JPH08119945A - New nucleotide analog - Google Patents

Abstract

PURPOSE: To obtain the subject compound, represented by a specific formula, hardly undergoing the hydrolysis with an enzyme in the living body, readily synthesized, suitable for anti-sense molecules and useful as medicines capable of inhibiting the function of a gene and treating diseases such as an antitumor agent. CONSTITUTION: This compound is expressed by formula I [B1 is pyrimidine, a purine nucleic aid base or its derivative; X and Y are each O or S; R is H, an alkyl or an acyl; W is H, an alkyl or an acyl or may be a (oligo) nucleotide or a polynucleotide through a phosphoric ester bond when X is O; (n) is 1-50]. Furthermore, the compound is preferably obtained by selecting, e.g. an optically active (S)-glycidol of formula II as a raw material, initially reacting the selected optically active (S)-glycidol with aqueous ammonia, subsequently adding triethylamine and di-t-butyl carbonate thereto, stirring the prepared mixture, further adding and reacting t-butyldiphenylsilyl chloride and 4-dimehylaminopyridine with the mixture and passing the reactional product through the resultant monomer unit intermediate, etc.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to novel nucleotide analogs, and more particularly to nucleotide analogs suitable for antisense molecules.

[0002]

2. Description of the Related Art The first report was made in 1978 that an antisense molecule inhibited the infection of influenza virus. Since then, it has been reported that it inhibited oncogene expression and AIDS infection. Since antisense oligonucleotides specifically control the expression of undesired genes, it is one of the most promising fields for pharmaceuticals in recent years.

The antisense method is based on the concept of controlling a series of so-called central dogma flow of DNA → RNA → protein using an antisense oligonucleotide.

However, when a natural type oligonucleotide was applied to this method as an antisense molecule, there were problems that it was hydrolyzed by an enzyme in the living body and cell membrane permeability was not high. In order to solve these problems, many nucleic acid derivatives have been synthesized and researched. For example, phosphorothioate in which oxygen atom on phosphorus atom is replaced by sulfur atom, methylphosphonate in which methyl group is substituted, and more recently, phosphorus atom is also replaced by carbon atom, or molecule in which ribose is acyclic skeleton. Have also been synthesized (F. Eckstein et al., Biochem., 18,
592 (1979), PSMiller et al., Nucleic Acids Res.,
11, 5189 (1983), P. Herdewijn et al., J. Chem. Soc.
Perkin Trans. 1, 1567 (1993), PE Nielsen et al.,
Science, 254, 1497 (1991)).

However, in any case, a derivative which is satisfactory in terms of stability in vivo or easiness of oligonucleotide synthesis has not been obtained.

[0006]

It is desired to provide a nucleotide analog for an antisense molecule which is less susceptible to enzymatic hydrolysis in vivo and is easy to synthesize.

[0007]

The inventors of the present invention designed and synthesized a nucleic acid derivative that would be useful in the antisense method, and confirmed its usefulness. The present invention will be described below.

The nucleic acid derivative directed to the antisense method was designed with the following three points in mind. 1. Substituting carbamate or thiocarbamate bonds for phosphodiester bonds that are hydrolyzed by enzymes in the body.

2. In order to eliminate the structural distortion caused by the carbamate or thiocarbamate bond at the time of double chain formation, make ribose an acyclic skeleton so that the molecule has flexibility.

3. The basic skeleton should be a glycerin derivative that is close to the biological constituents in order to suppress the development of toxicity when it is decomposed and metabolized in the body.

From the above points, an antisense DNA analog represented by the following general formula was designed and synthesized.

The nucleotide analog of the present invention can be represented by the following general formula.

[0013]

Embedded image [Wherein B ₁ is the same or different and is a pyrimidine or purine nucleobase or a derivative thereof, X and Y are independently oxygen or sulfur, R is hydrogen, an alkyl group, or an acyl group, W may be hydrogen, an alkyl group, an acyl group, or when X is oxygen, a nucleotide, an oligonucleotide or a polynucleotide via a phosphate bond. n is an integer of 1 to 50. However, when n is 2 or more, B ₁ is not limited to the same base. ]

[Chemical 4] [Wherein B ₁ and B ₂ are the same or different, each is a pyrimidine or purine nucleobase or a derivative thereof, X and Y are independently oxygen or sulfur, W is the same or different, and hydrogen, alkyl It may be a nucleotide, oligonucleotide or polynucleotide through a group, an acyl group, or a phosphate bond. m is an integer of 1 to 25. However, when m is 2 or more, B ₁ and B ₂ are not limited to the same base. ] In the present invention, the pyrimidine or purine nucleobase is
Thymine, uracil, cytosine, adenine, guanine and their derivatives.

The nucleotide analog of the present invention can be synthesized as follows. To simplify the explanation, first, a compound in which X and Y in the above formula are both oxygen will be described as an example.

(1) Synthesis of monomer unit

Embedded image First, since glycerin has a basic skeleton, optically active (S) -glycidol (Compound 1) is selected as a raw material, and this is reacted with aqueous ammonia to obtain Compound 2. Then t
The amino group was protected with an ert-butoxycarbonyl (Boc) group to give compound 3 as a colorless oily substance. Subsequently, the primary hydroxyl group was converted to tert-butyldiphenylsilyl (TB
The compound (silyl compound) 4 is obtained as colorless crystals by selective protection with a DPS group.

[0016]

[Chemical 6] Then, the secondary hydroxyl group is methylthiomethylated with dimethyl sulfoxide, acetic anhydride, and acetic acid to obtain compound 5. Further, the monomer unit (R-form) 6 is obtained by reacting with a nucleobase (B ₁ ) activated with a trimethylsilyl (TMS) group, for example, thymine · 2TMS.

Subsequently, in order to condense with a natural nucleoside (for example, thymidine or the like) or to polymerize the monomer unit, the TBDPS group is deprotected to remove the desilylated product 7 and the Boc group to obtain the deBoc product 8. .

[0018]

[Chemical 7] If the starting material is (R) -glycidol (compound 9), the S-form monomer unit 10 can be prepared by the same method.
Is obtained in the same manner, and deprotection is performed in the same manner to remove the desilylated product 11 and the B
The oc body 12 is obtained.

(2) Synthesis of homo-oligomer

Embedded image The desilylated product 7 of the monomer unit (R-form) 6 synthesized above and the de-Boc product 8 are condensed with N, N′-carbonyldiimidazole to obtain the homodimer 13. This is further deprotected to give the desilylated body 14 and the de-Boc body 1
Get 5.

Then, the homosilylated desilylated product 14 and the homodimer-free Boc product 15 are similarly condensed to obtain a homotetramer 16. Further, the homotrimer 17 can be synthesized by condensing the desilylated body 7 of the monomer unit and the deBoc body 15 of the homodimer in the same manner, or by condensing the deBoc body 8 of the monomer unit and the desilylated body 14 of the homodimer.

A homo-oligomer ([Chemical Formula 3]) can be obtained by condensing in the same manner as described above. By using the monomer unit (S-form) 10, an S-form homooligomer can also be synthesized.

(3) Synthesis of type I oligonucleotide analogue (hetero-oligomer)

[Chemical 9] And the 5'-side hydroxyl group is dimethoxytrityl (DMTr)
Group-protected nucleoside 18 (eg thymidine form)
Is reacted with N, N′-carbonyldiimidazole and condensed with the previously obtained de-Boc body (R body) 8 to obtain a dimer unit 19. The TBDPS group is deprotected to give compound 20, which is then led to amidite body 21.

Purification was performed by synthesizing antisense oligomer analogs of various sequences from Compound 21 using a DNA synthesizer and then purifying using a C18 reverse phase column, and analyzing the purity of the purified product by reverse phase C18 HPLC. It can be confirmed that the oligonucleotide analogue (type I R-isomer) is produced. In addition, instead of the de-Boc body (R body) 8, the de-Boc body is removed.
If the body (S body) 12 is used, an S-oligonucleotide analog can be obtained.

(4) Synthesis of type II oligonucleotide analogue (hetero-oligomer)

[Chemical 10] Like the de-Boc form (S form) 12 obtained by deprotecting the Boc group of the S form monomer unit 10 synthesized above and the 3'-O-acetyl nucleoside 22 (for example, 3'-O-acetyl thymidine, etc.) By condensing with natural nucleosides
The silyl compound 23 of the type II dimer is obtained. Then, the hydroxyl group of the desilylated dimer 24 is protected with a DMTr group to give a compound 25, which is deacetylated to 26, and then an amidite body 27 is obtained. Using this amidite with a commercially available natural nucleoside amidite, the type II oligonucleotide analog of the present invention can be obtained by a DNA synthesizer. If the Boc-free body (R body) 8 is used instead of the Boc-free body (S body) 12, the R body (II type) is obtained.
An oligonucleotide analog is obtained. Purification and identification are as described in (3) above.

The method for synthesizing the oligonucleotide analog of the present invention has been described. Among the compounds represented by the above formulas (formula 3 and formula 4), a thio derivative in which X and / or Y in the formula is sulfur. Is 3′-instead of the natural nucleoside
X or Y is sulfur by using a thionucleoside or a 5'-thionucleoside or an analog thereof, or by using N, N'-thiocarbonyldiimidazole instead of N, N'-carbonyldiimidazole. Thio derivatives can be synthesized. Further, by performing both simultaneously, a derivative in which both X and Y are sulfur can be obtained. For example, the type I and type II heterodimer thio derivatives can be produced by the following synthetic method.

