JPH04154794A - Antisensoligodeoxyribonucleotide and hiv activity inhibitor containing the same nucleotide as active ingredient - Google Patents

Abstract

NEW MATERIAL:An oligodeoxyribonucleotide expressed by the formula [P(S) is S atom bonded through a double bond to P atom of internucleotide part; P(O) is O atom bonded through a double bond to P atom of internucleotide part]. USE:A HIV activity inhibitor useful as a treating agent of AIDS. PREPARATION:An alcohol expressed by the formula [R<1> is C(CX3)3, CH(CX3)2 (X is halogen), etc.] is reacted with a compound expressed by the formula R<2>OPCl2 (R<2> is 1-6C alkyl) to afford a new phosphite compound expressed by the formula (R<1>O)2P(OR<2>). Then, a nucleotide unit having a base suitable for target sequence is introduced into a solid phase carrier on which nucleotide is fixed to carry out condensation reaction. Then, capping of unreacted 5'-OH and solid phase carrier is carried out using the above-mentioned phosphite compound as a capping agent. The reaction is successively repeated to synthesize the objective sequence having a base sequence and then sulfur oxidation and final deprotection are carried out.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an antisense oligodeoxyribonucleotide and an 1111 activity inhibitor containing the same as an active ingredient.

More specifically, AID having IIIV activity inhibitory effect
S (acquired!m5unodeflc
lency 5yndro+ac.

The present invention relates to antisense oligodeoxyribonucleotides useful as therapeutic agents for acquired immune deficiency syndrome (Acquired Immune Deficiency Syndrome).

[Problems to be solved by conventional technologies/inventions 31985
N11l (National In5titut)
e orIlcalth.

In vitro test II at National Institutes of Health)
lV(human 1ssundeficiency)
The mechanism of action of azidothymidine (^ZT), which has been proven to inhibit the growth of human immunodeficiency virus (Human Immunodeficiency Virus) and is the only drug in the world recognized as a treatment for AIDS, is that ^ZT has a strong effect on reverse transcriptase 111. They compete with each other and are mistakenly incorporated during DNA biosynthesis, stopping chain length formation. However, ^ZT is associated with severe bone marrow damage and gastrointestinal and neurological symptoms. Furthermore, lentlnan, which is currently being researched in Japan, is intended to have therapeutic effects by improving the decline in cell-mediated immunity that accompanies 111V infection, but there are still unknowns about the immunodeficiency in AIDS. many,
Furthermore, there is a risk that the immune enhancer stimulates infected T cells, thereby promoting the expression of the latent provirus. In addition, research has begun on monoclonal anti-idiotype antibodies to be applied to therapy with the aim of blocking IIIV, which is the initial infection route, from binding to CD4 molecules. Blocking reagents are being developed at various stages of the life cycle.

IIIV, which belongs to the Retroviridae family, utilizes a reverse transcription phenomenon that is different from the molecular biological cycle of its infected human, that is, it uses its own reverse transcriptase using RNA as a template for self-replication. It biosynthesizes DNA, proliferates, and follows the course of AIDS onset. If the reverse transcription phenomenon is affected, it will not affect human physiological functions in any way.
Only 11 ■ will die. In recent years, attention has been paid to the reverse transcription phenomenon, and the transcriptional inhibition effect of chemically synthesized oligonucleotides has been reported (P.C. Zamecnik, M.L. Stephenson, P.C.
Fist of the National Academy of Science Life of the Fist of America
P.C.

ZagecnlkSM le, 5tephenson,
Proc, Natl, ^cad.

Sci, USA), Vol. 75, No. 1, p. 280 (1978
) and P. C. Zamecnik, J. Gutschild, Y. Taguchi, P. Nis Sarin (P.

C, Zamecnlk, J, Goodchlld,
Y, Taguc old, p, s.

5arln), Proceedings of the National Academy of Sciences of the United States of America, Volume 83, No. 6, 41
(See page 43 (198B)). Therefore, the present inventors administered a complementary chemically synthesized oligodeoxynucleotide to form a hybrid duplex with RNA, which is a viral gene, in vitro, and interrupted reverse transcriptase reading at that site. By causing D to become a provirus.
As a result of extensive research with the aim of inhibiting NA biosynthesis, we discovered that the transcriptional transactivating gene tat-1l, which is a factor that promotes replication during provirus synthesis in living organisms,
We discovered an antisense deoxyribonucleotide having the RN^ sequence 5348-5367'i-9=get sequence containing a splice acceptor site in the center within 1, and completed the present invention.

