WO1993024512A1

WO1993024512A1 - Nucleic acid homologue

Info

Publication number: WO1993024512A1
Application number: PCT/JP1993/000684
Authority: WO
Inventors: Junichi Yano; Tadaaki Ohgi; Kouichi Ishiyama
Original assignee: Nippon Shinyaku Co., Ltd.
Priority date: 1992-05-25
Filing date: 1993-05-24
Publication date: 1993-12-09

Abstract

An object of the invention is to provide a novel antisense molecule utilizable as a medicine which suppresses infection with herpesvirus, influenza virus, human immunodeficiency virus or the like and the action of cancer genes. The nucleic acid homologue of the invention is one represented by general formula (I), wherein X and Y may be the same or different from each other and each represents S or O; B?1, B2, B3 and B4¿ may be the same or different from one another and each represents adenine, guanine, hypoxanthine, cytosine, thymine or uracil; and n represents an integer of 1 or above, provided that B?2 and B3¿ need not necessarily be one and the same base when n is 2 or above.

Description

1

Book 2

Nuclear acid homolog technology

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.

Background technology

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.

Weintrau h., Scientific American, January 1990, p. 34-40) Based on _zero or more principles, 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)).

(1) DNA type oligonucleotide derivative (Compound II) Two

(2) RNA type oligonucleotide derivative (Compound II)

(3) phosphoric acid ester type oligonucleotide derivative (compound ③)

(4) Methylphosphonate-type oligonucleotide derivative (Compound II)

(5) Phosphoramidate-type oligonucleotide derivative (Compound II)

(6) Phosphorothioate-type oligonucleotide derivative (Compound I)

(7) Phosphorodithioate-type oligonucleotide derivative (Compound II)

(8) Capillate-type oligonucleotide derivative (Compound I)

The structural characteristics of the above compound are shown below. Both aim to synthesize antisense molecules that function in vivo by modifying the natural nucleic acid structure (Compound II). One 0

0 one

df / JL3d

(Wherein, を represents a nucleobase, and R and R ″ represent hydrogen, alkyl, or other substituents.)

As a result of intensive studies, the present inventors have found that the following nucleic acid homologues have more excellent effects than those described above, and have filed a patent application (Japanese Patent Application No. 2 — 1452).

No. 16).

(9) Thiocarbamate-type oligonucleotide derivative (Compound II)

Five

(Wherein B represents a nucleic acid base, I? ^1, R ² represents hydrogen, an inorganic acid residue, organic acid residue, an alkyl or Ashiru.)

Many other nucleic acid homologs have been synthesized, including the above compounds.

(For example, nucleic acid homologs described in US Pat. No. 5,034,506). However, a satisfactory effect has not always been obtained, and a highly practical antisense molecule satisfying the following conditions has been obtained. It has not been developed.

(a) Stability of binding to DNA and RNA (complex forming ability)

(b) Resistance to nucleolytic enzymes

(c) Physicochemical stability to acids, alkalis, temperature and humidity

(d) Cell membrane permeability

(e) Nucleic acid safety

ぱ) Convenience of synthesis and low cost

(g) Solubility in water or buffer.

Therefore, 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.

Disclosure of the invention

The present inventors have conducted intensive studies and found that the following general formula [I]

(Wherein, X, Y are the same or different and each represents S or ^{^{0. Β], Β 3,}} Β 3, are the same or different, Ryo adenine, GuRyo Nin, Hipokisanchi down, collected by Singh, Η indicates an integer on 1 ^, provided that when η is on 2 J¾, 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.

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.

The 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.

When the double-stranded helical structure of the nucleic acid homologue of the present invention is analyzed by circular dichroism (Circular Dichroism), 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.

As described later, 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.

As a use of the nucleic acid homolog according to the present invention, ° ^{93 2 512} So-called image 0684

8

Since it inactivates gene information, it can infect foreign substances that have entered the living body, such as herpes virus, influenza virus, and human immunodeficiency virus (HIV, AIDS virus, etc.), and the function of oncogenes. It can be used as a medicine to suppress the disease. In addition, it can be used for IS for breeding and other technologies by actively controlling the gene transfer of animals and plants. In addition, it has the potential to be used as a DNA probe for analysis of gene function and for infections of infectious diseases, pneumococci, and viruses. Can be considered.

