JPH0967392A - Vinylated deoxyguanosine derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new derivative for production, etc., of antisenseologonucleotide having crosslink ability, which is a specific vinylated deoxyguanosine derivative, chemically stable and forms a covalent bond by reacting with a nucleophilic molecule such as cytidine. SOLUTION: This new vinylated deoxyguanosine derivative (salt) is expressed by formula I R<1> is H; R<2> is H, tert-butyldiphenylsilyl, a group expressed by formula II [R<4> is H or di(p-methoxyphenyl)phenyl-methyl]; R<3> is acetyl or R<1> is methyl; R<2> is tert-butyldiphenylsilyl; R<3> is acetyl or R<1> is trimethylsilyl; R<2> is tert-butyldiphenylsilyl; R<3> is H, acetyl or P(0) (OH)H} and useful as an intermediate for producing antisenseoligonucleic acid having crosslink ability. The compound is obtained by reacting a compound of formula III (TBDS is tert- butyldiphenylsilyl; X is a halogen; Ac is acetyl) with an allylmagnesium halide, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a vinylated deoxyguanosine derivative or a salt thereof. More specifically, the present invention relates to a vinylated deoxyguanosine derivative or a salt thereof useful for producing a nucleic acid cross-linking agent or an antisense oligonucleic acid having a cross-linking ability.

[0002]

2. Description of the Related Art Specific genes and messenger RNA (mR
A method is known in which an antisense oligonucleic acid having a nucleotide sequence complementary to the nucleic acid sequence is specifically bound to a nucleic acid such as NA) to suppress gene expression. This method has great interest in the fields of biochemistry and medicine / pharmaceuticals because it can be applied to full-scale gene therapy as well as being used as a biochemical experimental tool.

In order to carry out this method efficiently, it is necessary to stabilize the double-stranded or triple-stranded complex formed by complementary binding, and various means for stabilization have been proposed. There is. As a typical example thereof, for example, a functional group having an alkylating ability is introduced into an oligonucleic acid to generate a gene DN.
Antisense oligonucleic acids that have the ability to covalently bind to target nucleic acids such as A and mRNA (cross-linking ability) are known (eg, Volssov, VV et al., Nucleic Ac
id Res., 14, 4065, 1986; Webb, TR et al., J. Am.
Chem. Soc., 108, 2764, 1986; Baker, BF et al.,
J. Am. Chem. Soc., 111, 2700, 1989, etc.).

However, most of the antisense oligonucleic acids modified as described above have a problem that the cross-linking speed is slow, and those having a high reactivity have a drawback that the chemical stability is low. It is not desirable to apply such an antisense oligonucleic acid to a living body (R
okita, SE et al., J. Am. Chem. Soc., 116, 1690,
1994).

[0005]

An object of the present invention is to provide a compound useful for production in an antisense oligonucleic acid having a property of efficiently cross-linking with a target nucleic acid. Another object of the present invention is to provide a compound that is chemically stable as described above.
Still another object of the present invention is to provide a compound having the above characteristics, which is useful for producing an antisense oligonucleic acid applicable to a living body.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a vinylated guanosine derivative having the following structure is chemically stable and easy to react with a nucleophilic nucleotide. It was found that a strong crosslink can be formed in response to. Further, they have found that the above compound is useful for producing an antisense oligonucleic acid having a property of efficiently cross-linking to a target gene nucleic acid. The present invention has been completed based on these findings.

That is, the present invention provides the following formula (I):

Embedded image And an intermediate for producing an antisense oligonucleic acid comprising the above compound; and an intermediate for producing the above, which is used for producing an antisense oligonucleic acid having a crosslinkability.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION R ¹ , R ² , and R in the above formula (I)
³ is defined by any of the following (1) to (3). (1) R ¹ represents a hydrogen atom, R ² represents a hydrogen atom, a tert-butyldiphenylsilyl group, or the following formula:

Embedded image (In the formula, R ⁴ represents a hydrogen atom or a di (p-methoxyphenyl) phenylmethyl group), and R ³ represents an acetyl group; (2) R ¹ represents a methyl group, R ² represents a tert-butyldiphenylsilyl group, and R ³ represents an acetyl group; or (3) R ¹ represents a trimethylsilyl group, R ² represents a tert-butyldiphenylsilyl group, and R ³ represents hydrogen. Indicates an atom, an acetyl group, or -P (O) (OH) H.

The above compounds may form acid addition salts or base addition salts, and such salts are also included in the scope of the present invention. When the compound of the present invention itself is applied to a living body as a cross-linking agent, or when it is used as a production intermediate for producing a biocompatible antisense oligonucleic acid, a salt is physiologically acceptable. Is preferred. Examples of the base addition salt include triethylamine,
Mention may be made of salts of amines such as dimethylamine, ammonia, triethylamine or diethylamine; or salts of metals such as sodium, potassium, calcium or magnesium. As acid addition salts,
For example, salts of mineral acids such as hydrochloric acid, sulfuric acid, or perchloric acid; or salts with organic acids such as oxalic acid, fumaric acid, maleic acid, acetic acid, propionic acid, methanesulfonic acid, or p-toluenesulfonic acid. Can be mentioned.

