WO1996018640A9

WO1996018640A9 -

Info

Publication number: WO1996018640A9
Authority: WO
Publication date: 1998-07-16

Description

Specification

TECHNICAL FIELD The present invention relates to an antisense nucleic acid analogue useful as a pharmaceutical. More specifically, by specifically binding to DNA or RNA and forming a complex, transmission of gene information carried by the DNA or RNA is selectively inactivated, activated or The present invention relates to an antisense molecule having a function to control. BACKGROUND ART A gene usually has a form of double-stranded DNA consisting of complementary base sequences, and a coding sequence (sense sequence) having a meaning as amino acid sequence information is encoded on one of the strands. An oligonucleotide complementary to this sequence can specifically bind to the sense sequence, and in the case where this binding is strong, gene function is selectively inactivated by closing a target constant region. , Can be activated or controlled. A compound having such a function is called an antisense molecule.

Antisense molecules can thus bind to DNA and inhibit the process by which messenger RNA is generated. It can also bind to RNA and selectively inhibit the process of translation of protein from messenger RNA (Harold M. Weintraub, Scientific American, 34-40 1990). Based on the above principle, an antisense molecule can be useful for elucidating and controlling biological functions, and thus represented by the formulas [10 1] to [1 08] and [1 1 1] to [1 12] Among them, B is a nucleobase, R and R ′ each represent hydrogen, an alkyl group or another substituent). A nucleic acid analogue has been synthesized (Murakami et al., Synthetic Organic Chemistry, vol. 48, No. 3, 180-193. 1990; Chapter Murakami, Cell Engineering, Vol. 13, No. 4, 259-266 1994).

(A) DNA type oligonucleotide derivative (partial structure is shown in the formula [10 1]) (B) RNA type oligonucleotide derivative (a partial structure is shown in Formula [102])

(C) Phosphoric acid triester type oligonucleotide derivative (The partial structure is shown in Formula [103])

(D) Methylphosphonate type oligonucleotide derivative (The partial structure is shown in the formula [104])

(E) Phosphoroamidate type oligonucleotide derivative (The partial structure is shown in the formula [105])

(F) Phosphorothioate-type oligonucleotide derivative (a partial structure is shown in Formula [106])

(G) Phosphorus oxide type oligonucleotide derivative (The partial structure is shown in the formula [107])

(H) Force Lubamate-Type Oligonucleotide Derivative (The partial structure is shown in the formula [108])

(I) Thiocarbamate-type Oligonucleotide Derivative (The structure is shown in Formula [109]) (J) Thiocarbamate-phosphodiester-type oligonucleotide derivative (Structure is Formula

[1 10])

(K) Hi-aminoa type oligonucleotide derivative (partial structure is shown in formula [1 1 1]) (ぃ enantio type oligonucleotide derivative (partial structure is shown in formula [1 12]) (M) Other non-phosphate Bonded oligonucleotide derivative (partial structure is shown in formulas [1 13] to [1 16]

All of them are aimed at providing an antisense molecule that functions in vivo by modifying the natural nucleic acid structure (partial structural formula [1]).

Formula [101] Formula [102]

Moth

Next, structural formula [109] is represented. In the formula, Β represents a nucleobase, and Ri, R ₂ represents hydrogen, an inorganic acid residue, an organic acid residue, an alkyl group, or an asyl group. n represents a natural number.

ヽ 0 ·

8/96 O-I

J

Next, Formula [1 1 3], Formula [1 1 4], Formula [1 1 5], Formula [1 1 6] are shown. In the formula, B represents a nucleobase. R represents an alkyl group or a phenyl group.

Formula [1 1 3] Formula [1 1 4]

Formula [1 1 5] Formula [1 1 6] Other nucleic acid homologs have been reported (John A. Montgomery and Kathleen Hewson., J. Heterocycl. Chem. 7 Li. Apr. 443-445 1970; Antonium Chem. Co. im., 35 81-88 1970; Michael W. Winkley., Carbohyd. Res., 31 45-254 1973.), all of which are applicable to antisense. Not disclosed.

In addition, many other nucleic acid homologs have been synthesized including the above-mentioned compounds (for example, nucleic acid homologs described in US Pat. No. 5,034,506), but a satisfactory effect is necessarily obtained. It has not been developed to develop highly practical antisense molecules fully equipped with the following conditions 1) to 8). 1) Stability of binding to DNA or RNA (complex formation ability)

2) Resistance to nucleolytic enzymes

3) Physical and chemical stability against acid, alkali, temperature and humidity

4) Low cytotoxicity

5) Binding specificity to the recognition sequence

6) Simplicity of synthesis

7) Solubility in water or buffer

8) Preparation without generation of asymmetric phosphorus, that is, generation of an asymmetric phosphorastereomer accompanying modification of a phosphate ester moiety, which is a drawback of the conventional preparation method, causes more isomerism as the nucleic acid becomes longer. To eliminate the relative decrease in the content of useful molecules due to the formation of a body (2 isomers ^{per 1} n; here, n represents a natural number) Disclosure of the Invention A high level of practicality fully equipped with the above conditions 1) to 8).