When X is 0, the compound of the general formula [I] gives an optically pure active substance when Y is either S or 0. On the other hand, when X is S, when Y is 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.

When the nucleic acid homolog of the present invention is used as a medicament, the compound of the present invention is administered as it is or in a pharmaceutically acceptable nontoxic and inert carrier.

As 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)

Next, a general method for synthesizing the nucleic acid homolog of the present invention will be described.

* S solid phase synthesis by periestil method

Ten

In the formula, 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.

Procedure 1 Commercially available 5'-dimethoxytrityl derivative 1 of a nucleoside or a nucleotide protected in the base moiety was converted to 1, ^93/24512 contract

11

Treat with thiocarbonimidazole (H. Staab et al., Ann, 657, 98-103 (1962)) and heat at 50 t or 1,1'-carbonimidimidazole (JM Coull et 28, 745-748 (1987)), and reacted at room temperature for 4 hours to obtain an imidazolyl carbonyl derivative or imidazolyl carbonyl derivative __ ^. -5'-Deoxynucleoside or nucleoside derivative 3 whose base moiety is protected (T. Hata et al., Chem. Lett., 977-

When 980 (1975)) is catalytically reduced at room temperature in 10% palladium carbon methanol, the amino body j is easily obtained. This can be used for the next condensation reaction without purification.

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 _ ^.

Procedure 4 In order to use the so-called triester method (H. Ito et al., Nucleic Acids Res., 10, 1755-1769 (1982)) of the solid phase method, dimer 5 (X ⁰ = S) re phosphorylation agent (C. Broka et al., ucleic Acids Res., 8, by 5461-5472 (1980>), _P phosphodiester of the resulting Daimabu D click _ ^ directly solid phase Using 1% polystyrene as a support, the condensation of dimer block 6 is repeated, and the last step is a monomer block (deoxynucleotide 2-chloro). By condensing lipopolyester (manufactured by Sigma Co., Ltd.), a novel antisense molecule can be synthesized.

Operation 5 Among the solid-phase methods, the so-called Amidite method Die marb n_J can be obtained by treating j by the method described in the literature (oester et al., Nucleic Acids Res., 12, 4539-4557 (1984)). By using this 8 and using a DNA synthesizer (for example, AB clay), 9 types of intense molecules can be easily prepared.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail with reference to Examples, Reference Examples, Test Examples, and Comparative Examples, but the present invention is not limited thereto.

Example 1 - In general formula [I] X = S, Y = S, τι = 6, 5 'CTTC

Synthesis of substance j of the present invention which is TTTGAGCTTG ³ '

A die Maburo click (Dimer block) _ (X ° = S, B l = T, B 2 = T) 3 pieces, 8 (X ° = S, B '= G, B a = C) 1 piece, _ (X ° = S, ^Bl = G, ^Ba = A) and j (X ° = S, ^Bl = C, ^Ba = T) are prepared, and these are prepared by the amidite method. It was synthesized by condensation. T is thymine, G is guanine, C is cytosine, and A is rydenine. 1. The following Daimaburo click 8_ Synthesis of ^{(X ° = S, B]} = T, B 2 = T) [hereinafter (8TT) abbreviated]

13

^ Synthesis

5'-D-Dimethoxytrityl thymidine_1 (manufactured by Wako Pure Chemical Industries, Ltd.) 3.95 g is dissolved in 36 ml of pyridine, and 1, Γ-thiocarbyldimidazole is dissolved.

3.23 g was added, and the mixture was heated for 5 hours. After heating, the mixture was concentrated to dryness, and the residue was purified with silica gel chromatography (silica gel 170 g, methanol / dichloromethane), and 1 (X ° = S, B ' = T) was obtained in 4.16 g (88% yield). MS (PAB): 655 (MH +)

② Synthesis of J [

5'-Ryside-5'-Doxythymidine 3 (T. Hata et al. Chem.