The compounds of the present invention are specifically shown in Table 1 below. In the table, when R ² is shown as “Chemical 4”,
It is shown that R ² is a group of the thymidyl acid derivative (group represented by [Chemical Formula 4] above), and R ⁴ in the group is the one shown in the table. Also, in the table, Ac, Me, Et, TBDP
S, TMS, and DMTr each represent an acetyl group, a methyl group, an ethyl group, a tert-butyldiphenylsilyl group, a trimethylsilyl group, and a di (p-methoxyphenyl) phenylmethyl group (hereinafter, referred to in the present specification. The same).

[0011]

[Table 1]

Examples of the method for producing the compound of the present invention are explained according to the following schemes, but the method for producing the compound of the present invention is not limited to these methods. In the scheme, X is a chlorine atom, a bromine atom, a p-toluenesulfonyloxy group,
Alternatively, it represents a leaving group such as a trifluoromethanesulfonyloxy group, and Met represents a metal such as tributyltin or magnesium halide (the same applies in the following schemes).

[0013]

Embedded image

Compound (2): 2'-deoxyguanosine (1) is triethylamine in a suitable solvent such as a halogenated hydrocarbon solvent such as dichloromethane or chloroform, or an aprotic polar solvent such as dimethylformamide or dimethylsulfoxide. Compound (2) can be produced by reacting with 0.2 to 10 equivalents of tert-butyldiphenylsilane chloride in the presence of a base such as imidazole. Normally, the reaction is carried out at a temperature of -20 ℃ to + 150 ℃ 5
It should be done for 24 hours a minute.

Compound (3): in an aromatic hydrocarbon solvent such as benzene or toluene, a halogenated hydrocarbon solvent such as dichloromethane or chloroform, an ether solvent such as tetrahydrofuran or diethyl ether, or a mixed solvent thereof. Compound (2) is reacted with 0.2 to 100 equivalents of an acetylating agent such as acetic anhydride or acetyl chloride in the presence of a base such as pyridine or triethylamine, or by using the above acetylating agent also as a solvent, if necessary, the above base. Compound (3) can be produced by reacting in the presence. Normally,
The reaction may be performed at a temperature of 0 to 150 ° C. for 5 minutes to 24 hours.

Compound (4): aromatic hydrocarbon solvent such as benzene or toluene, halogenated hydrocarbon solvent such as dichloromethane or chloroform, ether solvent such as tetrahydrofuran or diethyl ether, dimethylformamide, water or methanol Compound (3) in a polar solvent of, or a mixed solvent thereof, for example, 0.2 to 100 equivalents of p-toluenesulfonyl chloride,
Is it reacted with a sulfonylating agent such as methanesulfonyl chloride, trifluoromethanesulfonyl chloride, p-toluenesulfonic anhydride, methanesulfonic anhydride, or trifluoromethanesulfonic anhydride in the presence of a base such as pyridine or triethylamine? , Or using the above sulfonylating agent also as a solvent,
If necessary, the compound (4) can be produced by reacting in the presence of the above base. Usually, the reaction may be performed at a temperature of 0 to 150 ° C. for 5 minutes to 24 hours. In the above reaction, a reaction accelerator such as dimethylaminopyridine is used.
0.01 to 10 equivalents may coexist.

Aromatic hydrocarbon solvents such as benzene or toluene, halogenated hydrocarbon solvents such as dichloromethane or chloroform, ether solvents such as tetrahydrofuran or diethyl ether, polar solvents such as dimethylformamide, water or methanol. Compound (3) is treated with a halogenating agent such as 0.2 to 100 equivalents of phosphorus oxychloride, phosphorus oxybromide, phosphorus pentachloride, or thionyl chloride in a mixed solvent thereof or in the absence of a solvent. The compound (4) can also be produced by this. Usually, the reaction may be performed at a temperature of 0 to 150 ° C. for 5 minutes to 24 hours.

Compound (Ia): an aromatic hydrocarbon solvent such as benzene or toluene, a halogenated hydrocarbon solvent such as dichloromethane or chloroform, an ether solvent such as tetrahydrofuran or dioxane, dimethylformamide, water or methanol Reaction of vinyl metal compound (5) such as vinyltributyltin or vinylmagnesium bromide (5) 0.2 to 100 equivalents with compound (4) in the presence of 0.0001 to 1 equivalent of a catalyst in a polar solvent or a mixed solvent thereof. Can produce the compound (Ia). As the catalyst, a palladium catalyst such as tetrakis (triphenylphosphine) palladium or bis (triphenylphosphine) palladium chloride or a nickel catalyst such as bis (acetylacetonato) nickel chloride can be used. The above reaction may be carried out in the presence of a base such as pyridine or triethylamine. Usually, the reaction may be performed at a temperature of 0 to 150 ° C. for 5 minutes to 24 hours.