As a result of intensive studies aimed at the development of molecules, the following partial structural formula [1]

[1]

(Wherein, B represents adenine-9-yl, guanine-9-yl, hypoxanthine-9-yl, thymine-1-yl, uracil-1-yl or cytosine-1-yl XY is the same or different and represents a nucleoside analogue represented by hydrogen, hydroxy, halogen or alkoxy), and a DNA RNA or phosphodiester bond is It has been found that an antisense molecule containing at least one or more in the same strand of a modified or substituted nucleic acid homologue solves the above purpose and has properties as an excellent antisense molecule, and the present invention has been completed. The

Examples of the halogen contained in the nucleoside represented by the partial structural formula [1] include chlorine, bromine and fluorine. Examples of lower alkoxy include linear or branched ones having 1 to 8 carbon atoms. Specifically, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy or t-butoxy can be mentioned.

The antisense molecule of the present invention can exhibit resistance to various nucleolytic enzymes by including a nucleoside having the partial structure represented by the partial structural formula [1]. In particular, molecules having the nucleoside at the 5 'end or 3' end or at both ends exhibit remarkable resistance to hydroxylase. Further, those containing a plurality of, for example, 2 to 9 residues of the nucleoside analogues continuously or intermittently near both ends can be expected to exhibit stronger resistance.

The nucleoside included in the present invention is particularly soluble in water or the like, as compared to a component of a conventional antisense molecule (e.g., a force carbamate-type nucleoside that constitutes a force carbamate-type oligonucleotide derivative (formula [10 8])). Sex is extremely high. Therefore, the solubility in water and the like does not significantly decrease even if the nucleic acid analogue in which the D NA, R N A or phosphodiester bond is modified or substituted is contained in an unlimited amount of one or more residues.

It is less preferred that a suitable number of the nucleoside is contained in natural D NA or R N A, since the degree of freedom in the steric structure is less limited, the association is stabilized and the function as an antisense molecule is easily exerted.

The content of the nucleoside is suitably 1 residue or more and 80% or less of all residues constituting the molecule in one antisense nucleic acid analogue. It is preferable to have one or more residues and an equal number of consecutive nucleosides at each end of 5 'and 3', and the composition ratio is preferably 60% or less of all nucleoside residues of the contained molecule. is there.

In addition, when the structure of the sugar moiety of the nucleoside is identical to that of the other nucleic acid (for example, ribose or deoxyribose) constituting the contained antisense nucleic acid analogue, the homogeneity of the steric helical structure of the side chain is In particular, binding to the recognition sequence It is expected to be superior to the opposite sex. On the other hand, resistance to degradative enzymes is considered to be larger if the structure of the sugar moiety is different from that of the natural form, and should be appropriately selected according to the necessity.

The antisense molecule of the present invention is compared with the conventional antisense molecule, while keeping the conditions of 1) above, 2), 3), 4), 5), 6), 7). It has extremely excellent properties.

The antisense molecule of the present invention has extremely high solubility in water etc., particularly compared to conventional antisense molecules (eg, carbamate-type oligonucleotide derivatives (Formula [10 8])). Antisense molecules of the present invention can be expected to be highly useful in the medical field because they have a high proportion of binding to highly water-soluble DNA or RNA.

When administered in vivo, the antisense nucleic acid analogue of the present invention, in particular, forms a complex with the cationic ribosome, thereby blocking its transfer into cells, and is equivalent to or more than a nucleic acid such as natural DNA. Demonstrate an antisense effect.

In cases where stronger resistance to degrading enzymes is required, for example,

It is also possible to combine with modification or substitution of [1 0 1] to [1 0 6] phosphodiester bonding moiety. The phosphorothioate bond, the alkyl phosphorothioate bond, the N-alkyl phosphoroamidate bond, the phosphorodithioate bond, the alkyl phosphoneate bond and the like as the modified or substituted form of the phosphate ester bond However, short chain alkyl or cycloalkyl structures are also useful. Of course, other bonds can be selected as needed.

Antisense molecules generally have higher activity and selectivity as the number of bases increases. The number of bases contained in one molecule of the antisense nucleic acid analogue of the present invention is preferably 1 to 50, more preferably 4 to 30.

The nucleotide sequence of the antisense nucleic acid homolog of the present invention is selected to be complementary to the nucleotide sequence of DNA or RNA to be inactivated, activated or controlled.

Antisense molecules of the invention are known to be substrates for aseH. This property is shown to be involved not only in gene inactivation but also in actively degrading hybrid RNAs when administered in vivo, and has high utility. As described later, since the antisense molecule of the present invention has a pharmacological action such as an inhibitory action on synthesis of a membrane receptor protein in HER-2 expressing cells, it is very useful as, for example, an anticancer agent.

Since the antisense molecule of the present invention inactivates the complementary genetic information of the antisense molecule according to the present invention, it is possible to use foreign substances that have invaded the living body, for example, hepes virus, influenza virus, human virus It can be used as a medicine to suppress the transmission of oncogenes and infections such as immunodeficiency virus (HIV, AIDS virus etc.). In addition, they can actively control animal and plant genes and be applied to techniques such as variety improvement. Furthermore, it has the potential as a DNA probe for analysis of gene function, infection check of hereditary diseases, bacteria and viruses, etc. It is also possible to find many other uses besides the above description.

The nucleoside, which is a main component of the antisense nucleic acid homologue of the present invention, is as low in cytotoxicity as natural nucleosides, and thus, the nucleic acid which is also naturally toxic to the antisense molecule in which the nucleoside is contained in DNA or RNA. Equally low.

When the antisense molecule of the present invention is used as a pharmaceutical, the compound of the present invention is administered as it is or in a pharmaceutically acceptable non-toxic and inert carrier. As the carrier, one or more of liquid, solid or semi-solid diluent, filler, and other processing aids are used. The pharmaceutical composition is preferably administered in dosage unit form. The antisense molecules of the present invention can be administered orally, systemically, topically or rectally.