Lett .. 977-980 (1975)) To 2.2 g of a 220 ml solution of methanol was added 0.66 g of 10% palladium carbon, and the mixture was catalytically reduced by M at room temperature for 3 hours. After the reaction,攄過through Celite DOO, The filtrate was concentrated and dried surface to give j (B ⁸ = T) and 1.67 g (84% yield).

Synthesis of ③

0.53 g of _J obtained in ② was added to a 30 ml solution of 21.31 g of DMP obtained in ① and treated at room temperature for 2 hours. After the reaction, concentrate with a vacuum pump to dryness, purify with silica gel chromatography (silica gel 70 g, methanol / dichloromethane), j (X ° = S, B ^ T , B ^a = T) 1.69 g were obtained as a white solid. MS (FAB): 828 (H ⁺ )

®_J synthesis

Immediately before use, 50.5 g obtained in ③ was azeotropically azeotroped with pyridine, azeotropic with toluene, and azeotroped with THF. The reaction system was replaced with nitrogen gas and finally dissolved in 5.0 ml of anhydrous THP. Add 0.42 ml of Ν, Ν-diisovir virethyl, and 2-cyanoethyl N, N-diisopropyl chloro ¹ α-phospho amide (Al 0.27 ml was added and the mixture was stirred at room temperature for 2 hours. ¾ The solution was concentrated to dryness. The residue was partitioned between ethyl acetate (saturated with nitrogen gas) and saturated aqueous sodium bicarbonate (saturated with nitrogen gas), and the organic layer was dried over sodium sulfate and concentrated to dryness again. When triturated with hexane the residue to n- (those saturated with nitrogen ^{gas), _ (X D = S} , B * = T, Β 2 = Τ) was obtained 0.54 g white solid. This _J was used directly in a dimer block for a DNA synthesizer (manufactured by Ryo Pride Systems, Inc. (hereinafter also referred to as ΓΑΒΙ)).

2. Synthesis of other dimer blocks _J

In the same manner as in 1., the dim block j (»= s, B '= G, B ² = C) required for the automatic surface phase synthesizer (manufactured by ABI) [hereafter (8GC)],

J (X ° = S, B ^ G, B a = A) [hereinafter (8GA)], and j were synthesized (, X ° = S B ' = C, B a = T) [hereinafter (8CT)] the .

Synthesis of 3.9 (X ° = S, n = 6, ⁵ 'CTTCTTTGAGCTTG ³ ')

(8TT) 537 mg to 5.21 ml anhydrous acetonitrile, (8GC) 192 mg to 1.60 ml anhydrous acetonitrile, (8GA) 287 mg to 2.30 ml anhydrous acetonitrile , (8CT) 190 mg in 1.70 ml of anhydrous acetonitrile, N ⁶ -benzoyl-3'-0-L2-cyanoethoxy- (N, N-diisop-D-piramino) phosphine ] -5'-0- (4.4'-dimethoxytrityl) -2'-deoxythymidine (manufactured by ABI) 500 rog was dissolved in 6.00 nil anhydrous acetonitrile. This was synthesized using an automatic DNA synthesizer (manufactured by ABI) according to a commercially available program, but the proprietary method was used for the following points.

(1) 3% tri-acetic acid / diacetic acid for removal of DMTr (dimethoxytrityl) group The reaction time was divided into 90, 60, and 30 seconds using 10% formic acid / dichloromethane in place of the cro-sigma methane. (2) A TBTD (tetraethyl thiuram disulfide) reagent was used in place of oxidization (reaction time: 900 seconds). ③ During condensation 縮合 was set to 600 seconds.

The reaction was carried out using two Gurunin SNAF / CPG columns (1Λ «Μ) (manufactured by ABI). After completion of the reaction, the resin was taken out of the column, treated with 5ϋ of a solution of DMF-concentrated ammonia water = 1: 1 at room temperature for 2 days at room temperature, and cut out from the resin and deprotected.