Compound (Ib): an ether solvent such as tetrahydrofuran or ether, dimethylformamide,
In a polar solvent such as water or methanol, or a mixed solvent thereof, the compound (Ia) is added to a fluorine-containing compound such as tetrabutylammonium fluoride or potassium fluoride 0.2 to
Compound (Ib) can be produced by reacting with 100 equivalents. Usually, the reaction may be performed at a temperature of 0 to 100 ° C. for 5 minutes to 24 hours.

Compound (Ic): 0.2 to 10 equivalents of β-cyanoethyl phosphoramidite in the presence of 0.2 to 100 equivalents of 1-tetrazole in a suitable solvent such as acetonitrile, dimethylformamide, or dimethylsulfoxide. After reacting deoxythymine (6) at 0 to 100 ° C for 5 minutes to 24 hours, the resulting product is reacted with a peroxide such as t-butyl hydroperoxide or cumene peroxide to give the compound (Ic ) Can be manufactured. Usually, the reaction may be performed at -20 to 50 ° C for about 5 minutes to 24 hours.

Compound (Id): aromatic hydrocarbon solvent such as benzene or toluene, halogenated hydrocarbon solvent such as dichloromethane or chloroform, ether solvent such as tetrahydrofuran or diethyl ether,
In a polar solvent such as dimethylformamide, water, or methanol, or a mixed solvent thereof, or in the absence of a solvent, compound (Ic) is added in an amount of 0.2 to 100 equivalents of an organic acid such as trifluoroacetic acid or dichloroacetic acid, or hydrochloric acid. Alternatively, the compound (Id) can be produced by treating with a mineral acid such as sulfuric acid. The reaction is generally 5 at a temperature of -20 to 50 ° C.
It should be done for 24 hours a minute.

[0022]

[Chemical 6]

Compound (IIa): A compound (4) and a compound (7) are reacted according to the reaction conditions for producing the compound (Ia) from the compound (4) and the compound (5) in Scheme 1. The compound (IIa) can be produced by the method (see Scheme 2 above).

[0024]

[Chemical 7]

Compound (IIIa) and compound (IIIb): Compound (4) and compound (8) according to the reaction conditions for producing compound (Ia) from compound (4) and compound (5) in scheme 1 Compound (IIIa) can be produced by reacting with (see Scheme 3 above). In an ether solvent such as tetrahydrofuran or ether, a polar solvent such as dimethylformamide, water, or methanol, or a mixed solvent thereof, compound (IIIa) is added in an amount of 0.2 to 100 equivalents of a metal water such as sodium hydroxide or potassium hydroxide. Compound (IIIb) can be produced by reacting with an oxide. The reaction may be generally performed at a temperature of 0 to 100 ° C. for 5 minutes to 24 hours.

Compound (IIIc): aromatic hydrocarbon solvent such as benzene or toluene, halogenated hydrocarbon solvent such as dichloromethane or chloroform, ether solvent such as tetrahydrofuran or diethyl ether, polar solvent such as dimethylformamide, or The compound (IIIb) is reacted with the phosphorus compound (9) in the presence of a base such as 0.5 to 50 equivalents of N-methylmorpholine or triethylamine in the mixed solvent, and then the compound is reacted with triethylamine. (IIIc) can be produced. The phosphorus compound (9) is, for example, 0.5 to 10 equivalents of phosphorus trichloride and 0.5 to 10 equivalents of 1,2,4-triazole.
It can be prepared by reacting at -50 ° C for 5 minutes to 24 hours. In the usual case, the above reaction may be carried out at a temperature of -100 to 50 ° C for 5 minutes to 48 hours.

Since the compound of the present invention can rapidly form an adduct with cytidine or guanosine, by using the compound of the present invention as a production intermediate,
Complementarily binds to target nucleic acids such as DNA and m-RNA,
Moreover, an antisense oligonucleic acid that forms a covalent crosslink can be produced. Therefore, according to another aspect of the present invention, there is provided an intermediate for producing an antisense oligonucleic acid, preferably an intermediate for producing an antisense oligonucleic acid having a crosslinkability, which comprises the compound of the present invention. Further, an antisense oligonucleic acid produced by using the above compound as an intermediate for production, preferably an antisense oligonucleic acid having a cross-linking ability is also included in the scope of the present invention. However, the use of the above compound of the present invention is not limited to these uses.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to the following Examples. Needless to say, the reagents and reaction conditions described in the above description of the general production method and the examples below can be appropriately modified or altered. The compound numbers in the examples correspond to the compound numbers in the above scheme.

[0029]

【Example】

Example 1: 5'-O-tert-butyldiphenylsilyl-2'-deoxyguanosine (2) 2'-deoxyguanosine (2.0 g, 7.
2 mmol) in anhydrous DMF (30 ml) in t-butylchlorodiphenylsilane (3.0 ml, 12 mmol) and imidazole.
(1.5 g, 21 mmol) was added, and the mixture was stirred at room temperature for 5 hours.
After 5 hours, water (30 ml) was added to the reaction layer and the obtained crystals were collected by filtration and washed with ethyl acetate. Recrystallization from methanol to give compound (2) as colorless powdery crystals (1.5 g,
3.0 mmol, yield 41%). mp: 163-166 ° C.