Needless to say, they are administered in dosage forms suitable for these administration methods, for example, various oral agents, injections, inhalants, eye drops, ointments, suppositories and the like. In particular, intra-tissue administration and local administration are preferred.

The dose varies depending on the type of disease, symptoms, age, body weight etc. For example, when used for the treatment of Herpes, topical administration of lmg to lg as an antisense molecule is suitable once a day for adults. . In addition, in the treatment of AIDS, it is general to give 1 mg to 10 g of intravenous drip infusion as an antisense molecule once a day to adults.

The administration method and dose depend on the therapeutic purpose and the type of the antisense molecule of the present invention to be used. It should be changed as appropriate.

Synthetic Methods Next, general methods for synthesizing antisense nucleic acid homologs of the present invention will be described.

In the following formula, B is adenine-9-yl, guanine-9-yl or cytosine-1-yl, and Bp is a protected adenine-9-yl, guanine-9-yl or cytosine- 1-yl or hypoxanthine-9-yl, thymine- 1-yl or uracil- 1-yl which is not protected.

(Nucleoside derivative)

(Amidite reagent)

[1 1] [12] [13] [14] (Preparation of controlled pore glass carrier)

(Solid phase synthesis by phosphate method:

ANTISENSE DNA

[14] C I 7] C I 8]

Procedure 1 Homonuclear nucleoside [11] prepared by the method of Ohki et al. (W 095/15964), and when B is adenine-9-yl or cytosine-1-yl, for example, in pyridine solution Nucleoside derivatives in which the base portion is protected through the process of reaction with benzyl chloride and, for example, with isobutyryl chloride when B is guanine-yl, hydrolysis with sodium hydroxide solution and then neutralization. 12] can be obtained. When B is hypoxanthine-9-yl, thymine-1-yl or uracil-1-yl, homotypic nucleosides prepared by the method of Oki et al. Can be used directly as Bp.

Operation 2 The reaction of [1 2] obtained in Operation 1 with, for example, 4, 4 -dimethoxytrityl chloride in a pyridine solution, to obtain a 5'-hydroxy protected dimethytril compound [13] Can. Operation 3 Among the solid phase methods, in order to use the reagent for automatic synthesis by the so-called amidite method, for example, a method such as phosphating agent and Kess Yu et al. (H. Koester et al., Nucleic Acids Res., 124539-4557 (1984) to obtain an amide reagent [14]. Operation 4 The dimethyloxytrityl compound [13] obtained in Operation 2 can be reacted, for example, with succinic anhydride in methylene chloride to obtain a succinic acid derivative [15]. Operation 5 The compound [15] obtained in Operation 4 is reacted with, for example, pentachlorophenol and dicyclohexylcarbodiimide (hereinafter abbreviated as DCC) in N, N-dimethylformamide to give an activated form [16 ] Can be obtained. Operation 6 The activated form [16] is reacted with, for example, a control depore glass carrier (hereinafter abbreviated as CPG carrier) in Ν, Ν-dimethyl formamide in the presence of triethlyamine to form nucleosideated CP G Carrier [17] can be obtained. Operation 7 For example, if a commercially available amidite reagent for DNA synthesis and amidite reagent [14] are appropriately combined and a CPG carrier [17] is used, the DNA synthesizer (eg, Perkin Elmer One) can be used to Antisense DNA homolog [18] containing the nucleoside derivative of the invention can be easily synthesized.

After deprotecting treatment according to the protective group used, purification with, for example, preparative HPLC or the like can be performed to obtain the antisense DNA homologue of the present invention of high purity.

Also shown are methods for preparing halogenated antisense nucleic acid homologs.

For example, the homotype nucleoside prepared by the method of Oki et al. (W095 / 15964) is tetraisopropyldisiloxylated at the 3, 5, -position of formula [19] as usual, and then [20] is synthesized, [20 Trifluoromesylation of the 2'-position of] can give cyclonucleoside [21]. By treating this with sodium halide, for example, sodium bromide in DMF, the 2-bromide [22] can be obtained. 2, -Bromide [22] as usual, It can be processed to give 2, -ha alpha genohomonucleoside [23].

: Preparation of nucleic acid homologs;

[1 9] [2 0] [2 1]

[2 2] [2 3] In addition to this, for example, an RNA type or phosphorothioate-linked antisense nucleic acid analogue can be synthesized by the method according to the above.

The compounds according to the invention can be used therapeutically as free phosphoric acid, but can also be used in the form of pharmaceutically acceptable salts according to known methods. As salts, sodium salts and potassium salts can be mentioned.

For example, the alkali metal salt of the compound having free phosphoric acid according to the present invention can be obtained preferably by adding sodium hydroxide or potassium hydroxide or the like in an alcoholic solvent.

The compounds according to the present invention can be obtained by conventional separation and purification means, for example, extraction, concentration, neutralization, It can be isolated and purified by means of filtration, recrystallization, column chromatography, reverse phase chromatography and the like.

Solvates (including hydrates) of the compounds according to the present invention or salts thereof are also included in the present invention. Solvates can generally be obtained by removing excess solvent from the corresponding solvent or a suitable mixed solvent containing the corresponding solvent. BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail by the following Examples, Reference Examples, Test Examples and Comparative Examples, but the present invention is not limited thereto. EXAMPLES Example 1 DNA-type antisense molecules containing nucleosides of the formula [1] in which X 2 H, Y = H: 5, aaaaaAAAAAAAAAAAAaa a3 '(a is a nucleoside analog of which the base is adenine-9- ones I Le, a is unless each represent. noted de O alkoxy adenosine, synthetic internucleoside linkages indicates a phosphodiester bond 3 '→ 5'. the following hA _{1 ()} referred to as a _15).