(Type I) Known 5'-monomethoxytrityl-3'-deoxy-3'-thiothymidine (R. Cro)
sstick, JS Vyle, J.Chem.Soc., Chem.Commun., 993 (1
988)) as a starting material and commercially available N, N′-thiocarbonyldiimidazole (W.Walter, KDBode, Angew.Chem., 7
9,285 (1967)) or N, N′-carbonyldiimidazole is used to condense with the de-Boc derivative of the monomer unit to obtain the desired type I heterodimer thio derivative.

(Type II) Literature known 3'-O-ter
t-Butyldimethylsilyl-5'-deoxy-5'-thiothymidine (SH Kawai, D. Wang, G. Just, Can. J. Chem.,
70, 285 (1967)) as a starting material, and commercially available N, N'-
Thiocarbonyldiimidazole (supra) or N, N '
The desired type II heterodimer thio derivative can be obtained by condensing the de-Boc derivative of the monomer unit using -carbonyldiimidazole.

According to the present invention, it is possible to synthesize an antisense molecule in which the nucleotide analog of the present invention is introduced at a required position in a required number (length). The length of the entire nucleotide analog is preferably 10 to 30 nucleoside units. Such an antisense molecule is hardly decomposed not only by exonuclease but also by endonuclease, and can remain in vivo for a long time after being administered to a living body. Then, for example, it forms a double strand with a sense strand RNA to inhibit the formation (translation) of an in vivo component (protein) that is a causative factor, or double strand D
It forms a triple chain with NA to inhibit transcription into mRNA. It is also considered to inhibit the growth of infected virus.

From these facts, the antisense molecule using the nucleotide analog of the present invention is useful as a drug for treating diseases by inhibiting the action of genes such as antitumor agents and antiviral agents. Is expected.

The antisense molecule using the nucleotide analog of the present invention can be made into a preparation for parenteral administration by incorporating a conventional auxiliary agent such as a buffer and / or a stabilizer. Further, as a topical preparation, a conventional pharmaceutical carrier can be blended to prepare an ointment, cream, liquid, salve or the like.

[0031]

EXAMPLES The synthesis of the nucleotide analogs of the present invention will be described in more detail with reference to Examples and Production Examples.

Example 1: Monomer unit intermediate (R
Body) 1-a: Synthesis of silyl body 4

[Chemical 11] By the method ¹⁾ known in the literature, (S) -glycidol
Concentrated aqueous ammonia (440 ml) was added to (Compound 1) (5.0 g, 68 mmol), and the mixture was stirred under ice cooling for 17 hours. After distilling off the aqueous ammonia at a low temperature, it was diluted with absolute ethanol (120 ml) and dried over anhydrous calcium carbonate, and the solvent was distilled off. DMF solution of the obtained residue (10
0 ml) with triethylamine (9.4 ml, 1e)
q. ) And di-t-butyl dicarbonate (15.5 m
1, 1 eq. ) Was added and the mixture was stirred overnight at room temperature. The residue obtained by distilling off DMF was added to a CH ₂ Cl ₂ solution (60 ml),
t-Butyldiphenylsilyl chloride (17.6 ml,
1 eq. ), 4-dimethylaminopyridine (0.83
g, 0.1 eq. ), Triethylamine (9.4 ml,
1 eq. ) Was added and stirred for 30 hours. After distilling off the solvent, a silica gel column (AcOEt: n-Hexane) was used.
= 1: 2), white powder 4 (21.3 g, 50
mmol, 73%) was obtained.

Mp 37-39 ° C. ¹ H-NMR (CDCl ₃ ) δ: 1.06 (9 H, s),
1.40 (9H, s), 2.88 (1H, m), 3.0
6-3.19 (1H, m), 3.32-3.40 (1
H, m), 3.55-3.68 (2H, m), 3.70.
-3.83 (1H, m), 4.82 (1H, br),
7.28-7.42 (6H, m), 7.60-7.71
(4H, m) 1-b: Synthesis of methylthiomethyl (MTM) body 5

[Chemical 12] 4 (10.0 g, 23.3 mmol) DMSO (60
ml, 35 eq. ) Solution, acetic anhydride (44 ml, 20
eq. ) And acetic acid (88 ml, 60 eq.) Were added dropwise, and the mixture was stirred at room temperature for 90 hours. The mixture was neutralized with saturated aqueous sodium hydrogen carbonate, and the aqueous phase was extracted with ethyl acetate. The organic phase was washed with water and saturated saline, and dried over anhydrous magnesium sulfate. Silica gel column (AcOEt: n-Hexane = 1: 1)
6) and colorless oily substance 5 (8.4 g, 17.
1 mmol, 73%) was obtained.

¹ H-NMR (CDCl ₃ ) δ: 1.05
(9H, s), 1.43 (9H, s), 2.12 (3
H, s), 3.21-3.23 (1H, m), 3.38.
-3.42 (1H, m), 3.69 (2H, d, J =
4), 3.83 (1H, m), 4.59 (1H, d, J
= 10), 4.69 (1H, d, J = 10), 4.91.
(1H, br), 7.38-7.45 (6H, m),
7.65-7.67 (4H, m) Example 2: Synthesis of monomer unit (R form)

[Chemical 13] 2-a: Methylthiomethyl (MTM) body 5 (2.7 in a synthetic nitrogen stream of the monomer unit 6a )
Molecular sieves (4 Å) (1 g) was added to a CH ₂ Cl ₂ solution of g, 5.6 mmol) and the mixture was stirred at room temperature for 10 minutes. O, O'-bistrimethylsilylthymine ²⁾ (3.0
g, 2 eq. ) Is added and the mixture is stirred for another 10 minutes, then N 2
-Bromosuccinimide (1.1 g, 1.1 eq.) Was added and stirred for 60 minutes. Saturated sodium bicarbonate water was added, and the aqueous phase was CH ₂
Extracted with Cl ₂ . The organic phase was washed with water and saturated saline,
It was dried over anhydrous magnesium sulfate. Silica gel column chromatography (CH ₂ Cl ₂ : MeOH = 30: 1)
In a colorless crystal, compound 6a (3.0 g, 5.3).
mmol, 94%) was obtained.

Mp 56-57 ° C. ¹ H-NMR (CDCl ₃ ) δ: 1.05 (9 H, s),
1.39 (9H, s), 1.87 (3H, s), 3.1
2 (1H, dt, J = 14, 6), 3.39 (1H,
m), 3.67 (2H, d, J = 4), 3.76 (1
H, m), 4.78 (1H, br), 5.10 (1H,
d, J = 10), 5.22 (1H, d, J = 10),
7.06 (1H, s), 7.40 (6H, m), 7.6
3 (4H, m), 8.11 (1H, br) [α] _D ²⁶ + 18 ° (c = 1.0, acetone) Anal. Calcd for C ₃₀ H ₄₁ N ₃ O ₆ Si
_{1 / 4H 2 O: C,} 62.97; H, 4.70; N,
7.34. Found: C, 62.86; H, 7.2.
6; N, 7.48. MS (EI) 568 (M ⁺ ) 2-b: Synthesis of monomer unit 6b 9-in place of O, O'-bistrimethylsilylthymine
Compound 6b was obtained using trimethylsilyl-6- (N-trimethylsilyl) benzamidopurine ³⁾ by the same method and conditions as in 2-a above.

Yield 19% mp 52-54 ° C. ¹ H-NMR (CDCl ₃ ) δ: 1.06 (9H, s),
1.38 (9H, s), 3.10 (1H, m), 3.4
1 (1H, m), 3.71 (2H, d, J = 6), 3.
87 (1H, m), 5.18 (1H, br), 5.74
(2H, dd, J = 9, 10), 7.38-7.66
(13H, m), 8.03 (3H, m), 8.79 (1
H, s), 9.34 (1H, s) [α] _D ²⁵ + ¹⁹ ° (c = 1.1, acetone) MS (EI) 681 (M ⁺ ) 2-c: Synthesis of monomer unit 6c O, O '-Bistrimethylsilyl thymine instead of 4-
Compound (6c) was obtained using (N-trimethylsilyl) benzamido-2- (trimethylsilyloxy) pyrimidine ⁴⁾ by the same method and conditions as in 2-a above.

Yield 46% mp 57-60 ° C. ¹ H-NMR (CDCl ₃ ) δ: 1.07 (9 H, s),
1.40 (9H, s), 3.21 (1H, m), 3.4
0 (1H, m), 3.73 (2H, d, J = 5), 3.
87 (1H, m), 4.86 (1H, br), 5.26
(1H, d, J = 10), 5.38 (1H, d, J = 1
0), 7.36-7.65 (16H, m), 7.92
(2H, m) [α] _D ²⁵ + 12 ° (c = 0.98, acetone) MS (CI) 657 (M ⁺ +1) Example 3: Polymerization of monomer unit (R form)
Synthesis of moligomers

Embedded image 3-a: Synthetic monomer unit (R form) 6a (500 mg, 0.88) of desilylated product 7a
(mmol) to a solution of anhydrous THF (14 ml).
0M tetrabutylammonium fluoride / THF
(0.88 ml, 1.0 eq.) Was added and the mixture was stirred at room temperature for 20 minutes, and the solvent was evaporated. Purified by silica gel column chromatography (chloroform: methanol = 20: 1), white powder desilylated product (R product) 7a (211m)
g, 0.64 mmol, 73%) was obtained.