¹ H-NMR (DMSO-d6) δ 10.60 (1H, brs),
7.80 (1H, s), 7.7-7.3 (10H, m), 6.48 (2H, s), 6.14 (1
H, t, J = 6.6Hz), 5.38 (1H, d, J = 4.0Hz), 4.50-4.40 (1H,
m), 3.95-3.65 (2H, s), 2.65-2.45 (1H, m), 2.35-2.20
(1H, m), 0.99 (9H, s) IR (cm ^-1 , neat): 3600-2700, 1680, 1630 FABMS (m / z): 506 (M + 1) ⁺ , 528 (M + 23) ⁺

Example 2: 3'-O-acetyl-5'-O-tert-butyldiphenylsilyl-2'-deoxyguanosine (3) Anhydrous pyridine of compound (2) (870 mg, 1.7 mmol) under a stream of argon. (35 ml) solution in acetic anhydride (0.8 ml, 8.5 mmo
l) was added, and the mixture was stirred at 85 ° C. After 5 hours, the reaction layer was diluted with ethyl acetate (50 ml), washed with water (20 ml x 2) and saturated saline (20 ml), dried over anhydrous sodium sulfate,
It was evaporated under reduced pressure. The residue is recrystallized from acetonitrile
Compound (3) was obtained as colorless powdery crystals (651 mg, 1.2
mmol, yield 70%). mp: 226-229 ° C.

¹ H-NMR (CDCl ₃ ) δ 12.01 (1H, brs), 7.8
0-7.30 (10H, m), 6.23 (3H, dd, J = 8.7,5.8Hz), 5.53 (1
H, d, J = 5.9Hz), 4.20 (1H, m), 3.89 (2H, d, J = 3.6Hz), 2.
85-2.70 (1H, m), 2.60-2.45 (1H, m), 2.10 (3H, s), 0.9
9 (9H, s) IR (cm ^-1 , neat): 3500-2600, 1740, 1680, 1600 FABMS (m / z): 548 (M + 1) ⁺ , 570 (M + 23) ⁺

Example 3: 3'-O-acetyl-5'-O-tert-butyldiphenylsilyl-6-Op-toluenesulfonyl-2'-deoxyguanosine (4) Compound (3) (625 mg) under an argon stream. , 1.14 mmol) in anhydrous dichloromethane (15 ml) was cooled to 0 ° C., triethylamine (0.5 ml, 3.59 mmol), p-toluenesulfonyl chloride (1.08 g, 5.65 mmol) and dimethylaminopyridine (43.3 mg, 0.355 mmol). Was added and the mixture was stirred. After 20 hours, the reaction layer was diluted with chloroform (15 ml), the organic layer was washed with water (10 ml), and the aqueous layer was chloroform.
Back-extract with (10 ml x 2). The organic layers were combined, dried over anhydrous sodium sulfate, and then evaporated under reduced pressure. The residue was purified by silica gel column chromatography (FL-60D, chloroform-ethyl acetate 9: 1 → 4: 1) to obtain compound (4) as a colorless caramel-like substance (800 mg, 1.14 mmol, yield).
100%).

¹ H-NMR (CDCl ₃ ) δ 8.02 (2H, d, J = 8.3H
z), 7.87 (1H, s), 7.70-7.60 (4H, m), 7.50-7.30 (8H,
m), 6.26 (1H, dd, J = 8.7, 5.4Hz), 5.55 (1H, m), 4.91
(2H, s), 4.25-4.15 (1H, m), 3.90 (2H, m), 2.90-2.75
(1H, m), 2.60-2.40 (1H, m), 2.45 (3H, s), 2.11 (3H,
s), 1.06 (9H, s) IR (cm ^-1 , neat): 3600-3000, 1740, 1620 FABMS (m / z): 702 (M + 1) ⁺ , 644 (M-57) ⁺ HR-FABMS (m / z): C ₃₀ H ₃₆ N ₅ O ₄ Si (M + 1) ⁺ : Calculated value 558.253 Measured value 558.2535

Example 4: 9- (3-O-Acetyl-5-O-tert-butyldiphenylsilyl-2-deoxy-β-D-ribofuranosyl) -2-amino-6-vinylpurine (Ia) under argon stream Lithium chloride (40 mg, 0.9 mg) in a solution of compound (4) (320 mg, 0.46 mmol) in anhydrous dioxane (5 ml).
4 mmol) and tetrakis (triphenylphosphine) palladium (0) (110 mg, 0.095 mmol) were added and stirred at room temperature. After 30 minutes, vinyltri (n-butyl) tin (760 μ
(1, 2.3 mmol) was added and the mixture was heated to reflux. After 90 minutes, the reaction layer was poured into ethyl acetate-10% aqueous ammonia (30 ml: 3 ml), and the organic layer was washed with 10% ammonium water (3 ml) and saturated saline.
The extract was washed with (3 ml × 2), dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform-ethyl acetate 4: 1) to obtain the compound (Ia) as a pale yellow caramel-like substance (150 mg, 0.
27 mmol, yield 59%).