1) Synthesis of amidite reagent

1-1) Synthesis of Compound [12] (wherein Bp is N ⁶ -benzoyladenine-9-yl)

Among the nucleotide analogues represented by the formula [11] obtained by the method of Ohki et al. (WO 95/15964), 2.60 g of adenine base (hereinafter referred to as homotypic adenosine) suspended in 30 ml of pyridine After stirring and adding 8.43 g of benzyl chloride under ice-cooling, the mixture was allowed to react at room temperature for 1 hour. The above reaction mixture was added to a mixture of 100 ml of chloroform, 70 g of ice and 5.48 g of sodium hydrogen carbonate, and the mixture was separated to separate an organic layer. The remaining aqueous layer is further extracted twice with 50 ml of methylene chloride, and the organic layer is After combining and drying sodium sulfate, the solvent was distilled off under reduced pressure.

To the residue were added 20 ml of pyridine and 30 ml of ethanol, and a mixture of 2N sodium hydroxide solution 4 (kl and ethanol 40 ml) was added under ice-cooling After stirring for 30 minutes at room temperature, 40 ml of 2N hydrochloric acid was added for neutralization After further adding 200 ml of water, the aqueous layer was extracted and partitioned with ether, and the aqueous layer was concentrated under reduced pressure overnight, which was allowed to stand overnight in a cold and dark place to obtain compound [12] (where Bp is N ^6- Benzyl adenine) was obtained as a white precipitate (3.11 g).

1-2) Synthesis of compound [13] (wherein Bp is N ⁶ -benzoyladenine-9-yl)

3.0 g of the compound [12] obtained in 1) was dissolved in 35 ml of pyridine, and 3.03 g of 4,4, -dimethyoxytrityl chloride (Wako Pure Chemical Industries, Ltd.) was added and reacted overnight at room temperature. After 5 ml of methanol was added, the solvent was evaporated under reduced pressure. The organic layer obtained by separation treatment with methylene chloride-water was dried over sodium sulfate and concentrated under reduced pressure to dryness. The residue was purified by silica gel column chromatography (silica gel-120 g, methanol / methylene chloride) to obtain 3.97 g of compound [13] as a white powder.卜 3) Synthesis of compound [14] (wherein Bp is N ⁶ -benzoyladenine-9-yl)

200 mg of the compound [13] obtained in 1-2) is azeotroped with pyridine immediately before use, then with toluene and azeotrope, then with tetrahydrofuran (hereinafter abbreviated as THF), and the inside of the vessel is purged with nitrogen gas Dissolved in 3 ml of anhydrous THF. To this solution was added 0.22 ml of Ν, ピル -diisopropyl ether pyrethlamine, and 0.15 ml of 2-cyanoethyl Ν, クロロ -diisopropyl chloroform (manufactured by Sigma), and the mixture was stirred at room temperature for 30 minutes. The precipitated salt was filtered off with a glass filter, and the filtrate was concentrated under reduced pressure to dryness. The residue was partitioned between ethyl acetate and saturated sodium hydrogen carbonate solution each saturated with nitrogen gas, and the obtained organic layer was dried over sodium sulfate and concentrated under reduced pressure to dryness. The residue was triturated with n-hexane saturated with nitrogen gas to obtain 174 mg of a compound [14] (wherein Bp is for N ⁶ -benzyladenine-9-yl) as a white powder. The molecular weight was measured by high-speed atomic impact mass spectrometry (hereinafter abbreviated as FAB method). The amount was 871. This compound [14] was used below as a reagent for an automatic DNA synthesizer (Perkin-Elmer One).

2) Preparation of CPG carrier for DNA synthesizer

In 3 ml of methylene chloride, 470 mg of the compound [13] obtained in 1-2) was dissolved, 23 mg of 4-dimethylaminopyridine and 105 mg of succinic anhydride were added, and the mixture was stirred at room temperature for 3 hours. 10 ml of methylene chloride and 10 ml of 0.5 M potassium dihydrogen phosphate solution were added thereto to separate, and the organic layer was separated. The organic layer was washed with water, dried over sodium sulfate and evaporated to dryness under reduced pressure. As a result, 509 mg of succinic acid derivative [15] (Bp in the case of N ⁶ -benzyladenine-9-yl) was obtained as a white powder.

Dissolve 500 mg of this succinic acid derivative [15] (provided that Bp is N ⁶ -benzyladenine-9-yl) in anhydrous N, N-dimethylformamide, and 190 mg of pentachlorophenol and DC 200 mg of C was added and stirred overnight at room temperature. The precipitate was filtered off with a glass filter, and the filtrate was concentrated to dryness under reduced pressure. A small amount of benzene was added to the residue and the insolubles were filtered off again. The filtrate was concentrated to dryness under reduced pressure, and powdered with n-pentane to obtain 492 mg of an activator compound [16] as a white powder. The molecular weight was measured by the FAB method to be 1020.

A mixture of 412 mg of this activator compound [16] and 2 g of aminoized CPG (Long chain amino-alkyl CPG 500 onc, steam, Funakoshi Co., Ltd.), and 10 ml of anhydrous Ν, Ν-dimethyl formamide, The solution became turbid and 0.44 ml of triethylamine was added and shaken at room temperature for 3 days. The CPG carrier was collected by filtration, washed with N, N-dimethylformamide, then with pyridine and then with methylene chloride, and dried under reduced pressure.