¹ H-NMR (CDCl ₃ ) δ: 1.43
(9H, s), 1.93 (3H, s), 2.72 (1
H, br), 3.35 (1H, m), 3.48 (1H,
m), 3.61 (2H, m), 3.71 (1H, m),
5.10 (1H, br), 5.22 (2H, m), 7.
19 (1H, s), 9.38 (1H, s) [α] _D ²⁵ + 9 ° (acetone.c = 1.0) 3-b: Synthetic monomer unit (R form) 6a (372 mg, 0) of homodimer 13a .65
mmol, 1.1 eq. ) In methylene chloride (5.5
(ml), trifluoroacetic acid (0.5 ml) was added dropwise under ice cooling, the mixture was stirred at room temperature for 120 minutes, and the solvent was distilled off to obtain B-free
The oc body 8a was used for condensation without purification. Desilylated body 7
a (200 mg, 0.61 mmol, 1.0 eq.) was azeotropically distilled with anhydrous pyridine three times to dehydrate it to give an anhydrous pyridine solution (5.5 ml). N, N'-Carbonyldiimidazole (198 mg, 2.0 eq.) Was added, and the mixture was stirred at room temperature for 150 minutes under a nitrogen stream. Water (0.13 ml, 12
eq. ) Is added and the mixture is stirred for 30 minutes to obtain an excess of N, N '.
After decomposing carbonyldiimidazole, the solvent was distilled off. It was dehydrated by azeotropic distillation with anhydrous pyridine three times to give an anhydrous pyridine solution (5.5 ml). To this was added a solution of 8a in anhydrous pyridine (5.5 ml) and 4-dimethylaminopyridine (DMAP) (7 mg, 0.06 mmol, 1.0e).
q. ) Was added, and the mixture was stirred under a nitrogen stream for 2 days at room temperature. After distilling off the solvent, the mixture was diluted with ethyl acetate, washed 4 times with water and 2 times with saturated saline, and the organic layer was dried over anhydrous sodium sulfate, and then the solvent was distilled off. After purification by silica gel column chromatography (chloroform: methanol = 30: 1), white powder of homodimer (R form) 13a (320 m)
g, 0.39 mmol, 64%) was obtained.

Mp 80-83 ° C. ¹ H-NMR (CDCl ₃ ) δ: 1.06 (9 H, s),
1.43 (9H, s), 1.87 (3H, s), 1.9
1 (3H, s), 3.00 (1H, m), 3.17 (1
H, m), 3.32 (1H, m), 3.38 (1H,
m), 3.67 (2H, m), 3.84-3.95 (3
H, m), 4.14 (1H, m), 5.08-5.26.
(5H, m), 5.55 (1H, br), 6.98 (1
H, s), 7.20 (1H, s), 7.40 (6H,
s), 7.65 (4H, m), 9.55 (1H, s),
9.96 (1H, s) [α] _D ²⁸ + 25 ° (c = 1.0, acetone) 3-c: Synthesis of homotetramer (R isomer) 16a Homodimer (R isomer) 13a (119 mg, 0.145)
mmol, 1.1 eq. ) Methylene chloride solution (1.0
(ml) was added dropwise with trifluoroacetic acid (0.11 ml) under ice-cooling and stirred at room temperature for 120 minutes, and then the solvent was distilled off to obtain homo-dimer de-Boc derivative 15a which was used for condensation as it was without purification.

Homodimer 13a (111 mg, 0.1
35 mmol, 1.0 eq. ) To anhydrous THF (3.0 m
1), 1.0 M tetrabutylammonium fluoride / THF (0.14 ml, 1.0 eq.) was added, and the mixture was stirred at room temperature for 120 minutes. After distilling off the solvent, silica gel column chromatography (chloroform: methanol = 3)
The resulting homodimeric desilylated product 14a was azeotroped with anhydrous pyridine three times to dehydrate it to obtain an anhydrous pyridine solution (0.6 ml). N, N'-Carbonyldiimidazole (44 mg, 2.0 eq.) Was added, and the mixture was stirred at room temperature for 150 minutes under a nitrogen stream. Water (30 μl, 1
2 eq. ) Is added and the mixture is stirred for 30 minutes, whereby excess N,
After decomposing N'-carbonyldiimidazole, the solvent was distilled off. It was dehydrated by azeotropic distillation with anhydrous pyridine three times to obtain an anhydrous pyridine solution (0.6 ml). To this was added a solution of 15a in anhydrous pyridine (0.6 ml) and 4-DMAP (7 m
g, 0.06 mmol, 1.0 eq. ) Was added, and the mixture was stirred under a nitrogen stream for 2 days at room temperature. After the solvent was distilled off, it was diluted with methylene chloride, washed twice with water and once with saturated saline, and the organic layer was dried over anhydrous sodium sulfate, and then the solvent was distilled off. Reprecipitation with methanol / water gave a white powder of homotetramer (R form) 16a (95 mg, 0.071 mmo).
1, 53%).

Mp84-86 ° C Example 4: Synthesis of IR heterodimer unit
(I)

[Chemical 15] 4-a: Heterodimer unit / silyl body 19a
Synthesis of 5'-O- (4,4'-dimethoxytrityl) thymidine 18a (440 mg, 0.81 mmol) in pyridine (5 ml) was added with N, N'-carbonyldiimidazole (260 mg, 2 eq.). Stir for hours. The reaction was stopped with water (15 μl, 1 eq.) And dehydrated by azeotropic distillation with pyridine to give a solution of anhydrous pyridine (5 ml). To this, compound 6a (500 mg, 0.87 mmo
1, 1.1 eq. ) To a CH ₂ Cl ₂ solution (7 ml), trifluoroacetic acid (TFA) (1 ml) was added, the mixture was stirred at room temperature for 3 hours, and then the solvent was distilled off to obtain the de-Boc body 7 a (410
mg, 0.87 mmol) in pyridine (3 ml) and 4-dimethylaminopyridine (110 mg, 1.1).
eq. ), Triethylamine (120 μl, 1.1e
q. ) Was added. After stirring for 100 hours at room temperature, pyridine was distilled off. Pyridine was removed by azeotropic distillation with benzene, the residue was diluted with ethyl acetate, and washed with water and saturated brine. The organic phase was dried over anhydrous sodium acetate, and the solvent was distilled off, followed by silica gel column chromatography (AcOEt: n).
-Hexane = 5: 1) and purified as a white powder I
-R-type silyl compound 19a (660 mg, 0.64 mmo
1, 79%).

Mp112-116 ° C. ¹ H-NMR (CDCl ₃ ) δ: 1.06 (9 H, s),
1.34 (3H, s), 1.86 (3H, s), 2.4
0 (2H, m), 3.13 (1H, m), 3.42 (1
H, m), 3.46 (2H, dd, J = 9, 17),
3.67 (2H, d, J = 6), 3.79 (1H,
m), 3.79 (6H, s), 4.18 (1H, m),
5.04 (1H, d, J = 10), 5.16 (1H,
d, J = 10), 5.28 (1H, t, J = 5), 5.
32 (1H, m), 6.44 (1H, t, J = 7),
6.83 (4H, AB, J = 9), 6.97 (1H,
s), 7.23-7.64 (19H, m), 8.47.
(1H, s), 8.85 (1H, s), 10.3 (1
H, br) MS (FAB) 1036.5 (M ⁺ ) heterodimer unit / silyl compound 19b to 19f
The following heterodimer unit was synthesized by the method and conditions described above.

The bases bonded to the synthesized heterodimer unit / silyl compounds 19b to 19f are as follows.

