d'obtenir un rendement élevé d'un oligonucléotide H-phosphonate de synthèse, tout en évitant la décomposition de celui-ci en présence de conditions basiques, lors de la synthèse. Ce dérivé peut former un double brin avec le gène cible, il est résistant aux phosphodiestérases et il s'incorpore efficacement dans des cellules. On a notamment l'intention d'utiliser ce dérivé comme un antisens.
PCT/JP1997/004128 1996-11-13 1997-11-12 Derive oligonucleotidique h-phosphonate et procede de production associe WO1998021226A1 (fr)

Priority Applications (1)

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AU49648/97A AU4964897A (en) 1996-11-13 1997-11-12 H-phosphonate oligonucleotide derivative and process for producing the derivative

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JP8/301430 1996-11-13
JP30143096 1996-11-13

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995023160A1 (fr) * 1994-02-23 1995-08-31 Isis Pharmaceuticals, Inc. Nouveaux composes oligomeres de phosphoramidate et de phosphorothiomidate
WO1996022297A1 (fr) * 1995-01-18 1996-07-25 Pharmagenics, Inc. Oligomeres d'ester de phosphore non nucleotidiques

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995023160A1 (fr) * 1994-02-23 1995-08-31 Isis Pharmaceuticals, Inc. Nouveaux composes oligomeres de phosphoramidate et de phosphorothiomidate
WO1996022297A1 (fr) * 1995-01-18 1996-07-25 Pharmagenics, Inc. Oligomeres d'ester de phosphore non nucleotidiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NUCLEIC ACIDS RESEARCH, Vol. 18, No. 8, (1990), A. WILK et al., "Backbone-Modified Oligonucleotides Containing a Butanediode-1,3 Moiety as a 'Vicarious Segment' for the Deoxyribosyl Moiety-Synthesis and Enzyme Studies", p. 2065-2068. *
TETRAHEDRON LETTERS, Vol. 36, No. 46, (1995), K. SCHUTZ et al., "Synthesis of Oligonucleotides Labeled with a Novel Type of Chemically Stable Acridine Dye", p. 8407-8410. *

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