¹ H-NMR (CDCl ₃ ) δ 7.93 (1H, s), 7.75-
7.30 (10H, m), 7.13 (1H, dd, J = 17.6, 10.7Hz), 6.86 (1
H, dd, J = 17.6, 1.9Hz), 6.34 (1H, dd, J = 8.9, 5.6Hz), 5.
85 (1H, dd, J = 10.7, 1.9Hz), 5.56 (1H, m), 4.87 (2H,
s), 4.20 (1H, m), 3.90 (2H, m), 2.95-2.75 (1H, m), 2.
60-2.50 (1H, m), 2.12 (3H, s), 1.07 (9H, s) IR (cm ^-1 , neat): 3600-3100, 1740, 1600 FABMS (m / z): 558 (M + 1 ) ⁺ , 500 (M-57) ⁺ HR-FABMS (m / z): C ₃₀ H ₃₆ N ₅ O ₄ Si (M + 1) ⁺ : Calculated value 558.2537 Measured value 558.2535

Example 5: 9- (3-O-acetyl-2-deoxy-β
-D- Ribofuranosyl) -2-amino-6-vinylpurine (Ib) A solution of compound (Ia) (25 mg, 46 μmol) in anhydrous tetrahydrofuran (0.1 ml) was cooled to 0 ° C. under an argon stream, and tetrabutylammonium fluoride was added. Mode (100 μl, 100 μmo
l, 1M solution in THF) was added dropwise. After 1 hour, the reaction layer was purified by silica gel column chromatography (FL-60D, hexane-ethyl acetate 9: 1) to obtain the compound (Ib) as a colorless oily substance (14 mg, 44 μmol, yield 98%). .

¹ H-NMR (CDCl ₃ ) δ 7.77 (1H, s), 7.12
(1H, dd, J = 17.5, 10.9Hz), 6.89 (1H, dd, J = 17.6, 1.8H
z), 6.21 (1H, dd, J = 9.9, 5.6Hz), 5.89 (1H, dd, J = 10.6,
2.0Hz), 5.53 (1H, d, J = 5.6Hz), 5.07 (2H, brs), 4.25
(1H, m), 3.20 (1H, dd, J = 9.2, 5.0Hz), 2.38 (1H, dd, J = 1
4.0, 5.4Hz), 2.13 (3H, s) IR (cm ^-1 , neat): 3350, 3200, 1730 FABMS (m / z): 320 (M + 1) ⁺ , 550 (M-31) ⁺

Example 6: 5'-O-di (paramethoxyphenyl)
Phenylmethylthymidylyl- (3'-5 ')-9- (3-O-acetyl-
2-Deoxy-β-D-ribofuranosyl) -2-amino-6-vinylpurine-3 '-[P-β- (cyanoethyl) ester (Ic) Compound (Ib) (24 mg, 75 μmol) under an argon stream. To a solution of T-beta amidite (84 mg, 110 μmol) in anhydrous acetonitrile (0.5 ml), 1-tetrazole (6) (73 mg, 90 μm)
ol) was added and the mixture was stirred at room temperature. After stirring for 2 hours, the reaction layer was cooled to 0 ° C. and t-butyl hydroperoxide was added.
(16 μl, 86 μmol, 5.4M solution in toluene) was added and stirring was continued. After 2 hours, the insoluble matter was filtered off and the solvent was distilled off under reduced pressure. Silica gel column chromatography of the residue
(FL-60D, chloroform-methanol 99: 1 → 95: 5) was purified to obtain compound (Ic) as a colorless oily substance (29 mg,
30 μmol, yield 40%).

¹ H-NMR (CDCl ₃ ) δ 7.86 (1H, s), 7.53
(0.5H, d, J = 0.99Hz), 7.46 (0.5H, d, J = 13Hz), 7.42-7.17
(10H, m), 7.10 (1H, ddd, J = 17.5, 10.9, 2.6Hz), 6.90-
6.77 (4H, m), 6.44-6.33 (1H, m), 6.30-6.23 (1H, m),
5.84 (1H, ddd, J = 10.9, 3.6, 1.98Hz), 5.47 (1H, dd, J = 1
5.8, 5.3Hz), 5.35-5.25 (3H, m), 5.22-5.10 (1H, m),
4.60-4.05 (6H, m), 3.79 (6H, s), 3.55-3.27 (3H, m),
2.72-2.24 (5H, m), 2.12 (1.5H, s), 2.11 (1.5H, s), 1.
41 (1.5H, d, J = 0.99Hz), 1.38 (1.5H, d, J = 0.99Hz) IR (cm ^-1 , neat): 3480, 3340, 3200, 1740, 1690, 160
0 FABMS (m / z): 979 (M + 1) ⁺

Example 7: Thymidylyl- (3'-5 ')-9- (3-O-acetyl-2-deoxy-β-D-ribofuranosyl) -2-amino-6-
Vinyl purine-3 '-[P-β- (cyanoethyl) ester (Id) To compound (Ic) (2.0 mg, 2.0 μmol), trifluoroacetic acid (0.2 ml, 3 μmol) in 0.1 ml CHCl ₃ was added dropwise. . 5
After minutes, ether (ca. 1 ml) was added to the reaction layer, and the precipitated crystals were collected by filtration to obtain the compound (Id) as a colorless powdery substance.
(0.6 mg, 0.9 μmol, yield 45%).