[Means for Solving the Problems] The present invention provides base sequence: d-Ap(s)Cp(s)Ap(o)Cp(o)Cp(
o)Cp(o)Ap(o)-Ap(o)Tp(o)Tp
(o)Cp(o)Tp(o)Gp(o)Ap(o)Ap
(o)-Ap(o)Ap(o)Tp(s)Gp(s)G
(In the sequence, p(s) represents that the atom bonded to the phosphorus atom of the internucleotide portion by a double bond is a sulfur atom, and p(o) represents the atom bonded to the phosphorus atom of the internucleotide portion by a double bond. (representing that the atom is an oxygen atom) is provided.

Furthermore, the present invention provides the base sequence: d-Ap(x)Cp(c)Ap(x)Cp(x)Cp(
x) Cp(x)Ap(x)-Ap(x)Tp(x)Tp
(x)Cp(x)Tp(x)Cp(x)Ap(x)Ap
(x)-Ap(x)Ap(x>Tp(x)Gp(x)G
(In the sequence, p(x) is independently p(s) or p(0), and p(s) represents that the atom bonded to the phosphorus atom of the internucleotide part by a double bond is a sulfur atom. , p(o) indicates that the atom bonded to the phosphorus atom of the internucleotide moiety through a double bond is an oxygen atom) containing as an active ingredient an antisense oligodeoxyribonucleotide. I will provide a.

[Example] - The gene sequence site (see Figure 1) which is the target sequence of the antisense oligodeoxyribonucleotide of the present invention consists of a total of 111 VRN^ consisting of approximately 10,000 bases.
It is a sequence that is repeated several times in the sequence, and forms base pair bonds at other gene sequence sites as well.
There is a possibility that an inhibitory effect on activity may be observed (El Ratner, W Haserchin, R Batalka, K Jie φ Rybak, B K Starsich, Nis φ F Josephs, E R Tran, Jie Ni φ Raf Alski, E-ken-nee-ken Filehorn, K. Barameister, El Ivanov, Niss R. Petteway Kai, Jr., M. L. Pearson, J.N.C. Rothenberger, TΦ Nis Babas, J.C. Guraieb, F.T. Chan, R.C. Gallo, F.W. Staal, Naitsch + - (L, Ratn
er, W., Haseltlne SR, Patarc.
a.

K,J,l,Ivak,B,5tarcich,
S, F, Josephs SE, R.

Doran % LA, Rafalskl, E, ^J
hllehorn s K.

r3aumelstor, L, 1vano “f SS
, R.Petteway Jr.

M,I,,Pearson, J,^,1. aUte
nbergor, T., S., Papas.

J., Ghrayeb, N., T., Chang, R.
, C., Ga1lo, P.J.

5Laal, Nature) Volume 313, No. 24, 2
77 (1985)).

In addition, the antisense oligodeoxyribonucleotide of the present invention uses a sulfur oxidation approach to introduce a phosphorothioate bond into the internucleotide moiety, thereby evading the recognition of degrading enzymes in vivo and becoming a physiologically stable oligomer. IIIV activity inhibition effect is sustained.

Although the method of synthesizing the compound of the present invention is not particularly limited, it can be produced, for example, by the following method.

The following general formula (1) is used as a capping agent: (wherein, R
'! ;i −C(CX ) , −CIl (CX
3) This invention is based on the phosphite method using a new bosphite compound represented by 2 or -C112CX3 (in the formula, X represents a halogen atom and R2 represents an alkyl group having 1 to 6 carbon atoms). 1 shows a method for synthesizing compounds of the invention.

Note that the above compound (1) can be obtained, for example, by the following method.

R0+1+ R0PCI →(RO) I'OR
2(m (II+) (1)■ (wherein R and R2 are the same as above) That is, the above compound (1) can be converted by reacting the alcohol (11) with the alkylphosphorodichloridite (II+). Alcohols as compounds (II+)
in the presence of 3 to 4 equivalents of a tertiary base such as triethylamine or pyridine in a solvent inert to the reaction compound such as ether, tetrahydrofuran (TIIF), acetone, or dichloromethane at room temperature (
The phosphite compound (1) can be converted in high yield by carrying out the reaction at a temperature of 10 to 35°C for 2 to 5 hours.