Acetic anhydride (2 ml) and pyridine (6 ml) were added to the CPG carrier for suspension, and the mixture was shaken overnight at room temperature. The CPG carrier was collected by filtration, washed with pyridine and then with methylene chloride, and dried under reduced pressure to obtain 2 g of CPG carrier [17].

3) Synthesis of 5, aaaaa AAA AAA AAA AAA AAA AAA aaa

About 25 mg of CPG carrier [17] (where Bp is N ⁶ -benzyladenine-9-yl) corresponding to nucleic acid base l ^ M was loaded on a column for automatic DNA synthesizer. 170 mg of the amidite reagent [14] was dissolved in 3.4 ml of anhydrous acetonitrile. Commercially available amidite reagent, [N ⁶ -benzyl-5,0- (4,4, -dimethyxitylytyl) -2,2-deoxyadenosine 3'-0- (2-cyanoethyl N, N-diisopropylphosphoamida It) (Perkin-Elmer One) 500 mg was also dissolved in 5 ml of anhydrous acetonitrile, mounted on an automatic DNA synthesizer (Perkin-Elmer One) with these two reagents, and subjected to automatic synthesis according to the program of the synthesizer.

After completion of the reaction, the sample solution (conc. Ammonia solution) in the collection vial was treated at 55 ° C. for 18 hours, and then concentrated under reduced pressure to dryness. The residue was dissolved in 5 ml of 50 m acetic acid-triethyl ammonium buffer (PH 7.0) (hereinafter abbreviated as TEM buffer), and preparative reverse-phase chromatography (Preparative RP-18 (55-105 m), 125 Onk, Preparation and purification were carried out using a stream, 1 mm x 100 mm column Waters Co., Ltd.). Solution A Solution B was eluted with a concentration gradient of 0 to 100% using a solution of 50 mM TEAA and solution B = 40% acetonitrile (in 50 mM TEAA buffer). Among the fractionated fractions, an orange-colored fraction was collected when acetic acid was added, the solvent was distilled off under reduced pressure, 5 ml of 80% acetic acid was added, and the mixture was left at room temperature for 15 minutes. After confirming by TLC that the protective group had been removed, the solvent was evaporated under reduced pressure. Liquid separation extraction was performed with ethyl acetate and water, and the aqueous layer was evaporated to dryness to obtain 80 OD (260 nm) of the desired product. Example 2 DNA-type antisense molecule containing a nucleoside of the formula [1] in which X 2 H, Y = H: 5, t CCGGT C CCAATGGAGGGGAAt 3 (where t is a nucleoside analogue and the base is thymine- 1- C represents decipherin, C represents deoxy guanosine, G represents deoxy thymidine, T represents deoxy thymidine, and A represents deoxy adenosine. Unless otherwise specified, the internucleoside linkage is a 3 'to 5' phosphodiester. Showing a bond)).

1) Synthesis of amidite reagent

In the same manner as described in 1) of Example 1, 260 mg of amidite reagent [14] (wherein Bp is in the case of thymine-1-yl) was obtained as a white powder. The molecular weight was measured by the FAB method to be 758.

2) Preparation of CPG carrier for DNA synthesizer In a similar manner to that shown in 2) of Example 1, 2 g of CPG carrier [17] (wherein Bp is in the case of thymine-1-yl) was obtained.

3) Synthesis of 5, t CCGGTCCAATGGAGGGGAAt3,

About 70 mg of CPG carrier [17] (wherein Bp is thymine-1-yl) corresponding to nucleic acid base 2〃M was loaded on a column for an automatic DNA synthesizer.

Amidite reagent [14] (provided that Bp is thymine-yl), a commercially available amidite reagent for DNA synthesis, [N ⁶ -benzoyl-5,-0- (4,4,-dimethyxitylytyl] )-2,-deoxyadenosine 3,-0-(2-cyanoethyl Ν, Ν -diisopropylphosphoramide 卜)], [Ν ² -isopyryl-5,-0-(4,4,-dimethyxitylytyl) -2, -deoxyguanosine 3'- 0- (2-cyanoethyl Ν, Ν-diisopropylphosphoramide 卜)], [Ν ⁴ -benzyl -5,-0- (4,4,-dimethyxitylytyl) -2, -Deoxycytidine 3, 0-(2-cyanoethyl Ν, Ν-diisopropylphosphoramidite)] and [5, 0-(4, 4,-dimethyloxytrityl)-thymidine 3, 0-(2-cyanoethyl) , [Dimethylphosphoramidite]] (Perkin Elmer One) in the same manner as in Example 1 3). Was Tsu. The purification was also conducted in the same manner to obtain 120 OD (260 nm) of the desired product. Example 3 DNA A type antisense molecule containing a nucleoside of the formula [1] in which X 2 H, Y = H: 5 'aaaaaaaaaaaaaaaaaaaaa aaaa3' (a is a nucleoside analog of which the base is adenine-9-yl Unless otherwise stated, the internucleoside linkage indicates a 3 'to 5' phosphodiester linkage, hereinafter referred to as hA ₂₅ ) synthesis.

By the same procedure as in Example 1, 52 OD (260 nm) of the desired product was obtained. Example 4 DNA-type antisense molecules containing nucleosides which are X 2 H, Y 2 H in the formula [1]: Of the homologs, those of which the base is adenine-9-yl, A is deoxyadenosine Each represents. Unless otherwise stated, the internucleoside linkage is 3 'to 5' Stel bond is shown. The following composition of hA ₂ A ₂₃ ).