After evaporating the solvent, dissolve in 5 ml of 50 mM triethylammonium acetate (pH 7) buffer, and apply to Waters Prep RP-18 (55 to 105 um) 125 A reverse phase chromatography. Preparative purification (0 = 10 mm x 100 mm force ram).

Solution A = 50 mM TBAA (PH 7) buffer 100 ml, Solution B = 40% cetonitrile / 50 mM TEAA (pH 7) buffer 100 ml Β¾'0 → 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. After evaporating the solvent under reduced pressure, the residue was purified again by the above-mentioned reversed-phase column to obtain 20.6.D. (measured at 260 nm). Fig. 1 shows the chromatogram of this target product by high-performance liquid chromatography (HPLC). Analysis by HPLC was performed on a Lichrospher 100 RP-18 (5 jura, 4 mml.D. x 125 mm) column, solution A = 5% acetate nitrile Z50 mM TEAA (PH 7.0), solution B = 80% Tonitrile ^ OmM TEAA (pH 7.0 ), B¾: 0 → B0 (15 minutes) Gradient, flow rate 1 rolZ minutes. The solubility in water was above 0.5raMJil.

Example 2 - general formula [I] in X = 0, Y = S, n = 5, 5 'TCTT TGAGCTTG 3' in which the present invention material J [Synthesis of

Dimer one block ^{(B '= T, B 2} = T) 2 ^{amino, _i (B l = G,} B 2 = C) 1 piece, _ (B' = G, B 2 = A) 1 piece, and (B ^J = C, B = T) were prepared and synthesized by condensing them by the phosphotriester method.

1. The following Daimaburo click j Synthesis of ^{^{(B l = C bz, B}} a = T) [hereinafter (6TT) abbreviated]

C ^bz represents N-pentylcytosine.

① Synthesis

N ⁶ -Benzoyl-5'-0-dimethoxytrityl-2'-deoxycytidine_11.90 g (manufactured by Wako Pure Chemical Industries, Ltd.) 1.34 g was added, and the mixture was heated at 40 for 5 hours. The same treatment as in ① of Example 1 was carried out to obtain 1.92 g of _ (X ° = S, B ¹ = C ^a ) which was the target substance. 86% yield. MS (PAB): 745 (MH +)

② Synthesis of 5 0.40 g of j (B ³ = T) was added to 0.89 g of the DMP 16 ml solution obtained in ①, and the mixture was treated at room temperature for 2 hours. After the reaction, the mixture is treated in the same manner as in 3 of Example 1, and purified by silica gel chromatography (silica gel 40 g).

^{(X ° == S, B 1} = C b B a = T) was obtained in white facepiece (quantitative).

S (FAB): 918 (ΜΗ +)

®_I synthesis

2.70 ml of a phosphorylation reagent 0.28M (C. Broka et al., Nucleic Acids Res., _8_, 5461-5472 (1980)) was added to 0.46 obtained in ②, and the mixture was stirred at room temperature for 50 minutes. After the reaction, 0.9 ml of 50% pyridine solution was added, and the mixture was partitioned between π-methan pentamethine and a 0.5 M aqueous solution of triethylammonium bicarbonate, and the organic layer was concentrated to dryness. This residue is dissolved in 5.0 ml of dichloromethane and powdered with 45 ml of _n- hexane (0.1% triethylamine). Quantitatively, j (B ^ C ^b B ² = T) I got

2. Synthesis of other dimer block 6

In the same manner as in 1., the dimer α j j (B ^ = T, B ² = T) [^ lower C6TT), _6 (B ^J = G ^ib , B ^a = C ^bz ) [the following (6GC >>)] and _J (B '= G ^ib , B ² = A ^bz ) [the following (A)] were synthesized, where G ^ib was N-isobutyril the Nin, a ^bz is N ⁶ - represents a Penzoi Ruadenin.

3. Synthesis of J [

The manual procedure for phosphotriester solid phase synthesis was performed according to the literature (H. Ito et al., Ucleic Acids Res., 10, 1755-1769 (1982)).

① 榭 d (DMTr) G ^ib- polystyrene (manufactured by Wako Pure Chemical Industries) 25 mg The plate was washed with a solution of methanol and methanol (7/3).