[0044] B ₁ B ₂ 19b thymine (T) adenine which the amino group is protected with benzoyl (A ^BZ) 19c 〃 cytosine amino group is protected by a benzoyl (C ^BZ) 19d 〃 amino group is protected with an isobutyryl guanine G ^iBu 19e benzoylated adenine (A ^BZ) thymine (T) 19f benzoyl cytosine (C ^BZ) 〃 compound 19b yield ^{84% mp96-99 ℃ 1 H-NMR} (CDCl 3) δ: 1.07 (9H , S),
1.90 (3H, s), 2.61 (1H, m), 3.0
4 (2H, m), 3.46 (3H, m), 3.67 (2
H, m), 3.76 (6H, s), 3.87 (1H,
m), 4.45 (1H, m), 5.01 (1H, d, J
= 11), 5.25 (2H, m), 5.39 (1H,
m), 6.61 (1H, t, J = 5), 6.79 (4
H, AB, J = 7), 6.98 (1H, s), 7.17
-7.72 (22H, m), 8.04 (2H, d, J =
7), 8.25 (1H, s), 8.84 (1H, s),
9.18 (1H, s), 9.52 (1H, br) Compound 19c Yield 92% mp116-118 ° C ¹ H-NMR (CDCl ₃ ) δ: 1.06 (9H, s),
1.85 (3H, s), 2.29 (1H, m), 2.7
9 (1H, m), 3.16 (1H, m), 3.44 (3
H, m), 3.68 (2H, d, J = 6), 3.77.
(3H, s), 3.78 (3H, s), 3.81 (1
H, m), 4.33 (1H, br), 5.06 (1H,
d, J = 10), 5.20 (1H, d, J = 10),
5.31 (2H, m), 6.32 (1H, t, J =
7), 6.85 (4H, m), 7.16-7.72 (2
5H, m), 7.90 (2H, d, J = 7), 8.10
(1H, d, J = 7), 9.00 (1H, br) Compound 19d Yield 95% mp124-126 ° C ¹ H-NMR (acetone-d ₆ ) δ: 1.06 (9
H, s), 1.21 (6H, d, J = 6), 1.83
(3H, s) 2.55 (1H, m) 2.85 (1H,
m), 2.98 (1H, m), 3.25 (1H, m),
3.31 (1H, m), 3.43 (1H, m), 3.5
0 (1H, m), 3.78 (6H, s), 3.78 (2
H, m), 3.96 (1H, m), 4.30 (1H,
m), 5.26 (3H, m), 6.27 (1H, dd,
J = 5, 5), 6.46 (1H, m), 6.82 (4
H, m), 7.17-7.33 (7H, m), 7.45
(8H, m), 7.73 (4H, m), 7.95 (1
H, s), 10.17 (1H, br), 10.68 (1
H, br) 12.10 (1H, br) compound 19e Yield 50% mp 102-105 ° C ¹ H-NMR (CDCl ₃ ) δ: 1.09 (9H, s),
1.30 (3H, s), 2.34 (2H, m), 3.0
6 (1H, m), 3.41 (2H, m), 3.42 (1
H, m), 3.72 (2H, m), 3.75 (6H,
s), 3.87 (1H, m), 3.94 (1H, s),
5.24 (1H, m), 5.52 (1H, br), 5.
73 (2H, dd, J = 11, 15), 6.32 (1
H, t, J = 8), 6.80 (4H, d, J = 9),
7.20-7.69 (23H, m), 8.03 (2H,
d, J = 7), 8.10 (1H, s), 8.84 (1
H, s), 10.11 (1H, br), 10.42 (1
H, s) Compound 19f yield 70% mp 115-118 ° C ¹ H-NMR (CDCl ₃ ) δ: 1.07 (9H, s),
1.27 (3H, s), 2.34 (2H, m), 3.0
7 (1H, m), 3.44 (1H, m), 3.46 (2
H, m), 3.69 (2H, d, J = 5), 3.77.
(6H, s), 4.02 (1H, m), 4.21 (1
H, s), 5.19 (1H, d, J = 10), 5.33.
(1H, d, J = 5), 5.42 (1H, d, J = 1
0), 5.65 (1H, br), 6.42 (1H, t,
J = 7), 6.82 (4H, d, J = 9), 7.22-
7.67 (25H, m), 7.90 (2H, d, J =
7), 8.76 (1H, s), 9.55 (1H, br) 4-b: heterodimer unit / desilylated product 20a
Synthesis of

Embedded image IR type silyl compound 19a (180 mg, 0.17 mmo
1) Tetrahydrofuran (THF) solution (1 ml)
1.0M tetrabutylammonium fluoride / TH
F (25 μl, 1.3 eq.) Was added dropwise, and the mixture was stirred at room temperature for 1 hour. The solvent was distilled off, and the product was purified by silica gel column chromatography (CHCl ₃ : MeOH = 20: 1) to give a white powder of IR desilylated product 20a (130 mg, 9).
5%).

Mp95-98 ° C., ¹ H-NMR (CDCl ₃ ) δ: 1.34 (3H,
s), 1.72 (1H, br), 1.89 (3H,
s), 2.43 (2H, m), 3.32 (1H, m),
3.39 (1H, m), 3.44 (2H, m), 3.6
2 (2H, m), 3.75 (1H, m), 3.78 (6
H, s), 4.15 (1H, m), 5.18 (1H,
d, J = 10), 5.23 (1H, d, J = 10),
5.35 (1H, m), 6.04 (1H, t, J =
6), 6.38 (1H, t, J = 6), 6.82 (4
H, d, J = 8), 7.18 (1H, s), 7.22-
7.30 (8H, m), 7.36 (2H, d, J =
7), 9.7 (1H, br), 10.3 (1H, br) [α] _D ²¹ + 7 ° (c = 1.0, acetone) heterodimer unit / desilylated form 20b to 20f
The following IR desilylated products 20b to 20f were synthesized by the method and conditions described above.

Compound 20b Yield 58% mp 123-126 ° C. ¹ H-NMR (CDCl ₃ ) δ: 1.85 (3H, s),
2.00 (1H, br), 2.63 (1H, m), 2.
97 (1H, m), 3.27 (1H, m), 3.43
(3H, m), 3.59 (1H, m), 3.66 (1
H, m), 3.75 (6H, s), 3.78 (1H,
m), 4.33 (1H, s), 5.19 (1H, d, J
= 10), 5.23 (1H, d, J = 10), 5.42
(1H, m), 5.76 (1H, br), 6.50 (1
H, t, J = 7), 6.77 (4H, d, J = 7),
7.11 (1H, s), 7.15-7.37 (9H,
m), 7.49 (2H, m), 7.57 (1H, m),
8.04 (2H, d, J = 8), 8.23 (1H,
s), 8.75 (1H, s), 9.47 (1H, br) [α] _D ²⁷ -2 ° (c = 0.53, acetone) Compound 20c Yield 88% mp 126-128 ° C ¹ H- NMR (CDCl ₃ ) δ: 1.87 (3H, s),
2.27 (1H, m), 2.79 (1H, m), 3.4
0 (4H, m), 3.66 (2H, m), 3.78 (6
H, s), 3.90 (1H, m), 4.30 (1H,
s), 5.22 (2H, m), 5.33 (1H, m),
6.04 (1H, br), 6.28 (1H, t, J =
6), 6.82 (4H, m), 7.16-7.64 (1
5H, m), 7.92 (2H, m), 8.09 (1H,
d, J = 8), 9.56 (1H, br) compound 20d Yield 65% mp128-131 ° C ¹ H-NMR (acetone-d ₆ ) δ: 1.22 (6
H, d, J = 6), 1.86 (3H, s), 2.59
(1H, m), 2.83 (1H, m), 2.96 (1
H, m), 3.21 (1H, m), 3.33 (2H,
m), 3.48 (1H, m), 3.57 (1H, m),
3.65 (1H, m), 3.78 (6H, s), 4.0
0 (1H, s), 4.29 (1H, m), 5.26 (3
H, m), 6.27 (1H, dd, J = 5, 5), 6.
47 (1H, m), 6.82 (4H, d, J = 7),
7.18-7.32 (7H, m), 7.44 (2H,
d, J = 7), 7.54 (1H, s), 7.95 (1
H, s), 10.19 (1H, br), 10.72 (1
H, br), 12.10 (1H, br) [α] _D ²⁷ + 9 ° (c = 0.98, acetone) Compound 20e Yield 54% mp 100-102 ° C. ¹ H-NMR (CDCl ₃ ) δ: 1 .31 (3H, s),
2.36 (2H, m), 2.76 (1H, br), 3.
22 (1H, m), 3.34 (1H, m), 3.42
(2H, s), 3.65 (2H, m), 3.76 (6
H, s), 3.83 (1H, m), 4.04 (1H,
s), 5.30 (1H, m), 5.80 (2H, m),
5.99 (1H, br), 6.34 (1H, t, J =
9), 6.81 (4H, d, J = 9), 7.20-7.
56 (14H, m), 8.01 (2H, d, J = 7),
8.24 (1H, s), 8.76 (1H, s), 9.7
9 (1H, br) compound 20f Yield 32% mp143-145 ° C. ¹ H-NMR (CDCl ₃ ) δ: 1.28 (3H, s),
2.38 (2H, m), 3.27 (1H, m), 3.4
2 (2H, m), 3.63 (1H, m), 3.69 (2
H, m), 3.76 (6H, s), 3.88 (1H,
m), 4.13 (1H, s), 5.33 (2H, m),
5.42 (1H, s), 6.19 (1H, br), 6.
35 (1H, t, J = 10), 6.81 (4H, d, J
= 9), 7.17-7.78 (18H, m), 9.55
(1H, br) 4-c: heterodimer unit / amidite body 21a
Synthesis of

[Chemical 17] IR type desilylated product 20a (400 mg, 0.50 mm
ol) and diisopropylammonium tetrazolide (6
5 mg, 0.75 eq. ) Azeotroping the mixture of
Further, benzene was azeotropically distilled for dehydration. C of this mixture
2-cyanoethyl-N, N, N ′, N′-tetraisopropylphosphorodiamidite (210 μl, 1.3e) was added to a H ₃ CN / THF = 3/1 (v / v) solution (4 ml).
q. ) Was added dropwise at room temperature and stirred for 1 hour. The solvent was evaporated, diluted with CH ₂ Cl ₂ and washed with saturated aqueous sodium hydrogen carbonate, water and saturated brine. After that, it was dried over anhydrous magnesium sulfate, the solvent was distilled off, and silica gel column chromatography (AcO) was performed.
It was purified by Et: Et ₃ N = 100: 1). White powder IR type amidite body 21a (304 mg, 0.30 m
mol, 61%) was obtained.