By the same procedure as in Example 1, 50 OD (260 nm) of the desired product was obtained. Example 5: DN A type antisense molecule comprising a nucleoside of the formula [1] wherein X 2 H, Y = H: 5, aaaAAAAAAAAAAAAAAAAaa3, (a is a nucleoside analog of which the base is adenine-9-yl) And A represent deoxyadenosine, respectively. Unless otherwise specified, the linkage between nucleosides indicates a 3 'to 5' phosphodiester linkage, hereinafter referred to as hA ₆ A ₁₉ ).

By the same procedure as in Example 1, 44 O.D. (260 dishes) of the desired product was obtained. Example 6 A DNA template comprising a nucleoside of the formula [1] in which X = H, Y = H. Antisense molecule: 5 'aaaaaaaAAAAAAAAAAaaa aaa a3, (a is a nucleoside analog of which adenine-9-yl is a base, A is deoxyadenosine, respectively. Unless otherwise specified, the linkage between nucleosides is 3 The '→ 5' phosphodiester bond is shown, hereinafter referred to as hAwA) synthesis.

By the same procedure as in Example 1, 90 OD (260 nm) of the desired product was obtained. Example 7 DNA-type antisense molecule containing a nucleoside of the formula [1] in which X 2 H, Y = H: 5, aaaaaaaaAAAAAAaaaaaa aaaa3 '(a is a nucleoside group of which the base is adenine-9-yl And A represents deoxyadenosine, respectively. Unless otherwise specified, the linkage between nucleosides indicates a phosphodiester linkage of 3, → 5, below, which is hereinafter referred to as “hA ₁₈ A?” Synthesis.

The same procedure as in Example 1 was carried out to obtain 58 OD (260 dishes). EXAMPLE 8 A DNA-type antisense molecule comprising a nucleoside that is X 2 H, Y 2 H in Formula [1]:

5, aaaaaaaaaaaAAAAaaaaaaaaa ^ '(a is a nucle Among the homologues, those of the bases adenine-9-yl and A respectively represent deoxyadenosine. Unless otherwise stated, linkages between nucleosides indicate 3, → 5, phosphogether linkages. The following composition of hA ₂₂ A ₃ ).

The same procedure as in Example 1 was performed to obtain 61 O.D. (260 nm) of the desired product. Test Example Test Example 1 Toxicity test on mouse fibroblasts

Cytotoxicity to mouse fibroblasts expressing the HER-2 gene of 5, t CCGGTCCCAATGGAGGGGAAt3 obtained in Example 2, NIH 3T 3 -HER 2 cells (Difore et al., Methods in Enzymol., 198 272-277, 1991) In comparison with the currently most advanced phosphorothioate oligonucleotides in clinical research. The sequence of the phosphorothioate type oligonucleotide used as a control was 5, CGGTCCCAAT GGAGGGGAAT3 ', and was prepared by the method of Shiutain et al. (Shityne et al., Nucleic Acid Res., 16 3209-3221 1988). In order to increase the intracellular permeability of each test substance, a complex with a cationic ribosome was also used in the test. The components of the cationic ribosome are 3-0- (2-jetyl aminothiol) power lvamoyl-1,2- 0-dioleyl glycerol (see W094 / 19314 published) and egg yolk phosphatidylethanolamine (Nippon Yushi (stock) The mixing ratio of 3: 1 was used. Each test substance was added to the culture medium at a nucleoside concentration of 10, 3, 1 or 0.1 zM, respectively. The mixing ratio between the cationic liposome and the antisense oligonucleotide was 2: 1.

The NIH3T3-HER2 cells were seeded at a density of 10 ⁵ cells / well in a Koning's 24-well plate, and then cultured overnight at 37 ° C.-5% CO ₂ in Dulbecco's modified Eagle medium. .

After replacing the medium with a fresh one, each test substance was added to the supernatant and cultured overnight. The test substance in which the cell morphology was determined under a microscope, the test substance in which the cells were significantly atrophy was cytotoxic (indicated by + in the table), and the test substance in which the cell morphology was not changed was not cytotoxic (in FIG. Was represented. The results are shown in Table 1. Table 1. Cytotoxicity test

Cytotoxicity (

Molecule 10 3 1 0.1 Phosphoπ π-type o r: r Nucleotide + phospho D-t o -type Orico ,, Nucleo 'NT NT +

+ Katchi II, Sokrijo. Realms Antisense molecules of the invention Antisense molecules of the invention NT

+ Power Chio 3, Rikuriho. Reem

NT; unexamined phosphorothioate type oligonucleotide alone has relatively high cytotoxicity, and its toxicity is further enhanced when complexed with dynamic ribosome, while the antisense molecule of the present invention is not effective. Even when added alone, it did not show toxicity up to a concentration of ΙΟ ^ Μ, and even when administered as a complex with a cationic ribosome, up to a concentration of 1 〃M.

In addition, all the nucleoside analogues obtained in Example 3 of the present invention

It has also been confirmed that the concentration of the molecule does not show toxicity. Test Example 2 HER-2 synthesis inhibitory activity and toxicity test of 5 ′ t CCGGTCCCAATGGAGGGGA At 3 ′ synthesized in Example 2

The inhibitory effect on the protein synthesis in cells of 5, t CCGGTCCCAATGGAGGGGAAt3 'obtained in Example 2 was observed. This nucleotide sequence shows an antisense sequence near the 5, cap region of the HER-2 gene, which is one of the EGF receptor family and considered to be involved in malignancy of breast cancer (Woolrich et al., Science, 230 1132-1139 1985). As a control, a DNA of the same sequence and a phosphorothioate type oligonucleotide of the sequence of 5 'CGG TCCCAATGGAGGGGAAT 3' were used. The DNA was prepared by the method described in Test Example 1 according to a conventional method using a DNA synthesizer (Applied Biosystems Inc. Model 380 B) and phosphorothioate type oligonucleotides.