(2) The resin was quickly detritylized with M for a short period of time until the color disappeared with a solution of 2.pi.-methanol in methanol.

③ Replace the fat with viridine, azeotropically dry to make it anhydrous. To this was added a dimer block pyridin solution, and after azeotropic drying, MSNT and anti-applied viridine 150〃1 were added. The mixture was allowed to stand at room temperature for 40 minutes, and then washed with pyridine.

溶液 A solution of 0.1 dimethylamino viridine viridine in the resin

Add 1.8ml and 0.2ml anhydrous anhydride. After leaving for 2 minutes at room temperature, wash with viridine / ".

Repeat the above operation 5 times, and finally dimer block 20 mg of 5'-0-Dimethoxytritylthymidine-1,3- (2-chlorophenyl) phosphate (Sigma). In addition to the above, the process up to ③ was completed to complete the condensation reaction.

Average yield 88%.

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.

The reaction solution is subjected to open click Roma preparative reversed phase (Preparative C. _e 125A 55-105 manufactured »IO U O Ta Inc.) to afford the pure desired product 50 OD. Fig. 2 shows the HPLC chromatogram of this target compound. HPLC analysis was performed on a Lichrospher 100 RP-18 (5 im, 4 difficult ID x 125 mm) column, Solution A = 5% cetonitrile << 50mM TEAA (PH 7.0), Solution B = 80% acetonitrile Z50mM ΤΒΑΑίρΗ 7.0), Β¾: 0-50 (15 minutes) Gradient, flow rate The test was performed under the condition of 1 ralZ. The solubility in water was 1.0 mM or more.

Example 3 - general formula in [I] X - 0, Y = 0, η = 7, S Synthesis of 'TGCT TCTTTGAGCTTG ^3' in which the present invention substances j

J (X ° = 0, B '= T, B ^a = T) 3 j, X (X ° = 0, B ^l = G, B ² = C) 2 j (X ° = 0, B ' = G, B 2 = a) 1 piece, and j (X ° = D, B l = C, B = T) number to use Thanked by these a Mi Daito method It was synthesized by condensation. T is thymine; G is guanine; C is cytosine; and A is adenine.

1. Synthesis of the following Daimaburo click J (X ° = Q, B ^ T, B 3 = T) [hereinafter (B'TT) abbreviated]

Synthesis of

0.65 g of 5'-0-dimethoxytritylthymidine_1 (manufactured by Wako Pure Chemical Industries, Ltd.) is dissolved in 5 ml of pyridin, and 0.23 g of 1, Γ-carbonyldiimidazole is added. Reacted for 4 hours. After evaporating the solvent under reduced pressure, the residue was partitioned with a ₅ % solution of dichloromethane in sodium dihydrogen phosphate to give dichloromethane. The methane solution was washed with water, dried over sodium sulfate, and concentrated to dryness to obtain 0.70 g (91% yield) of (X ° = 0, B ′ = T).

② Synthesis of j

5'-Ryside-5'-Doxythymidine 3 (T. Hata et al., Chem.

Lett., 977-980 (1975)) To 2.2 g of a 220 ml solution of methanol was added 0.66 g of 10% palladium carbon, and the mixture was subjected to catalytic reduction at room temperature for 3 hours. After the reaction was filtered through Celite the windows, and the concentrated dry surface滤液, "the (B ^a = T) to give 1.67 g (84% yield).

③ Synthesis of j

0.35 g of _J obtained in ② was added to a solution of 0.70 g of DMF in 8 ml obtained in ① and reacted at room temperature for 20 hours. After the reaction, the solution is concentrated to dryness using a vacuum pump and purified using silica gel chromatography (silica gel 40 g, methanol / dichloromethane). J (X ° = D, B ' = T, B ³ = T) and 0.39 g was obtained in white color facepiece (40% yield). MS (PAB): 812 (MH ⁺ )

