WO1993024512A1 - Homologue d'acide nucleique - Google Patents

Homologue d'acide nucleique Download PDF

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Publication number
WO1993024512A1
WO1993024512A1 PCT/JP1993/000684 JP9300684W WO9324512A1 WO 1993024512 A1 WO1993024512 A1 WO 1993024512A1 JP 9300684 W JP9300684 W JP 9300684W WO 9324512 A1 WO9324512 A1 WO 9324512A1
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Prior art keywords
nucleic acid
solution
present
synthesis
dna
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PCT/JP1993/000684
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English (en)
Japanese (ja)
Inventor
Junichi Yano
Tadaaki Ohgi
Kouichi Ishiyama
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Nippon Shinyaku Co., Ltd.
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Publication date
Application filed by Nippon Shinyaku Co., Ltd. filed Critical Nippon Shinyaku Co., Ltd.
Publication of WO1993024512A1 publication Critical patent/WO1993024512A1/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

Definitions

  • the present invention relates to nucleic acid homologs having a so-called antisense function. More specifically, the present invention relates to a nucleic acid homolog that specifically binds to a nucleic acid of DNA or RNA to form a complex, thereby selectively inactivating, activating, or controlling their genetic information.
  • the gene is a double-stranded DNA consisting of a complementary base sequence, and a significant coding sequence (sense sequence) is coded on one of the strands. Oligonucleotides complementary to this sequence can specifically bind to the sense sequence, thereby blocking gene function in a manner that covers the desired region of interest. . Compounds having such a function are called antisense molecules.
  • Antisense molecules act not only on such DNA binding but also on RNA, and can selectively inhibit the process of protein translation from messenger RNA (Harold M.
  • antisense molecules can be used to elucidate and control biological functions, and to date, the following types of nucleic acids Homologs have been synthesized (Murakami et al., Synthetic Organic Chemistry, Vol. 48, No. 3, P1BO-193 (1990)).
  • ⁇ represents a nucleobase
  • R and R ′′ represent hydrogen, alkyl, or other substituents.
  • R 2 represents hydrogen, an inorganic acid residue, organic acid residue, an alkyl or Ashiru.
  • nucleic acid homologs have been synthesized, including the above compounds.
  • the inventors of the present invention have conducted studies for the purpose of finding a novel and useful synthetic antisense molecule that solves the above problems.
  • ⁇ indicates an integer on 1 ⁇ , provided that when ⁇ is on 2 J3 ⁇ 4, B 2 and B 3 are not limited to the same base.
  • the present inventors have found that a nucleic acid homolog to be solved has solved the above-mentioned problems and has properties as an excellent antisense molecule, and has finally completed the present invention.
  • the antisense molecule of the present invention has extremely excellent properties under the conditions described in (a) above, as compared with the conventional antisense molecule.
  • the nucleic acid homologues of the present invention have extremely good solubility in water and the like, in particular, as compared with conventional antisense molecules (for example, potassium-oligonucleotide derivative (compound I)). Therefore, the nucleic acid homologue of the present invention has a high binding ratio to highly water-soluble DNA or RNA. Therefore, high utility in the medical field can be expected.
  • conventional antisense molecules for example, potassium-oligonucleotide derivative (compound I)
  • Structural features of the nucleic acid homologs of the present invention are characterized by In the case there is an alternating presence of a calcium or thiocarbamate group and a phosphoric or thiophosphate group.
  • the solubility of the nucleic acid homolog of the present invention is considered to be due to this structural feature.
  • the gist of the present invention resides in the structural features of the nucleic acid homolog of the present invention described above.
  • Antisense molecules generally have higher activity and selectivity as the number of bases increases. However, conversely, the greater the number of bases, the stronger the toxicity.
  • N relating to the nucleic acid homolog of the present invention is 1 or more, but in consideration of the balance between activity and toxicity, 1 to 20 is preferable, and 3 to 10 is more preferable.
  • nucleotide sequence of the nucleic acid homolog of the present invention that is, the type of bases of BB 2 , B 3 , differs depending on the nucleotide sequence of DNA or RNA to be inactivated, activated or controlled.
  • the double-stranded nucleic acid derivative shows a characteristic C-type compared to many double-stranded nucleic acid derivatives having a B-type I have.
  • the reason for this is not clear, but the structural transition from type B to type C occurs only when natural DNA with random nucleotide sequence is placed under special conditions (such as high salt concentration or low humidity). Therefore, it is inferred that they form a three-dimensional structure under similar conditions.
  • the nucleic acid homologue of the present invention has a pharmacological action such as a P protein synthesis inhibitory action in HelaS3-MDR1 cells, and is therefore extremely useful as, for example, an anticancer agent.
  • the compound of the general formula [I] gives an optically pure active substance when Y is either S or 0.
  • the compound has two optical isomers (diastereomers) for each thiophosphoric acid bond, and any compound is included in the present invention. is there.
  • the nucleic acid homologs of the invention have been found to be less toxic.
  • the nucleic acid homolog of the present invention When used as a medicament, the compound of the present invention is administered as it is or in a pharmaceutically acceptable nontoxic and inert carrier.
  • the carrier one or more liquid, solid, or semi-solid diluents, fillers, and other prescription auxiliaries are used.
  • the pharmaceutical compositions are preferably administered in dosage unit forms.
  • the nucleic acid homologs of the present invention can be administered orally, intraosseously, topically, or rectally. Needless to say, the composition is administered in a dosage form suitable for these administration methods, for example, various oral preparations, injections, inhalants, eye drops, softeners, suppositories, and the like. In particular, intra-tissue administration and local administration are preferred. (Synthesis method)
  • B ′, B 2 and B 3 are the same or different and are hypoxanthine, thymine, peracyl, N-benzoyladenine having a protected base, and N-di having a base protected. It represents sobutyrylguanine or N-benzoylcytosine whose base moiety is protected.
  • X 0 represents S or 0.
  • n represents an integer of 0 or more. However, when n is 2 or more, B 3 and B 3 are not determined by the same base.
  • Step 3 When _ obtained in Step 1 and _ obtained in Step 2 are mixed in a dimethylformamide or pyridine solution at room temperature, the mixture is quantitatively condensed to obtain dimer _ ⁇ .