As the cells, the NIH3T3-HER2 cells shown in Test Example 1 were used.

In order to increase the intracellular permeability of each test substance, a complex with a cationic ribosome was administered to the cells. The cationic ribosome used and the method of addition were the same as in Test Example 1. Each test substance was added to the culture medium to give a nucleoside concentration of 1, 0.1 or 0.01 iM, respectively.

NIH3T3-HER2 cells were seeded at a density of 10 ^s cells / well in a Corning 24-well plate, and then cultured overnight in Dulbecco's modified Eagle's medium at 37 ° C.-5% CO ₂ .

The medium was changed to a medium containing 0.2% calf serum, and after 7 to 8 hours after the first addition of the test substance, the calf serum concentration was increased to 5% overnight. The medium was changed to a medium containing 0.2% calf serum not containing methionine, and then each test substance was added a second time. The added concentration is the same as the first time. After 7 to 8 hours, ³⁵ S methionine (Amersham, 3TBq / olol) was added to culture the calf serum concentration to 5% overnight.

The test substance in which the cell morphology was determined under a microscope, the test substance in which the cells were significantly atrophy was cytotoxic (indicated by + in the table), and the test substance in which the cell morphology was not changed was not cytotoxic (in FIG. Was represented.

The cells were recovered, and HER-2 protein was immunoprecipitated by an ordinary method using an anti-human HER-2 antibody (Nichirei).

After performing a 7% polyacrylamide gel SD S electrophoresis, image analyzer one (FU JIXB AS 2000) to ³⁵ S by measuring the radioactivity of main Chionin HER-2 protein of the HER-2 protein using The combined amount was calculated. The amount of synthesis of HER-2 protein in cells to which no test substance was added was 100%, and the reduction rate of the amount of synthesis of HER-2 protein when each test substance was added was expressed as the inhibition rate. The toxicity test is the same as in Test Example 1. The results are shown in Table 2. Table 2 Suppressive effect and toxicity of HER-2 protein expression of molecule

Working concentration (M) Inhibition rate (%) Cytotoxicity Phospho D D-Alicole "nucleotide

0.01 0

0.1 0

1.0 +

DNA

1.0 42.7 The present invention 7 antisense molecules

1.0 57.8

The antisense molecule of the present invention showed a synthetic inhibitory activity of ΗΕβ-2 protein equal to or higher than DNA at the same concentration as DNA.

Phosphorothioate type oligonucleotides have no effect at 0.01 to 0.1%, and cytotoxicity is expressed at a concentration of 1.0 〃M at which the antisense molecule of the present invention or DNΑ exhibits effective activity, and the activity can be measured. won.

In addition, the sense sequence near the 5 'cap region of the HER-2 gene of the compound and DNA of the present invention does not show synthetic inhibitory activity of HER-2 protein. Resistant compounds of the present invention hA ₂₅ against nucleolytic enzymes of Test Example 3 present compound (Nuclease SI) was the test substance. As controls opening one Le, using natural DN A oligomer dA ₂₅ to have the same sequence.

Add 50 mM sodium chloride and 30 mM zinc sulfate to 30 mM acetate buffer (pH 4.5), and make each test substance a final concentration of 4 OD (260 nm) in this buffer. I added it. Furthermore, a nucleolytic enzyme Nuclease SI (Boehringer 'Mannheim Yamanouchi) was added to a final volume of 2000 U / ml (final volume 501) and incubated overnight at 37 ° C. The reaction solution was sampled, and the degree of degradation was calculated by measuring the degree of decrease of the peak of the test substance by HPLC using a Lichrospher RP-18 (4 mml. D. x 125 mm) column. The results are shown in Table 3. Table 3. Resistance of the substance of the present invention to Nuclease SI

Degree of decomposition (%)

Test substance 0.7

Control> 62.9 While 60% or more of the control was degraded, the substance of the present invention remained over 99% undegraded even after overnight reaction. Test Example 4 Resistance of the substance of the present invention to snake venom phosphodiesterase

The 5 't CCGGTCCCAATGGAGGG GAAt3' of the substance of the present invention synthesized in Example 2 was used as a test substance. As a control, a natural D NA Aori mer having the same sequence was used.

Each sample was prepared in lOOmM Tris-HCl buffer solution (pH 8.9), lOOm sodium chloride, 14mM magnesium chloride solution to a final concentration of 4 OD (260nra), and snake venom phosphodiesterase (Co-ho Lit) was added as 3.6 The solution was added to a U / ml concentration (final volume 0.1 ml). After incubating for 1 hour at 37 ° C., the reaction was stopped by adding a final concentration of 50 mM of EDTA. The reaction solution was sampled, and the degree of degradation was calculated by measuring the degree of decrease of the peak of the test substance by HPLC using a Lichrospher RP-18 (4 ml. D. x 125) column. The results are shown in Table 4. Table 4. Resistance of the substance of the present invention to snake venom phosphodiesterase

Degree of decomposition (%)

Test substance 10.2

Control> 62.7 While the control was degraded by 60% or more, the substance of the present invention was degraded only by about 10% and showed strong resistance to snake venom phosphodiesterase. Test Example 5 Resistance of the substance of the present invention to calf spleen phosphodiesterase

The 5 't CGGTCC C CAATGGAGGG GAAt3' of the substance of the present invention synthesized in Example 2 was used as a test substance. As a control, natural DNA oligomers having the same sequence were used.