④ j synthesis

Immediately before use, 0.36 g of j obtained in ③ was azeotropically azeotroped with pyridine, azeotropic with toluene, and azeotroped with THP. The reaction system was replaced with nitrogen gas, and finally dissolved in 4 ml of anhydrous THF. To this was added 0.31 ml of N, N-diisopropyrethyl and 0.20 ml of 2-cyanoethyl N, N-diisoprovirk π-phosphoamidite (manufactured by Rydritz), and the mixture was added at room temperature. Reacted. After the filtrate was concentrated and dried, the residue was partitioned between ethyl acetate (saturated with nitrogen gas) and saturated aqueous sodium bicarbonate (saturated with nitrogen gas), and the organic layer was dried over sodium sulfate and concentrated again to dryness. When triturated with hexane the residue into .pi. (those saturated with nitrogen gas), 8 (X ° = 0 , B '= T, B 2 = T) 0.40s ( (90% yield) as a white solid. This j was used directly in a dimer book for a DNA synthesizer (manufactured by ABB) (hereinafter also referred to as “ABI”).

2. Synthesis of other dimer blocks 8

1. automated solid phase synthesizer Daimabu required by (Arufabetaiota Co.) eta click 8 by the same method as (X ° = 0, B ^ G, B a = C) [hereinafter (8'GC)], J ^{(X ° = 0, B l} = G, B a = A) [hereinafter (8'GA)], and j (X ° = D, B '= C, B 2 = T) [hereinafter (8'CT) ] Was synthesized.

3. 9 (X ° = Q, n = 7 5 'TGCTTCTTTGAGCTTG 3') Synthesis of

(8, TT) 400 mg to 7.96 ml of anhydrous acetonitrile, (8'GC)

313 tng to 5.23 ml of anhydrous acetonitrile, (8 'GA) 180 rag to 2.95 ml of anhydrous acetonitrile, and (8'CT) 213 mg to 3.87 ml of anhydrous acetonitrile , 3, -0- [2-Synoethoxy- (N-diisop-D-bilamine) phosphine] -5 '-(]-(4.4'-Dimethoxytrityl) thymidine (ABI) 500 tng were dissolved in 6.00 nil anhydrous acetonitrile, respectively, and synthesized using an automatic DNA synthesizer (ABI) according to a commercial program. The method was taken.

(1) Use 10% formic acid / dichloromethane instead of 2% trichloroacetic acid / dichloromethane to remove the DMTr (dimethoxytrityl) group, and reduce the reaction time to 90 seconds, 60 seconds, Divided into 3 times of 30 seconds. ② During condensation ¾ was divided into 600 seconds and 600 seconds.

The reaction was carried out using three columns of GUNIN SNAP / CPG columns (1 (manufactured by ABI). After the reaction was completed, the resin was removed from the column, and a DMF-concentrated aqueous ammonia = 1: 1 solution lOtnl For 3 days at room temperature and cut out from resin And deprotection.

After distilling off the solvent, 50 mM TBAA (triethylammonium phosphate) (pH 7) buffer solution was dissolved in 5 parts, and Waters Prep RP-1 55 ~: 105 m)

Preparative purification was performed by 125 A reverse phase chromatography (0 = 10 × 100 mm column).

A solution = 100 mM TBAA (pH 7) buffer 100 ml, B solution = 35% acetonitrile Z50 mM TBAA (pH 7) buffer 100 ml B¾: 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. Separation and extraction was performed with ethyl acetate-water, and the aqueous layer was separated to obtain the desired product (110 O.D. (measured at 260 nm)). Fig. 3 shows a chromatogram of the target product by high performance liquid chromatography (HPLC). HPLC analysis was performed on a Lichrospher 100 RP-18 (5 / m, 4 mml. D. 125 mm) column, solution A = 5% cetonitrile Z50 mM TBAA (pH 7.0), solution B = 80% acetate. The test was carried out under the conditions of 50 mM trinitrile TBAA (PH 7.0), B¾: 0 → 50 (15 minutes), gradient, and flow rate of 1 mlZ. The solubility in water was 5 tnM or more.