  • 5'-D-Dimethoxytrityl thymidine_1 (manufactured by Wako Pure Chemical Industries, Ltd.) 3.95 g is dissolved in 36 ml of pyridine, and 1, ⁇ -thiocarbyldimidazole is dissolved.
  • Solution A 50 mM TBAA (PH 7) buffer 100 ml
  • Solution B 40% cetonitrile / 50 mM TEAA (pH 7) buffer 100 ml ⁇ 3 ⁇ 4'0 ⁇ 100 .
  • Only the fractions that develop the color of the DMTr group (the color becomes orange due to the acid added) are collected, the solvent is distilled off under reduced pressure, treated with 80% acetic acid (5 ml), and treated at room temperature for 15 minutes. After standing, confirm that the DMTr group has been removed by TLC check, and then distill off the solvent under reduced pressure. Separate and extract with ethyl acetate-water, and separate the aqueous layer.
  • C bz represents N-pentylcytosine.
  • the resin was then deprotected with a 0.3 M aqueous solution of dioxane (CB Reese et al. Tetrahedron Lett., 2727 (1978)) in dioxane at room temperature for 24 hours at room temperature. After washing with, the washing solution was concentrated to dryness, 5.0 ml of concentrated ammonia water ZDMF (1/1) was added, and the solution was deprotected again at room temperature for 24 hours.
  • dioxane CB Reese et al. Tetrahedron Lett., 2727 (1978)
  • Fig. 2 shows the HPLC chromatogram of this target compound.
  • a solution 100 mM TBAA (pH 7) buffer 100 ml
  • B solution 35% acetonitrile Z50 mM TBAA (pH 7) buffer 100 ml
  • Elution was performed with a gradient of 0 ⁇ 100. Only the fractions that develop the color of the DMTr group (it becomes orange due to acetic acid) are collected, the solvent is distilled off under reduced pressure, treated with 80% acetic acid (5 ml), and left at room temperature for 30 minutes. After checking that the DMTr group had been removed by a check, the solvent was distilled off under reduced pressure.
  • test was carried out under the conditions of 50 mM trinitrile TBAA (PH 7.0), B3 ⁇ 4: 0 ⁇ 50 (15 minutes), gradient, and flow rate of 1 mlZ.
  • the solubility in water was 5 tnM or more.
  • HeLaS3-MDRl cells are seeded on a 6-well plate at a density of 6 ⁇ 10 5 cells / well, and after 24 hours and 48 hours, the test substance is lipofuctin (BRL, registered trademark) (3 jug). / ml final concentration) and added to the cells. After 72 hours, the cells were lysed with a detergent (Triton X-100 NP40), and radioimmunoassay was performed using a monoclonal antibody C219 against human P protein (manufactured by Centco Ryo). The amount of the synthesized P protein was measured by radioactivity using an image analyzer (BAS2000). table 1
  • Antisense nucleic acid consisting of only phosphothioate in "": the same sequence (references Stec. W. J. et al., J. Am. Chem. Soc.
  • As the control a natural DNA oligomer having the same sequence was used.
  • Each sample was prepared at a concentration of 25 / xM in 30 mM acetate buffer ( ⁇ 4.6), 50 mM sodium chloride, and Im-zinc chloride solution.
  • the present invention substance "to form a complex of (Example 2 materials) and complementary thereto native Doki Shi Li Boo Li Gonuku Reochi de (3 'AGAAACTCGAAC 5'), ' a ⁇ by that shown in FIG. 9 Melting curves were obtained. This melting curve was measured under the conditions of a sample concentration of 65 ⁇ , 0.15% sodium chloride, 10M sodium phosphate (PH 7.0), and a heating rate of 0.5 minutes.
  • the substance of the present invention strongly formed a complex (Tm 40.9 t).
  • the compound When the right-handed circularly polarized light and the left-handed circularly polarized light have different molar absorption coefficients in the absorption band of the compound, the compound is said to exhibit circular dichroism (referred to as “CD”). Determine the three-dimensional structure and conformation, especially for nucleic acids
  • Natural DNA is known to take Form B under physiological conditions from the results of CD measurement (VI Ivanov et al., Biopolymer 12, 89-100 (1973)) ⁇
  • the CD curve (FIG. 10) of the complex of the substance of the present invention and the complementary deoxyribonucleotide is shown in the figure for a conjugate having the same sequence instead of the substance of the present invention.
  • the case using natural DNA (Fig. 11) is also shown.
  • This CD curve was measured under the conditions of a sample concentration of 65%, 0.15M sodium chloride, 10mM sodium phosphate (PH 7.0), and a temperature lOt.
  • FIG. 1 shows a ⁇ -matogram of high-speed liquid chromatography (HPLC) of the substance of the present invention obtained in Example 1.
  • the vertical axis represents absorbance (260 nm), and the horizontal axis represents time (minutes).
  • FIG. 2 shows a high-speed liquid chromatography (HPLC) chromatogram of the substance of the present invention obtained in Example 2.
  • the vertical axis represents absorbance (260 nm), and the vertical axis represents time (minutes).
  • FIG. 3 shows a chromatogram of high-speed liquid chromatography (HPLC) of the substance of the present invention obtained in Example 3.
  • the vertical axis represents absorbance (260 nm), and the vertical axis represents time (minutes).
  • FIG. 4 shows a chromatogram of high-speed liquid chromatography (HPLC) of the substance of the present invention obtained in Example 4.
  • the vertical axis represents absorbance (260 ntn), and the vertical axis represents time (minutes).
  • FIG. 5 shows a chromatogram of high-speed liquid chromatography (HPLC) of the natural DNA oligomer before addition of the enzyme, which was checked in Test Example 2.
  • the vertical axis represents absorbance (260 nm), and the horizontal axis represents hours M (minutes).
  • Figure 6 shows the natural DNA polymerase after addition of the enzyme, which was checked in Test Example 2.
  • Figure 1 shows a high-performance liquid chromatographic (HPLC) chromatogram. The vertical axis represents absorbance (260nro), and the horizontal axis represents time (minutes).
  • FIG. 7 shows a high-speed liquid chromatographic (HPLC) chromatogram of the substance of the present invention before addition of the enzyme, which was tested in Test Example 2.
  • the vertical axis represents absorbance (260 nm), and the horizontal axis represents time (minutes).
  • FIG. 8 shows a high-performance liquid chromatographic (HPLC) chromatogram of the substance of the present invention after addition of the enzyme, which was checked in Test Example 2.
  • the vertical axis represents absorbance (260 nm), and the horizontal axis represents time (minutes).
  • FIG. 9 shows the melting curve measured in Test Example 3.
  • the vertical axis represents relative absorbance, and the horizontal axis represents temperature.
  • FIG. 10 shows a CD curve of a complex of the substance of the present invention and a complementary dextrin liponucleotide measured in Test Example 3.
  • the vertical axis represents the molecular ellipticity, and the horizontal axis represents the wavelength.
  • FIG. 11 shows the CD curve of the natural DNA duplex measured in Test Example 3.
  • the vertical axis represents the molecular ellipticity, and the horizontal axis represents the wavelength.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
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Abstract