Example 5: Synthesis of IR heterodimer
(II)

Embedded image 5-a: p-nitrophenyl 5'-O- (4,4'-dime
Toxitrityl) -N-benzoyl-2'-deoxyoxy
Synthesis of Tidine-3'-carbonate Bis (4-nitrophenyl) carbonate (380 m
g, 1.3 mmol) in anhydrous DMF solution (2 ml),
Triethylamine (0.10 ml, 0.69 mmol) and 5'-O- (4,4'-dimethoxytrityl) -N-benzoyl-2'-deoxycytidine (400 mg, 0.
63 mmol) (compound 18c) in anhydrous DMF (2
(ml) was added dropwise at room temperature, and the mixture was stirred for 5 hours. After the solvent was distilled off, the residue was diluted with methylene chloride and washed twice with a 0.01N aqueous sodium hydroxide solution, twice with water and once with a saturated saline solution. The organic phase was dried over anhydrous sodium sulfate and the solvent was distilled off, followed by silica gel column chromatography (C
It was purified with HCl ₃ : MeOH = 99: 1 to 97: 3). After distilling off the solvent, the residue was reprecipitated from toluene / n-hexane (1: 3) to obtain white powder of compound 28 (43).
0 mg, 0.54 mmol, 86%) was obtained.

Mp196-199 ° C. IRν (KBr): 1768,1663,1484,1
252,408 cm ^-1 [α] _D ²⁶ + 44 ° (c = 1.0, acetone) ¹ H-NMR (CDCl ₃ ) δ: 2.42 (1H, m),
3.04 (1H, m), 3.55 (2H, m), 3.7
9 (6H, s), 4.45 (1H, m), 5.40 (1
H, m), 6.36 (1H, t, J = 7), 6.86
(4H, d, J = 8), 7.20-7.42 (12H,
m), 7.53 (2H, m), 7.63 (1H, t, J
= 7), 7.90 (2H, d, J = 8), 8.15 (1
H, d, J = 7), 8.31 (2H, d, J = 9),
8.70 (1H, br) 5-b: Heterodimer unit / silyl compound 19g
Under formation of nitrogen stream, the compound 28 (50 mg, 0.063
mol) in DMF solution (0.5 ml), compound 7c
(42 mg, 0.076 mmol) in DMF (0.
5 ml) was added dropwise at room temperature. Subsequently, triethylamine (11 μl, 0.076 mmol) was added dropwise, and the mixture was stirred at room temperature for 1 hour.
After stirring for 9 hours and distilling off DMF, the residue was diluted with methylene chloride, and saturated aqueous sodium hydrogen carbonate solution was added once, water was added three times, and saturated brine was added once.
Washed twice. The organic phase is dried over anhydrous magnesium sulfate,
After distilling off the solvent, the residue was purified by silica gel column chromatography (CHCl ₃ : MeOH = 97: 3) to obtain 19 g (58 mg, 0.04 mg) of IR silyl compound as a white powder.
9 mmol, 77%) was obtained. mp113-115 ° C. IRν (KBr): 3247, 3070, 2932, 1
697, 1656, 1559, 1487, 1254, 1
112,703 cm ^-1 [α] _D ²⁷ + 44 ° (c = 1.0, acetone) ¹ H-NMR (CDCl ₃ ) δ: 1.07 (9H, s),
2.25 (1H, m), 2.75 (1H, m), 3.1
5 (1H, m), 3.41-3.50 (3H, m),
3.71 (2H, d, J = 10), 3.77 (3H,
s), 3.78 (3H, s), 3.96 (1H, m),
4.32 (1H, m), 5.19 (1H, d, J = 1
0), 5.33 (1H, m), 5.41 (1H, d, J
= 10), 5.52 (1H, br), 6.34 (1
H, t, J = 7), 6.82-8.04 (37H, m) MS (FAB) 1216 (M ⁺ +1) 5-c: 20 g of heterodimer unit / desilylated product
19 g of the above compound (140 mg, 0.12) under a synthetic nitrogen stream.
1.0 M tetrabutylammonium fluoride / THF solution (0.1 ml) in anhydrous THF solution (3 ml).
3 ml, 0.13 mmol) was added dropwise at room temperature. After stirring at room temperature for 7.5 hours, the solvent was evaporated, and the residue was subjected to silica gel column chromatography (CHCl ₃ : MeOH = 1).
0: 1) to obtain 20 g (98 mg, 0.10 mmol, 84%) of IR desilylated product as a white powder.

Mp136-140 ° C. IRν (KBr): 3314,1700,1654,1
559, 1486, 1314, 1253, 1072, 7
91,705 cm ^-1 [α] _D ²⁷ + 49 ° (c = 1.0, acetone) ¹ H-NMR (DMSO-d6) δ: 2.30 (1H,
m), 2.54 (1H, m), 3.00 (1H, m),
3.17-3.46 (5H, m), 3.62 (1H,
m), 3.73 (3H, s), 3.74 (3H, s),
4.15 (1H, m), 4.78 (1H, t, J =
6), 5.16 (1H, br), 5.27 (1H, d,
J = 10), 5.35 (1H, d, J = 10), 6.1
4 (1H, t, J = 6), 6.88 (4H, d, J =
7), 7.12-7.65 (17H, m), 7.95.
(2H, d, J = 8), 8.01 (2H, d, J =
8), 8.15 (1H, m), 11.22 (2H, b
r) MS (FAB) 978 (M ⁺⁺ 1) 5-d: Synthesis of heterodimer unit / amidite body

[Chemical 19] 20 g of IR type desilylated product (0.12 g, 0.12 mm
ol) and diisopropylammonium tetrazolide (2
1 mg, 0.12 mmol) of CH ₃ CN / THF (1 /
1) To the solution (4 ml), 2-cyanoethyl-N, N,
N ′, N′-Tetraisopropylphosphorodiamidite (90 μl, 0.14 mmol) was added at room temperature. After stirring at room temperature for 4 hours, the solvent was evaporated and the residue was diluted with ethyl acetate. This was washed once with saturated aqueous sodium hydrogen carbonate, once with water, and once with saturated brine, and dried over anhydrous magnesium sulfate. After evaporating the solvent, the residue was subjected to silica gel column chromatography (ethyl acetate: triethylamine = 9).
9: 1). This was re-precipitated from methylene chloride / n-hexane to give a white powder of IR type amidite body 2
1b (0.13 g, 0.11 mmol, 88%) was obtained.

Mp75-78 ° C MS (FAB) 1179 (M ⁺ +1) Example 6: Synthesis of IR hetero-oligomer 6-a: IR oligomer: 5'-GCGTTTT-Tt-GCT-3 '
Synthesis of (T4X-IR) Deprotection of the dimethoxytrityl group of 5'-dimethoxytritylthymidine (0.2 μmol) having the 3'-hydroxyl group bound to the support by trichloroacetic acid, and 5'-dimethoxy group Trityl-2'-deoxycytidine β-cyanoethylphosphoamidite derivative was condensed with tetrazole, and the unreacted 5'-hydroxyl group was acetylated with acetic anhydride, 4-dimethylaminopyridine and 2,4,6-collidine, and then iodine. And 2,4,6-collidine,
Oxidize phosphorus with water.

Similarly, deprotection, condensation, acetylation and oxidation are repeated. (The amidite derivative of the heterodimer could be used similarly to other amidite derivatives.) Obtained by condensing and oxidizing the final 5'-dimethoxytrityl-2'-deoxyguanosine β-cyanoethylphosphoamidite derivative. 12-mer oligomer (The process up to here is a DNA synthesizer manufactured by Pharmacia.
It was performed by Gene Assembler Plus. ) Is cleaved from the support with 1 ml of concentrated aqueous ammonia, the cyanoethyl group is removed from phosphorus, and the protective groups attached to adenine, guanine, and cytosine are removed.

The obtained 5'-dimethoxytrityl oligonucleotide was applied to a reverse phase column (Millipore, O).
5 ml of trifluoroacetic acid on Ligo-Pak ^™ SP)
The 5'-dimethoxytrityl group is removed by and the purification is continued. If necessary, the resulting oligonucleotide is purified by high performance liquid chromatography to obtain the desired 5'-G
CGTTTT- Tt- GCT-3 '(0.11 μmol) was obtained.

In the present specification, natural nucleosides are represented by capital letters such as T, C, A and G, and analogs of the present invention are represented by lower case letters such as t, c, a and g, for convenience. . The underline indicates the dimer unit.

6-b: Combination of other IR type oligomers
Adult · 5'-GCG- Tt -TTTTGCT-3 ' synthesized Similarly the (XT4-IR), the desired 5'-GCG- Tt -TTTTGCT-3' (0.06μmo
l) was obtained.

Synthesis of 5'-GCGTT- Tt -TTGCT-3 '(T2XT2-IR) Similarly, the desired 5'-GCGTT- Tt -TTGCT-3' (0.07 μm
ol) was obtained.

Synthesis of 5'-GCGTT- Tt - Tt- GCT-3 '(T2X2-IR) Similarly, the desired 5'-GCGTT- Tt - Tt- GCT-3' (0.15 μm
ol) was obtained.

Synthesis of 5'-GCG- Tt - Tt - Tt- GCT-3 '(X3-IR) Similarly, the desired 5'-GCG- Tt - Tt - Tt- GCT-3' (0.19 μm
ol) was obtained.

Synthesis of 5'-CTTTTTTTTT- Tt- G-3 '(T9X-IR) Similarly, the desired 5'-CTTTTTTTTT- Tt- G-3' (0.18 μm
ol) was obtained.

Synthesis of 5'-CTTTT- Tt -TTTTTG-3 '(T4XT5-IR) Similarly, the desired 5'-CTTTT- Tt -TTTTTG-3' (0.16 μm
ol) was obtained.