¹ H-NMR (CDCl ₃ ) δ 8.36 (0.5H, s), 8.32
(0.5H, s), 7.44 (0.5H, s), 7.43 (0.5H, s), 7.12-7.03
(1H, m), 6.35-6.26 (1H, m), 6.22-6.13 (1H, m), 6.06-
5.96 (1H, m), 5.58-5.48 (1H, m), 5.22-5.03 (1H, m),
4.63-4.17 (7H, m), 3.91-3.73 (3H, m), 3.54-3.19 (3H,
m), 2.82-2.72 (2H, m), 2.60-2.36 (2H, m), 2.16 (1.5
H, s), 2.15 (1.5H, s), 1.91 (1.5H, d, J = 0.99Hz), 1.89
(1.5H, d, J = 0.99Hz) FABMS (m / z): 677 (M + 1) ⁺

Example 8: 9- (3-O-Acetyl-5-O-tert-butyldiphenylsilyl-2-deoxy-β-D-ribofuranosyl) -2-amino-6-methylvinylpurine (IIa) Argon Stream Below, lithium chloride (42 mg, 1.0 mg) was added to a solution of compound (4) (350 mg, 0.5 mmol) in anhydrous dioxane (5 ml).
mmol) and tetrakis (triphenylphosphine) palladium (0) (116 mg, 0.1 mmol) were added and stirred at room temperature. After 30 minutes, methyl vinyl tri (n-butyl) tin (800
μl, 2.5 mmol, cis / trans = 2.5 / 1) was added, and the mixture was heated to reflux. After 6 hours, the reaction layer was poured into ethyl acetate (100 ml), and the organic layer was washed with 10% ammonium water (30 ml) and saturated saline.
The extract was washed with (30 ml × 2), dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform-methanol = 99: 1) to obtain the compound (IIa) as a pale yellow caramel-like substance (238 mg,
0.42 mmol, yield 84%).

¹ H-NMR (CDCl ₃ ) δ 7.90 (1H, s), 7.68-
7.63 (4H, m), 7.51-7.32 (6.6H, m), 6.87 (0.4H, dq, J = 1
1.2, 2.0Hz), 6.83 (0.6H, dq, J = 15.5, 1.6Hz), 6.28
(0.4H, dq, J = 11.5, 7.2Hz), 6.38 (1H, t, J = 6.3Hz), 5.55
(1H, d, J = 4.6Hz), 4.83 (1H, bs), 4.19 (1H, dd, J = 3.3,
2.0Hz), 3.91-3.89 (2H, m), 2.94-2.81 (1H, m), 2.56-
2.51 (1H, m), 2.25 (3H, dd, J = 1.7, 7.6Hz), 2.02 (3H, d
d, J = 1.7, 6.9Hz), 2.02 (3H, s), 1.06 (9H, s) IR (cm ^-1 , neat): 3500, 1740, 1600 FABMS (m / z): 572 (M + 1) ⁺ , 514 (M-57) ⁺

Example 9: 9- (3-O-acetyl-5-O-tert-butyldiphenylsilyl-2-deoxy-β-D-ribofuranosyl) -2-amino-6- (2-trimethylsilylvinyl) purine ( I
IIa) Lithium chloride (13 mg, 0.3 mg) was added to a solution of compound (4) (104 mg, 0.15 mmol) in anhydrous dioxane (2 ml) under an argon stream.
mmol) and tetrakis (triphenylphosphine) palladium (0) (35 mg, 0.03 mmol) were added and stirred at room temperature. After 30 minutes, trimethylsilyl vinyl tri (n-butyl)
Tin (1.7 ml, 1.5 mmol) was added, and the mixture was heated under reflux. After 1 hour, the reaction layer was poured into ethyl acetate (50 ml) and the organic layer was added to 1
The extract was washed with 0% ammonium water (10 ml) and saturated saline (10 ml × 2), dried over anhydrous sodium sulfate, and evaporated under reduced pressure.
The residue was purified by silica gel column chromatography (hexane-ethyl acetate 20: 1 → 10: 1) to obtain compound (IIIa) as a pale yellow caramel-like substance (80 mg, 0.13 mmol).
, Yield 87%).