Nouvelle molécule antisens utilisable comme médicament, pour combattre les infections causées par l'herpèsvirus, le virus de la grippe, le virus de l'immunodéficience acquise ou d'autres virus analogues et pour enrayer l'action des gènes du cancer. Cet homologue de l'acide nucléique est représenté par la formule générale (I), dans laquelle X et Y peuvent être identiques ou différents l'un de l'autre et représenter chacun S ou O; B?1, B2, B3 et B4¿ peuvent être identiques ou différents les uns des autres et représenter chacun l'adénine, la guanine, l'hypoxanthine, la cytosine, la thymine ou l'uracil; et n représente un entier égal à 1 ou plus, étant entendu que B2 et B3 ne constituent pas nécessairement une seule et même base lorsque n est égal à 2 ou plus.
PCT/JP1993/000684 1992-05-25 1993-05-24 Homologue d'acide nucleique WO1993024512A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP4/158652 1992-05-25
JP15865292 1992-05-25
JP4/231445 1992-08-05
JP23144592 1992-08-05

Publications (1)

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WO1993024512A1 true WO1993024512A1 (fr) 1993-12-09

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF ORGANIC CHEMISTRY, Vol. 42, No. 4, p. 703-706, (1977), W.S. MUNGALL et al., "Carbamate Analogues of Oligonucleotides". *
NUCLEOSIDE & NUCLEOTIDES, Vol. 9, No. 2, p. 259-273, (1990), M.J. DE VOS et al., "Solid Phase Nonisotopic Labeling of Oligodeoxy Nucleotides Using 5'-Protected Aminoalkyl Phosphoramidates". *
ORGANIC SYNTHETIC CHEMISTRY, Vol. 48, No. 3, pages 180 to 193, March 1990, MURAKAMI, MAKINO, "Chemical Synthesis of Functional Anti-Sense DNA". *

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