Example 4 - general formula [I] in Χ = 0, Υ = 0, η = 5, 5 Synthesis of 'ΤΤΤΤ TTTTTTTT ^3' in which the present invention substances j

Using the dimer block (Χ ⁰ = [), Β '= Τ, Β ² = Τ) and one thymine SNAP / CPG column, synthesis and purification were performed in the same manner as in Example 3, and the desired product was obtained. 55.D. (measured at 260 nm). Figure 4 shows a high-speed liquid chromatographic (HPLC) chromatogram of this target product. HPLC Analysis was performed using ichrospher 100 RP-18 (5 ^ m, 4 mml. D. 125 mm) column, solution A = 5% acetonitrile 50roM TBAA (pH 7.0) solution B-80% acetonitrile The reaction was performed under the following conditions: Ril / 50tnM TBAA (PH 7.0),% 0 → 50 (15 minutes) gradient, flow rate 1 ml / min. Test Example 1 Inhibitory effect of the substance of the present invention (the substance of Example 1) on the synthesis of P protein in HeLaS3-MDRl cells

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

n cells only

⁸⁵ plus Libofectin

^{35 2)} plus antisense nucleic acid 9

Antisense nucleic acid consisting of only phosphothioate in "": the same sequence (references Stec. W. J. et al., J. Am. Chem. Soc.

106, 6077-6079 (1984)) The substances of the present invention were more active than those currently promising as antisense nucleic acids.

Test Example 2 Resistance of the substance of the present invention to nuclease

The present invention substances (X = 0, Y = S , n - 3, S 'GATCGATC 3') was used as the test substance. 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 (ΡΗ4.6), 50 mM sodium chloride, and Im-zinc chloride solution.

S,) (Boehringer's Mannheim Yamanouchi) was added to a concentration of 10 U / ml. Incubated at 37t. The reaction solution was sampled, and the degree of decomposition was checked by HPLC. The results are shown in Fig. 5, Fig. 6, Fig. 7 and Fig. 8. Analysis by HPLC was performed using Lichrospher RP-18 (4 mml.

(125mm) Column, solution A = 5% acetonitrile ZlOmM TBAA (pH 7.0), solution B = 80% complete set nitrile ZlOmM TBAA (pH 7.0), see Figures 5 and 6 B%: 0-30 (15 minutes) gradient, and in FIGS. 7 and 8, ^: 0-50 (15 minutes) gradient, and the flow rate was 1 ml / min. While the control DNA almost disappeared in about 1 minute, the substance of the present invention remained almost 100% undegraded even after 1 hour reaction.

Test Example 3 Physicochemical properties

1. Melting curve (measurement of Tm value)

When nucleic acids form a complex, the absorbance increases rapidly around a certain temperature as the temperature is increased. This is the so-called light color of nucleic acids This is due to the decrease in effect (hypochroraicity). By measuring the temperature at which the absorbance increased to one half of the absorbance difference before and after this (hereinafter referred to as “ππ”), the complex with the nucleic acid was measured. You can know the forming ability.

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 ό by that shown in FIG. 9 Melting curves were obtained. This melting curve was measured under the conditions of a sample concentration of 65Μ, 0.15% sodium chloride, 10M sodium phosphate (PH 7.0), and a heating rate of 0.5 minutes.

As is clear from FIG. 9, the substance of the present invention strongly formed a complex (Tm 40.9 t).

2. Chemical structure of the complex (measurement of CD)

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

^ "る ί

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)) ο

This type I DNA has been shown to transfer to type C DNA only when exposed to special conditions (high salinity, low humidity) (AGW Lesic et al., J. Mol. Biol. 143, 49-72 (1980)).

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.

As is clear from FIG. 10, it was found that when the substance of the present invention was used, C-type DNA was extremely rare under normal physiological conditions. Using natural DNA under the same assay conditions indicated atypical B-type DHA.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a π-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.

Claims

28 Scope of Claim

1. The following general formula [I]

[I]

(Wherein, X and Y are the same or different and represent S or 0. BB ² , B ³ and B ⁴ are the same or different and represent adenine, guanine, hypoxanthine, cytosine, thymine or peracil N represents an integer of 1 or more, provided that when n is 2 or more, B ² and B ³ are not limited to the same base.