[0060] · 5'-C- Tt - Tt - Tt - Tt - Tt -TG-3 ' synthesis In the same manner of (X5-IR), 5'- C- Tt of purpose - Tt - Tt - Tt - Tt -TG-3 '(0.20
μmol) was obtained.

6-c: using IR type Cc heterodimer
Synthesis of IL-6 receptor antisense oligomer • Synthesis of 5′-GCAGCCTCCTTCCCATG-Cc-A-3 ′ (IL-6RAS1-IR) Using the amidite body 21b obtained in Example 5,
5'-GCAGCCTCCTTCCCATG-Cc-A-
3 '(11.3 nmol) was obtained.

5'-GCAG-Cc-TCCTTCCCATG-Cc-A-3 '(IL-
6RAS2-IR) Synthesis of 5'-GCAG-Cc-TCCTTCCCATG-Cc-A-3 '
(8.2 nmol) was obtained.

5'-CT-Cc-TTCCCATGCCAGC-Cc-A-3 '(IL-
6RAS3-IR) Synthesis of 5'-CT-Cc-TTCCCATGCCAGC-Cc-A-3 '(1
2 nmol) was obtained.

5'-AGCCTCCTTCCCATG-Cc-AGC-3 '(IL-6RA
S4-IR) Synthesis of 5'-AGCCTCCTTCCCATG-Cc-AGC-3 '(2
4.8 nmol) was obtained.

Example 7: II-S type heterodimer unit
Synthesis

Embedded image 7-a: Synthesis of heterodimer unit silyl compound 23a Monomer unit (S compound) 10a (2.02 g, obtained by the same method as in Examples 1 and 2 using (R) -glycidol (Compound 9) as a starting material 3.56 mmo
1, 1.1 eq. ) To a methylene chloride solution (30 ml) of TFA (2.8 ml) was added dropwise under ice cooling and the mixture was stirred at room temperature for 120 minutes, and then the solvent was distilled off to obtain the de-Boc body 12a which was used for condensation without purification. . Known 3'-O-acetylthymidine 22a (0.91 g, 3.20 mmol, 1.
0 eq) ⁵⁾ was dehydrated by azeotropic distillation with anhydrous pyridine three times to give an anhydrous pyridine solution (15 ml). N, N'-Carbonyldiimidazole (1.04 g, 2.0 eq.) Was added, and the mixture was stirred at room temperature for 150 minutes under a nitrogen stream. Water (0.7
0 ml, 12 eq. ) Was added and the mixture was stirred for 30 minutes to decompose excess N, N′-carbonyldiimidazole, and then the solvent was distilled off. It was dehydrated by azeotropic distillation with anhydrous pyridine three times to obtain an anhydrous pyridine solution (15 ml). 12 to this
a in anhydrous pyridine (15 ml) and 4-dimethylaminopyridine (4-DMAP) (0.39 g, 0.1
8 mmol, 0.5 eq. ) Was added, and the mixture was stirred under a nitrogen stream for 2 days at room temperature. After distilling off the solvent, the mixture was diluted with ethyl acetate, washed 4 times with water and 2 times with saturated saline, and the organic layer was dried over anhydrous sodium sulfate, and then the solvent was distilled off. After purification by silica gel column chromatography (ethyl acetate: n-hexane = 7: 1), reprecipitation was performed from methylene chloride / n-hexane, and white powder of II-S type silyl compound 23a (2.
10 g, 2.70 mmol, 84%) was obtained.

Mp 96-98 ° C. ¹ H-NMR (CDCl ₃ ) δ: 1.07 (9 H, s),
1.87 (3H, s), 1.89 (3H, s), 2.1
2 (3H, s), 2.47 (1H, m), 2.60 (1
H, m), 3.08 (1H, m), 3.46 (1H,
m), 3.67 (2H, d, J = 6), 3.74 (1
H, m), 4.24 (1H, dd, J = 3, 11),
4.28 (1H, m), 4.33 (1H, dd, J =
3, 11), 4.97 (1H, d, J = 9), 5.12.
(1H, d, J = 9), 5.27 (1H, d, J =
5), 6.02 (1H, m), 6.20 (1H, t, J
= 7), 6.89 (1H, s), 7.36 (1H,
s), 7.38-7.66 (10H, m), 9.29.
(1H, br), 9.56 (1H, br) [α] _D ²⁵ -18 ° (c = 1.0, acetone) MS (FAB) 778.4 (M ⁺ +1) 7-b: heterodimer unit .Of desilylated product 24a
Synthetic II-S type silyl compound 23a (716 mg, 0.92 mmo
l) as a solution of anhydrous THF (14 ml), and 1.0 M tetrabutylammonium fluoride / THF (0.95
ml, 1.0 eq. ) Was added and stirred at room temperature for 20 minutes,
The solvent was distilled off. Reverse-phase medium pressure column (30% CH ₃ CN i
n H ₂ O) to obtain a white powder of II-S type desilylated product 24a (350 mg, 0.65 mmol, 71%).

Mp163-166 ° C. ¹ H-NMR (C ₅ D ₅ N) δ: 1.89 (3 H, s),
1.97 (3H, s), 1.99 (3H, s), 2.4
9 (2H, m), 3.69 (1H, m), 3.82 (1
H, m), 4.01 (1H, dd, J = 6, 12),
4.07 (1H, dd, J = 5, 12), 4.30 (1
H, m), 4.44 (1H, m), 4.60 (1H, d
d, J = 6, 12), 4.69 (1H, dd, J = 4)
12), 5.49 (1H, m), 5.49 (1H, d,
J = 10), 5.58 (1H, d, J = 10), 6.7.
0 (1H, t, J = 6), 7.39 (1H, s), 7.
62 (1H, s), 8.42 (1H, br), 13.2
3 (2H, s) [α] _D ²¹ -11 ° (c = 1.0, acetone) Anal. Calcd for C ₂₂ H ₂₉ N ₅ O ₁₁・ H ₂
O: C, 47.40; H, 5.60; N, 12.56.
Found: C, 47.47; H, 5.48; N, 1
2.59 MS (FAB) 540.5 (M ⁺ +1) 7-c: of the heterodimer unit trityl body 25a
Synthetic II-S type desilylated product 24a (0.97 g, 1.80 mm
ol) was azeotroped with anhydrous pyridine three times to give anhydrous pyridine (1
0 ml) solution. DMTrCl (1.00 g,
1.5 eq. ) Was added and the mixture was stirred at room temperature for 90 minutes. After the solvent was distilled off, the residue was diluted with chloroform, washed twice with water and twice with saturated saline, and the organic layer was dried over anhydrous sodium sulfate. After distilling off the solvent, re-precipitation was performed from methylene chloride / n-hexane to obtain a white powder of II-S type trityl compound 25a (1.27).
g, 1.51 mmol, 84%) was obtained. ⁶⁾ mp125-129 ° C ¹ H-NMR (CDCl ₃ ) δ: 1.89 (6H, s),
2.12 (3H, s), 2.43 (1H, m), 2.5
8 (1H, m), 3.09 (1H, m), 3.18 (2
H, m), 3.41 (1H, m), 3.79 (1H,
m), 3.79 (6H, s), 4.23 (1H, dd,
J = 4, 12), 4.26 (1H, m), 4.33 (1
H, dd, J = 2,12), 5.08 (1H, d, J =
10), 5.17 (1H, d, J = 10), 5.27.
(1H, d, J = 6), 5.88 (1H, m), 6.2
1 (1H, t, J = 6), 6.83 (4H, d, J =
8), 7.03 (1H, s), 7.25-7.41 (1
0H, m), 9.13 (1H, s), 9.41 (1H,
s) [α] _D ²¹ -17 ° (c = 1.0, acetone) Anal. Calcd for C ₄₃ H ₄₇ N ₅ O ₁₃ : C,
61.35; H, 5.63; N, 8.32. Foun
d: C, 61.29; H, 5.69; N, 7.94MS
(FAB) 842.2 (M ⁺ +1) 7-d: heterodimer unit / deacetylated product 26a
Synthesis of II-S type trityl compound 25a (392 mg, 0.466 m)
mol) as a solution of EtOH (1.2 ml) and anhydrous pyridine (0.6 ml), and NaOH in 5 at room temperature
0% EtOH (equal volume of EtO in 2N NaOH aqueous solution)
H was added. ) 1.2 ml was added and stirred for 10 minutes at room temperature. After neutralizing with 2% hydrochloric acid, dilute with methylene chloride,
It was washed twice with water and twice with saturated saline, and the organic layer was dried over anhydrous sodium sulfate. After distilling off the solvent, methylene chloride /
Re-precipitation from n-hexane was performed to obtain a white powder of II-S type deacetylated product 26a (372 mg, 0.465 mmol, 10).
0%).