¹ H-NMR (CDCl ₃ ) δ 7.95 (1H, s), 7.73
(1H, d, J = 19.1Hz), 7.68-7.64 (4H, m), 7.45-7.33 (6H,
m), 7.36 (1H, d, J = 19.1Hz), 6.34 (1H, dd, J = 8.9, 5.3H
z), 5.57 (1H, bd, J = 6.3Hz), 4.97 (2H, b), 4.91 (1H, d, J
= 2.0Hz), 3.92-3.90 (2H, m), 2.88-2.79 (1H, m), 2.56-
2.49 (1H, m), 2.12 (3H, s), 1.08 (9H, s), 0.21 (9H, s) IR (cm ^-1 ,, neat): 3300, 1740, 1595, 1570 FABMS (m / z): 630 (M + 1) ⁺

Example 10: 9- (5-O-tert-butyldiphenylsilyl-2-deoxy-β-D-ribofuranosyl) -2-amino-6-
(2-Trimethylsilylvinyl) purine (IIIb) Compound (IIIa) (72 mg, 0.11 mmol) in methanol (1.0
(ml) solution, potassium carbonate (110 mg, 0.8 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. Reaction layer was methylene chloride (30 ml)
The mixture was diluted with, washed with saturated aqueous ammonium chloride solution (10 ml) and saturated brine (10 ml), dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform-methanol = 98: 2 → 95: 5) to obtain compound (IIIb) as a pale yellow oily substance (67 m
g, 0.11 mmol, yield 100%).

¹ H-NMR (CDCl ₃ ) δ 7.90 (1H, s), 7.71
(1H, d, J = 19.1Hz), 7.68-7.64 (4H, m), 7.45-7.33 (6H,
m), 7.34 (1H, d, J = 19.1Hz), 4.81 (2H, bs), 4.70 (1H, d
t, J = 3.0,2.6Hz), 4.10 (1H, dd, J = 7.3, 4.3Hz), 3.87 (1
H, dd, J = 10.9, 5.3Hz), 3.81 (1H, dd, J = 11.2, 4.3Hz),
2.70 (1H, dt, J = 13.5, 7.3Hz), 2.44 (1H, ddd, J = 13.2,
5.9, 3.3Hz), 1.07 (9H, m), 0.20 (9H, s) IR (cm ^-1 ,, neat): 3500, 1605, 1590 FABMS (m / z): 588 (M + 1) ⁺ , 530 ( M-57)

Example 11: 9- (5-O-tert-butyldiphenylsilyl-2-deoxy-3-OH-phosphonate-β-D-ribofuranosyl) -2-amino-6- (2-trimethylsilylvinyl) purine Triethylammonium salt (IIIc) Under an argon stream, phosphorus trichloride (4.4 μl, 0.05 mmol) was dissolved in methylene chloride (1.5 ml), and N-methylmorpholine (4.4 μl, 0.05 mmol) 1,2,4- Triazole was added and the mixture was stirred at room temperature for 30 minutes. The reaction solution was cooled to -78 ° C, and compound (IIIb) (11 mg, 0.03 mmol) in methylene chloride was used.
(0.5 ml) solution was added dropwise, and the mixture was stirred at -60 ° C for 10 hours, poured into TEAB buffer (10 ml) for separation, and the aqueous layer was diluted with methylene chloride.
(30 ml) extracted 4 times, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. Silica gel column chromatography of the residue (chloroform-triethylamine = 98: 2 → chloroform-methanol-triethylamine = 88: 10: 2)
The compound (IIIc) was obtained as a pale yellow oily substance.
(14 mg, 0.02 mmol, yield 73%).

¹ H-NMR (CDCl ₃ ) δ 7.91 (1H, s), 7.68
(1H, d, J = 19.1Hz), 7.68-7.64 (4H, m), 7.45-7.33 (6H,
m), 7.33 (1H, d, J = 19.1Hz), 6.37 (1H, dd, J = 8.2, 6.3H
z), 5.12-5.10 (1H, m), 4.93 (2H, bs), 4.30 (1H, dd, J =
6.3, 3.6Hz), 3.91 (1H, dd, J = 11.2, 4.3Hz), 3.82 (1H,
dd, J = 11.2, 4.0Hz), 3.05-2.73 (2H, m), 3.07 (6H, q, J =
7.3Hz), 1.34 (9H, t, J = 7.3Hz), 1.03 (9H, s), 0.18 (9
H, s) IR (cm ^-1 , neat): 3500, 1605, 1590 FABMS (m / z): 652 (M + 1) ⁺ , 594 (M-57)

Example 12: Function of the compound of the present invention as a cross-linking agent The function of the vinylated deoxyguanosine derivative of the present invention as a cross-linking agent was evaluated. According to the following scheme, the compounds (IIa) and (IIIa) of the present invention are used to react a vinylated deoxyguanosine derivative with hydroxylamine to obtain a half-life of the vinylated deoxyguanosine derivative.
(t _1/2 ) was calculated. Details of the reaction conditions are as follows. As a result, t _1/2 of Compound IIa is 1 hour, t _1/2 of Compound (IIIa) was more than 18 hours.