Each test substance was prepared in 100 mM ammonium succinate buffer solution (pH 8.9) and 1 mM EDTA solution to a final concentration of 4 OD (260 nm), and calf spleen phosphodiesterase (Boehringer) was adjusted to a concentration of 0.2 U / ml. Was added (final volume 0.1 ml). Incubate at 37 ° C. for 1 hour, sample the reaction solution, and use the Lichro-spher RP-18 (4 thighs; LD. X 125%) column as a high-performance liquid chromatograph (hereinafter abbreviated as “HP LC”) as the test substance. The degree of degradation was calculated by measuring the degree of reduction of the peak of The results are shown in Table 5. Table 5. Resistance of the substance of the present invention to calf spleen phosphodiesterase

Degree of decomposition (%)

Test substance <0.9

Control 27.6 While the control is degraded by nearly 30%, the degree of degradation of the substance of the present invention is 1% or less, showing strong resistance to calf spleen phosphodiesterase. Test Example 6 Substrate Specificity of RNase H for the Substance of the Present Invention

The substance of the present invention formed a hybrid with natural RNA, was recognized as a substrate for RNase H, and it was examined whether or not the RNA was degraded.

As a test substance

And hA ₂ A ₂₃ was used as a control and dA ₂₅ was used. As a RNA forming a Haipuriddo _{poly (U) (S 2 o} , w 6.6, Yamasa Shoyu Co.) was used.

40mM Tris-HCl buffer solution (pH 7.7), 1mM dithiothreous acid, 4m mag chloride E. coli RNase H (Takara Shuzo Co., Ltd.) is added with poly (U) and each test substance at a final concentration of 4 OD (260 nm) respectively to a solution of nesium, 4% (w / v) glycerol and 0.003% (w / v) calf serum albumin. ) Was added to a concentration of 100 U / ml (final volume 0.1 ml). Incubate at 20 ° C. for 18 hours and freeze at 20 ° C. to stop the reaction. Immediately after thawing the reaction solution, the degree of degradation was calculated by measuring the degree of reduction of the poly (U) peak by HP LC using a TSK G4000PWXL (7.8 mm ID × 300 mm) gel filtration column. The results are shown in Table 6. Table 6. Substrate Specificity of RNase H for the Substances of the Invention

Degree of decomposition (%)

dA ₂₅ (control) 100

hA ₂ A ₂₃ 98.3

hA ₆ A 100

hAioAis 98.3

hA ₁₄ An 98.3

hAisA 96.6 hAi ₈ A7, hAi 4 Ai

And hA ₂ A ₂₃ were shown to be RNase H substrates to the same extent as dA ₂₅ used as a control.

Test Example 7 Binding stability to I3NA or RNA

When the nucleic acids form a complex, the absorbance increases rapidly before and after a certain temperature as the temperature is raised. This is due to a decrease in the so-called hypochromicity of the nucleic acid, but by measuring the temperature (hereinafter referred to as Tm) when the absorbance has increased to half the absorbance difference before and after this, the nucleic acid and Know the complex formation ability of

HA25 as the test _{substance, hA 2 2 A3, i8A7,} hAi4Ai i, the hAi oAi 5, hA ₆ Ai ₉ and hA ₂ A _23, was used dA ₂₅ as a control. Poly (U) (S ₂₀ , w 6.6, Yamasa Syoyu Co., Ltd.) was used as the RNA for forming the hybrid.

A test substance or control is complexed with Poly (U), and the test substance concentration is 100 / M, 0.15 M sodium chloride, 10 mM sodium phosphate (pH 7.0), heating rate The Tm values were determined under the conditions of 0.5 ° C./min. Table 7. Tm values of the substances of the present invention to Poly (U)

Tm value (.C)

dA ₂₅ (control) 46.7

hAeA 47.4

hAioAis 43.3

hA ₂₅ 27.7 The substance of the present invention had a good ability to form a complex with natural RNA. The Tm value tended to decrease as the content of nucleoside homologs increased, but it was also shown that the content up to 40% (hAioA ₁₅ ) is comparable to natural DNA. From these results, the antisense molecule of the present invention has low toxicity, and can be used as a medicine for suppressing infection with herpes virus, influenza virus, human immunodeficiency virus and the like and the function of oncogenes.

Claims

l. The following partial structural formula [l]

[1]

(Wherein, B represents adenine-9-yl, guanine-9-yl, hypoxanthine-9-yl, thymine-1-yl, uracil-1-yl or cytosine-1-yl X, Y are the same or different and each represents a hydrogen, hydroxy, halogen or lower alkoxy)), a nucleoside represented by the following formula: DNA, RNA or a nucleic acid analogue in which a phosphodiester bond is modified or substituted; Or an antisense nucleic acid homolog or at least one salt thereof or a solvate thereof.

2. The modified phosphodiester bond of the nucleic acid homologue is selected from phosphorothioate bond, alkyl phosphorothioate bond, N-alkyl phosphoramidate bond, phosphorophordothioate bond or alkyl phosphonate bond The antisense nucleic acid homolog according to claim 1, which is one or more.

3. A medicament comprising the antisense molecule according to claim 1 or 2 as an active ingredient.