Mp135-139 ° C¹ H-NMR (CDCl₃) Δ: 1.88 (6H, s),
2.31 (1H, m), 2.51 (1H, m), 3.0
6 (1H, m), 3.16 (2H, m), 3.35 (1
H, m), 3.79 (6H, s), 3.85 (1H,
m), 4.10 (1H, dd, J = 4, 12), 4.1
4 (1H, m), 4.28 (1H, dd, J = 4, 1
1), 4.42 (1H, m), 5.08 (1H, d, J
= 10), 5.26 (1H, d, J = 10), 5.97.
(1H, br), 6.23 (1H, t, J = 6), 6.
82 (4H, AB, J = 9), 7.05 (1H, s),
7.25-7.30 (8H, m), 7.39 (2H,
d, J = 7), 9.8 (1H, br), 10.3 (1
H, br) [α]_D ^{twenty one}-11 ° (c = 1.0, acetone) MS (FAB) 800.2 (M⁺+1)7-e: Heterodimer unit / amidite body 27a
Synthesis of II-S type deacetylated product 26a (200 mg, 0.250
mmol) to CH₃2.5 ml CN, 2.5 m THF
l-cyanoethyl-N, N, N ', N'
-Tetraisopropyl phosphorodiamidite (90m
1, 1.1 eq. ) And diisopropylammonium
Tetrazolid (21 mg, 0.5 eq.) Was added, and room temperature
And stirred for 4 hours. After concentration, dilute with ethyl acetate, water,
Wash with 5% sodium bicarbonate solution twice, water, and saturated saline solution twice, in this order.
Was. The organic layer was dried over anhydrous sodium sulfate and the solvent was distilled off.
did. Silica gel flash column (3% Et₃N / A
After purification with cOEt), from methylene chloride / n-hexane
The white powder was re-precipitated and was a II-S type amidite 27a (1
52 mg, 0.155 mmol, 62%) was obtained.⁷⁾  mp95-98 ° C MS (FAB) 1000.4 (M⁺+1)Example 8: Synthesis of II-S type hetero-oligomer 8-a: Synthesis of II-S type oligomer II-S type amidite body 27a (300 mg, 0.30 m
(mol) in acetonitrile solution (2.5 ml)
Used with a natural nucleoside amidite on the market, D
II-S type oligomers are individually produced by NA synthesizer.
Was synthesized on a 0.2 μmol scale (5′-DMT
r: ON). Ammonia treatment at 70 ℃ for 3 hours, cut out
The liquid is a simple reverse-phase column (Millipore, Olig
o-Pak^TMSP) and further by reverse phase HPLC.
Refined. (Column: TOSOH, TSKgelOD
S-120T (3.2 mm ID x 1.5 cm (gar
De) +7.8 mmI. D.x30 cm), eluent: A;
0.1 M TEAA buffer (pH 7.0), B; CH₃
CN, A / B (v / v) 90/10 to 80/20
Linear gradient (30 min), flow rate; 2.0 ml
/ Min, temperature: 21-23 ° C., detection: UV (254n
m)8-b: Synthesis of II-S type oligomer II-S type oligomer: 5'-GCGTTTT-tT-GCT-3 '(T4X-II
S) Synthesis The following oligomer was synthesized by the above method, purified and isolated.
Objective 5'-GCGTTTT-tT-GCT-3 '(0.02 μmol) was obtained.

Synthesis of 5'-GCG- tT -TTTTGCT-3 '(XT4-IIS) In the same manner, the desired 5'-GCG- tT -TTTTGCT-3' (0.03 μmo
l) was obtained.

Synthesis of 5'-GCGTT- tT- TTGCT-3 '(T2XT2-IIS) In the same manner, the desired 5'-GCGTT- tT- TTGCT-3' (0.07 μmo
l) was obtained.

Synthesis of 5'-GCGTT- tT - tT- GCT-3 '(T2X2-IIS) Similarly, the desired 5'-GCGTT- tT - tT- GCT-3' (0.01 μmo
l) was obtained.

Synthesis of 5'-GCG- tT - tT - tT- GCT-3 '(X3-IIS) Similarly, the desired 5'-GCG- tT - tT - tT- GCT-3' (0.02 μm
ol) was obtained.

Example 9: II-S type heterodimer unit
Measurement of the molar extinction coefficient of preparative epsilon II-S type deacetylated body 26a (150 mg, 0.188
Trifluoroacetic acid (0.1 m1.7 eq.) was added to a methylene chloride (5 ml) solution of (mmol) and stirred at room temperature for 2 minutes, and then concentrated. Water was added, the mixture was filtered, and the filtrate was concentrated. column chromatography _{(CH 3 CN: H 2 O} =
2: 8) and then recrystallized from CH ₃ CN to give a white powder diol (46 mg, 0.092 mmol, 49).
%) Was obtained. Then, ultraviolet absorption at 260 nm at 90 ° C. was measured in a 10 mM sodium phosphate buffer (pH 7.2) to calculate the molar extinction coefficient ε of the II-S type heterodimer unit. The value of the literature ⁸⁾ was used as the molar extinction coefficient of the natural nucleoside.

Ε: A, 15000; G, 12500;
C, 7500; T, 8500, tT, 14900 Example 10: Synthesis of IS type Tt heterodimer

[Chemical 21] 10-a: combination of heterodimer unit / silyl compound 29
Forming 5'-O-(4,4'-dimethoxytrityl) - thymidine (910 mg, 1.60 mmol) (Compound 18c) in anhydrous pyridine solution (6 ml), 1,1'-carbonyldiimidazole (487 mg, 3. 20 mmol) was added, and the mixture was stirred at room temperature for 150 minutes under a nitrogen stream. Water (0.2
2 ml, 12 eq. ) Is added, and the mixture is stirred for 30 minutes, and excess 1,
After decomposing 1'-carbonyldiimidazole, the solvent was distilled off. It was dehydrated by azeotropic distillation with anhydrous pyridine three times to obtain an anhydrous pyridine solution (6 ml). Compound 12a (3
67 mg, 1.65 mmol) of anhydrous pyridine (6
ml) and 4-dimethylaminopyridine (91 mg,
0.5 eq. ) Was added and the mixture was stirred under a nitrogen stream for 4 days at room temperature. After distilling off the solvent, it was diluted with ethyl acetate and washed 4 times with water.
The organic phase was washed twice with saturated saline and dried over anhydrous sodium sulfate, and then the solvent was distilled off. It was purified by silica gel column chromatography (CHCl ₃ : MeOH = 20: 1). Reprecipitation from methylene chloride / n-hexane gave a white powder of the IS silyl compound 29 (900 mg, 0.87 m).
mol, 58%) was obtained.

Mp115-119 ° C. IRν (KBr): 3069, 2931, 1685, 1
509, 1253, 1112 cm ^-1 [α] _D ²⁸ -13 ° (c = 0.64, acetone) ¹ H-NMR (CDCl ₃ ) δ: 0.99 (9H, s),
1.27 (3H, S) 1.79 (3H, s), 2.32
(1H, m), 2.46 (1H, m), 2.92 (1
H, m), 3.32 (1H, m), 3.38 (2H,
m), 3.60 (2H, m), 3.71 (6H, m),
3.82 (1H, s), 4.03 (1H, m), 4.9
6 (1H, d, J = 10), 5.15 (1H, d, J =
11), 5.28 (2H, m), 6.36 (1H,
m), 6.76 (4H, d, J = 8), 6.91 (1
H, s), 7.14-7.59 (20H, m), 8.5.
4 (1H, br), 9.29 (1H, br) Anal. Calcd for C ₅₇ H ₆₃ N ₅ O ₁₂ Si
_{· 1 / 3H 2 O: C} , 65.56; H, 6.15; N,
6.71. Found: C, 65.60; H, 6.4.
1; N, 6.59 MS (FAB) 1038 (M ⁺ +1) 10-b: heterodimer unit / desilylation product 30
Synthesis 1.0M tetrabutylammonium fluoride / THF solution (0.68 ml, 0.10 mL) in anhydrous THF solution (10 ml) of compound 29 (700 mg, 0.67 mmol).
68 mmol) was added and the mixture was stirred at room temperature for 20 minutes, and then the solvent was evaporated. The residue was purified by silica gel column chromatography (CHCl ₃ : MeOH = 20: 1). Reprecipitation from methylene chloride / n-hexane gave a white powder I
-S-type desilylated product 30 (487 mg, 0.61 mmo
1, 90%).

Mp115-120 ° C. IRν (KBr): 3479, 3064, 2930, 2
837, 1695, 1510, 1465, 1253, 1
^{177,1103,668,412cm -1 1 H-NMR (CDCl} 3) δ: 1.36 (3H, s),
1.92 (3H, s), 2.41 (1H, m), 2.5
1 (1H, m), 2.70 (1H, m), 3.23 (1
H, m), 3.44 (2H, m), 3.64 (2H,
m), 3.79 (7H, s), 4.14 (1H, m),
5.12 (1H, d, J = 10), 5.17 (1H,
d, J = 11), 5.38 (1H, m), 5.54 (1
H, m), 6.41 (1H, m), 6.83 (4H,
d, J = 9), 7.13 (1H, s), 7.24-7.
61 (10H, m), 8.72 (1H, br) 9.18
(1H, br) Anal. Calcd for C ₄₁ H ₄₅ N ₅ O ₁₂ · H ₂
O: C, 60.21; H, 5.79; N, 8.56. F
found: C, 60.29; H, 5.80; N, 8.4
3 MS (FAB) 800 (M ⁺ +1) 10-c: Combination of heterodimer unit and amidite form
Success

[Chemical formula 22] An IS type amidite body 31 was obtained from the IS type desilylated body 30 in the same manner as in Example 5-b. (Yield 58%) mp 104-107 ° C. (CH ₂ Cl ₂ / n-hexane) ³¹ P-NMR (CDCl ₃ ) δ: 149.48 Anal. Calcd for C ₅₀ H ₆₂ N ₇ O ₁₃ P:
C, 60.05; H, 6.25; N, 9.80. Fou
nd: C, 59.48, H, 6.52, N, 9.65 MS (FAB) 1000 (M ⁺ +1) Experimental Example 1: Measurement of melting temperature (Tm) Various kinds synthesized in Examples 6 and 8 The hybridizing ability of the antisense strand was examined by measuring the Tm of an annealed oligomer strand (antisense strand) which is an antisense molecule and a sense strand.