[0052]

Embedded image

Example 13: Adduct of Compound (IIa) with Hydroxylamine Hydrochloride (10) Compound (IIa) (30 mg, 70 μmol) in ethanol (7.0 ml)
Hydroxylamine hydrochloride in solution (24 mg, 350 μmol)
Was added and stirred at room temperature. After 24 hours, the solvent was distilled off under reduced pressure. Silica gel column chromatography of the residue
(FL-60D, chloroform → chloroform-methanol 9
5: 5) to obtain the adduct (10) as a yellow oily substance.
(16 mg, 26 μmol, yield 38%).

¹ H-NMR (CDCl ₃ ) δ 7.93 (1H, s), 7.68-
7.63 (4H, m), 7.51-7.32 (6H, m), 6.33-6.27 (1H, m),
5.75 (2H, bs), 5.53 (1H, d, J = 5.9Hz), 4.18 (1H, t, J = 1.
7Hz), 3.92-3.85 (3H, m), 3.52 (1H, d, J = 16.5Hz), 3.36
-3.28 (1H, m), 2.78-2.71 (1H, m), 2.57-2.49 (1H, m),
2.11 (3H, s), 1.52 (3H, d, J = 6.6Hz), 1.02 (9H, s), IR
(cm ^-1 ,, neat): 3500, 1720, 1600 FABMS (m / z): 605 (M ⁺ +1), 572 (M ⁺ -33) HR-FABMS (m / z): C ₃₁ H ₄₁ N ₆ O ₅ Si (M ⁺ +1): Calculated value 605.2908 Measured value 605.2910

Example 14: Adduct of Compound (IIIa) with Hydroxylamine Hydrochloride (11) Compound (IIIa) (10 mg, 1.6 μmol) in ethanol (1 m
l) Hydroxylamine hydrochloride (5.5 mg, 8 μmol) in solution
, Ethanol 0.5 ml) was added, and the mixture was stirred at room temperature. After 24 hours, the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography (FL-60D, chloroform →
Chloroform-methanol 95: 5) and purified adduct (11)
Was obtained as a colorless oily substance (mixture of cis form and trans form)
(4.0 mg, 0.68 μmol, yield 44%).

¹ H-NMR (CDCl ₃ ) δ 7.92 (1H, s), 7.18
(1H, t, J = 4.9Hz), 7.75 (1H, t, J = 5.9Hz, minor), 7.68-7.
63 (4H, m), 7.51-7.32 (6H, m), 6.31 (1H, dd, J = 5.6, 8.
9Hz), 5.56 (1H, d, J = 5.9Hz), 5.01 (2H, bs), 4.19 (1H,
dd, J = 3.3, 2.0Hz), 4.15 (2H, d, J = 4.9Hz), 3.94 (2H, d,
J = 6.3Hz, minor), 3.91-3.89 (2H, m), 2.94-2.81 (1H,
m), 2.56-2.51 (1H, m), 2.12 (3H, s), 1.07 (9H, s), IR (cm ^-1 ,, neat): 3600-3300, 1605, 1590 FABMS (m / z): 589 (M ⁺ +1), 573 (M ⁺ -15) HR-FABMS (m / z): C ₃₀ H ₃₇ N ₆ O ₅ Si (M ⁺ +1): Calculated value 589.2600 Measured value 589.2595

[0057]

The compound of the present invention is chemically stable,
It also has the property of easily reacting with nucleophilic molecules such as cytidine and guanosine to form a covalent bond. By using the compound of the present invention as a production intermediate, an antisense oligonucleic acid having a crosslinkability can be produced.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Minor Maeda 3-24-21 Kazumigaoka, Higashi-ku, Fukuoka-shi, Fukuoka (72) Inventor Shigeki Sasaki 1-3-6-301 Chidori, Koga-cho, Kasuya-gun, Fukuoka

Claims (5)

[Claims] 1. The following formula (I): [Wherein R ¹ , R ² and R ³ are defined by any of the following (1) to (3): (1) R ¹ represents a hydrogen atom, R ² represents a hydrogen atom, tert-butyl A diphenylsilyl group or the following formula: (In the formula, R ⁴ represents a hydrogen atom or a di (p-methoxyphenyl) phenylmethyl group), and R ³ represents an acetyl group; (2) R ¹ represents a methyl group, R ² represents a tert-butyldiphenylsilyl group, and R ³ represents an acetyl group; or (3) R ¹ represents a trimethylsilyl group, R ² represents a tert-butyldiphenylsilyl group, and R ³ represents hydrogen. An atom, an acetyl group, or -P (O) (OH) H]] or a salt thereof. 2. An intermediate for producing an antisense oligonucleic acid, which comprises the vinylated deoxyguanosine derivative according to claim 1. 3. The production intermediate according to claim 2, which is used for producing an antisense oligonucleic acid having a cross-linking ability. 4. An antisense oligonucleic acid produced by using the compound according to claim 1. 5. The antisense oligonucleic acid according to claim 4, which has a cross-linking ability.