A sample solution having a final concentration of 100 mM NaCl, 10 mM sodium phosphate buffer (pH 7.2), 4 μM antisense strand and 4 μM sense strand was bathed in boiling water and cooled to room temperature over 10 hours. A nitrogen stream was passed through the cell chamber of the ultraviolet spectrophotometer to prevent condensation, and the sample solution was slowly cooled to 0 ° C.
Hold at 0 ° C for 0 minutes. The temperature is increased by 0.2 ° C./min to reach 0.
Ultraviolet absorption at 260 nm was measured at 5 ° C. intervals, and the temperature at the inflection point of the sigmoid curve was taken as the Tm value. In order to prevent the concentration from changing due to evaporation due to temperature rise, a cell with a lid was used, and one drop of mineral oil was placed on the surface of the sample solution. The sequences of the antisense strand and the sense strand used for the measurement are shown below.

In the present specification, natural nucleosides are represented by capital letters such as T, C, A and G, and analogs of the present invention are represented by lower case letters such as t, c, a and g, for convenience. To do.

[0079]

[Chemical formula 23] The measurement results are shown in the following table.

[0080]

[Table 1] In the case of the IR hetero-oligomer, the influence of the mismatch of the heterodimer unit ( Tt ) on the analog (t) was measured, and this value is the same as that of the II-S heterodimer unit II-S ( tT ). Since the influence of the mismatch on the nucleoside (T) is shown, direct comparison is not possible, but it is shown as reference data. Experimental Example 2: Measurement of enzyme resistance (1) With respect to the following natural type and non-natural type oligonucleotides, the resistance to exonuclease that decomposes the oligonucleotide from the 3'side was examined.

A snake venom phosphodiesterase was added to the oligonucleotide solution, and the increase in ultraviolet absorption due to the decomposition of the oligomer was measured with time at 37 ° C. and 260 nm. (Sample solution final concentration: oligomer 5 μM, Tris
HCl (pH 8.6) 0.1M, NaCl 0.1
M, MgCl ₂ 14 mM, snake venom phosphodiesterase 0.006 U) The sequence of the oligonucleotide used for the measurement is shown below.

[0082]

[Chemical formula 24] The time course of the relative absorbance at 260 nm is shown in FIG.

Since the non-natural oligomer has a change in absorbance halfway as compared with the natural one (T11), it is considered that the enzymatic degradation stops halfway. From this, the heterodimer introduced into the oligonucleotide is resistant to enzymatic degradation, and the oligomer of the present invention satisfies the properties required for the antisense molecule.

Experimental Example 3: Measurement of enzyme resistance (2) The following oligonucleotides of natural type and non-natural type were examined for resistance to exonuclease that decomposes the oligonucleotide from the 3'side.

Snake venom phosphodiesterase was added to the oligonucleotide solution and incubated at 37 ° C. (final concentration of sample solution: oligomer (as described below), Tr
isHCl (pH 8.0) 0.1M, NaCl 0.1
M, MgCl ₂ 14 mM, snake venom phosphodiesterase 0.00135 U). The reaction was stopped in boiling water over time, 20% polyacrylamide (7M Urea) was electrophoresed, and then stained with StainsAll.

T11 (85.4 μM) 5'-CTTTTTTTTTTTG-3 'T9X-IR (84.3 μM) 5'-CTTTTTTTTT Tt G-3' T4XT5-IR (85.8 μM) 5'-CTTTT Tt TTTTTG-3 ' Shown in 2.

It is considered that the non-natural type oligomer is terminated at the portion of the introduced heterodimer by the enzymatic degradation, as compared with the natural type (T11). From this, the heterodimer introduced into the oligonucleotide is resistant to enzymatic degradation, and the oligomer of the present invention satisfies the properties required for the antisense molecule.

Experimental Example 4: Human soluble IL-6R (sIL
-6R) expression suppressing effect (1) CHO. Preparation of SR344 cells pBSF2R. 236 (Science, 241, 825-828 (198
8)) was cleaved with SphI to give 1205 bp of IL-6Rc.
The DNA fragment was inserted into mp18 (manufactured by Amersham). sIL-6R cDNA is 5'-ATATTCTAGAGAGCTTCT
In vitro mutagenesis
It was prepared using a system (manufactured by Amersham).
As a result, the stop codon was at position 345 in the amino acid sequence.

The dhfr-cDNA is the plasmid pECE (C
ell, 45, 721-735 (1986)) and inserted into the PvuII site,
The plasmid pECEdhfr was created. Hin of sIL-6R
The dIII-SalI fragment was inserted into the plasmid pECEdhfr,
Soluble IL-6R expression vector plasmid pECEdhfr34
4 was produced.

PECEdhfr344 was dhfr-CHO cell D
XB-11 (Pro. Natl. Acad. Sci. USA, 77, 4216-4
220 (1980)) by the calcium phosphate method and MT
It was amplified at X. Finally 200nMMTX resistant sIL
-6R-producing CHO cells (CHO.SR344) were prepared (J. Biochem., 108, 673-676 (1990)). Normal culturing of cells was performed in IMDM medium (manufactured by Gibco) containing 5% FCS (manufactured by Xavier Investments) and 200 nMMTX.

(2) CHO. SIL-of SR344 cells
IL-6R antisense oligomers for 6R production
Effect CHO. SR344 cells were trypsin-EDTA (Gi
bco) to remove from the culture dish, wash with culture solution, and further wash with serum-free medium non-serum (Nippon Zenyaku Kogyo),
The cells were suspended in serum-free medium non-serum containing 200 nMMTX. Add CHO. 2 μM IL-in 100 μl of SR344 cell suspension (5 × 10 ⁴ cells / ml)
Add 100 μl of 6R antisense oligomer, and add 37
The cells were cultured in an incubator at 5 ° C and 5% CO ₂ .

After culturing for 24 hours, the soluble I of the culture supernatant was
Sandwich E using a mouse anti-IL-6R monoclonal antibody (MT18) (JP-A-2-288898) and a rabbit anti-IL-6R polyclonal antibody as the amount of L-6R
It was measured by the LISA method.

As a control, the sense oligomer derivative 5'-GCTGGGCATGGGAAGGAGGCT- with respect to the antisense oligomer IL-6RAS4-IR was used.
It measured using 3 '(IL-6RSE1). The synthesis of the sense oligomer (IL-6RSE1) was performed in Example 6
Can be obtained by a similar method without using Cc heterodimer.

The inhibitory effect on human IL-6R expression using the IR type Cc heterodimer of the present invention was shown (FIG. 3).

(References) 1) JCSowden et al., J. Am. Chem. Soc., 64 , 1291 (194
2). 2) CHTann et al., J. Org. Chem., 50 , 3684 (1985). 3) H. Vorbruggen et al., Chem. Ber., 114 , 1234 (1981). 4) LBTownsend and RSTipson , Nucleic Acid Chemi
stry part 3,78. 5) AMMichelson et al., J. Chem. Soc., 951 (1953). 6) T. Neilson et al., Can. J. Chem., 49 , 493 (1971). 7) M.Mag et al., Nucleic Acids Res., 16 , 3527 (1988). 8) GAMarel et al., J. Mol. Biol., 213 , 833 (1990).

[Brief description of drawings]

1] Ultraviolet absorption (260 nm) when various oligonucleotides of the present invention were digested with exonuclease
Shows the change with time.

FIG. 2 is an electrophoretogram showing changes with time when the oligonucleotide of the present invention and a natural type oligonucleotide are decomposed with exonuclease.

FIG. 3 is a graph showing that the antisense oligonucleotides of the present invention (IL-6RAS1-4-IR) in Example 6 suppress the expression of soluble IL-6R.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C07D 473/34 321 C07H 19/073 19/173 21/00 // C07H 21/04 A

Claims (2)

[Claims] 1. A compound of the general formula: [Wherein B ₁ is the same or different and is a pyrimidine or purine nucleobase or a derivative thereof, and X and Y
Are independently oxygen or sulfur, R is hydrogen, an alkyl group, or an acyl group, W is hydrogen, an alkyl group, an acyl group, or, when X is oxygen, a nucleotide, an oligo, or a phosphate ester bond. It may be a nucleotide or a polynucleotide, and n is an integer of 1 to 50. However, when n is 2 or more, B ₁ is not limited to the same base. ] The nucleotide analog represented by this. 2. A general formula: [Wherein B ₁ and B ₂ are the same or different and each is a pyrimidine or purine nucleobase or a derivative thereof, and X and Y are independently oxygen or sulfur,
W is the same or different and is hydrogen, an alkyl group, an acyl group,
Alternatively, it may be a nucleotide, an oligonucleotide or a polynucleotide via a phosphate bond, m
Is an integer of 1 to 25. However, when m is 2 or more, B ₁ and B ₂ are not limited to the same base. ] The nucleotide analog represented by this.