Commercially available cross-linked polystyrene, silica gel,
A solid phase support in which an appropriate nucleoside is immobilized on a support such as porous glass is placed in a reaction column, and after detritylation reaction with a methylene chloride solution of an organic acid such as trichloroacetic acid, dichloroacetic acid, or trifluoroacetic acid, methylene chloride and acetonitrile are added. Wash thoroughly using something like After drying the solid phase support under reduced pressure, a pre-prepared nucleotide unit having a base suitable for the target sequence is placed in a reaction tube in an amount of 3 to 5 times the amount of the immobilized nucleoside, preferably 3 times the amount of the immobilized nucleoside. Dry again under reduced pressure. Then, 30 to 50 times equivalent, preferably 30 times equivalent, of an activating agent is introduced into the column together with a solvent such as acetonitrile, TIIP, methylene chloride, etc. to carry out a condensation reaction.

After washing with a solvent such as acetonitrile or methylene chloride, the capping agent and tertiary amine described above are added to the reaction tube to cap unreacted 5-hydroxyl groups and the solid support. The capping agent is used in an amount of 50 to IH times equivalent to the fixed nucleoside,
Preferably, 60 to 80 equivalents are added, and the tertiary amine as an activator is added in 3 to 6 equivalents, preferably 4 to 5 equivalents. Examples of tertiary amines used include pyridine, triethylamine, dimethylaminopyridine, N-
Examples include methylimidazole. The solvent used in this capping operation is not particularly limited as long as it is inert to the reaction compounds, and examples thereof include dichloromethane, ether, TIIF, acetone, dioxane, and acetonitrile. The reaction temperature is 5-40℃,
Preferably it is 30-37 degreeC. The reaction time is 5 to 20 minutes, preferably 10 to 15 minutes.

It is washed again with a solvent such as acetonitrile or methylene chloride, and then an oxidation reaction is performed to make it into a basic and stable state in living organisms as phosphate. As an oxidizing agent, iodine is a common oxidizing agent, with 0.05 to 0.1
A mixture of M, preferably 0.1 M of 1, and TIIP, pyridine, 1120, etc. is added and treated for 1 to 2 minutes, preferably about 1 minute. It is also possible to introduce a phosphorothioate bond into the ink-nucleotide moiety by oxidation with sulfur, for example, as follows.

After the capping operation is completed, add 3 to 10 sulfur to the reaction tube.
Add a mixture of carbon disulfide and a tertiary amine such as pyridine containing 4% to 5% by weight, and further add 3 to 10% by volume, preferably 4 to 5% by volume of the mixture.
Add 5% by volume of triethylamine for 10-30 minutes.
The sulfur oxidation operation is preferably carried out for 15 to 20 minutes.

However, in order to ensure that the atmosphere is prepared so that sulfur oxidation can proceed sufficiently, and to not affect the condensation reaction, sufficient cleaning must be performed with a mixed solution of carbon disulfide and the tertiary amine before and after sulfur oxidation. to remove residual sulfur. Then,
The obtained oxide was mixed with acetonitrile:pyridine-1:1
Wash thoroughly using (V/V), acetonitrile, methylene chloride, etc.

The above series of operations is repeated until the desired oligonucleotide is obtained. Incidentally, the above-mentioned oxidation reaction may be performed at the end after synthesizing the target base sequence.

At that time, a reaction time of about 1 hour is sufficient.

Then, the deprotection reaction of dimethoxytrityl (DMTr) M at the 5° end and the protecting group of the amino group at the base is carried out, for example, as follows. 55-60℃ with concentrated ammonia water
, preferably 4 to IO hours at 55°C, preferably 6 to 7
time treatment to desorb the product from the solid support, and
Deprotect the base part. Next, in order to separate only those with dimethoxytrityl groups, the peaks are purified using a reversed-phase C18 open column (for example, manufactured by Waters) using a TEAA (triethylammonium acetate) elution solvent containing 20 to 50% acetonitrile. The portion in which color development of the DMTr group is confirmed is fractionated. next,
This DMTr group-containing oligomer was dissolved in an aqueous solution of 80-90%, preferably 80% acetic acid at 5-37°C, preferably at 2.
Detritylation is performed by treating at 0 to 37°C for 5 to 15 minutes, preferably 10 minutes, and ammonium formate aqueous solution containing 20% acetonitrile (pl + 6.8.048M -1
, 38M) and is isolated and purified by ion exchange II PLC to obtain the deprotected target oligonucleotide. The reaction yield of each step in this series is a high yield of 95% or more.

Examples of the nucleotide unit used in the condensation reaction include a phosphite unit represented by -Fukudai (V): (in the formula, B represents a base), and in this case, as an activator, N
- Tertiary bases such as methylimidazole and dimethylaminopyridine are used.

The present invention will be explained in more detail below using Examples, but the present invention is not limited thereto.

Example 1 Base sequence: d-Ap(o)Cp(o)Ap(o)Cp(o)Cp(
o)Cp(o)Ap(o)-Ap(o)Tp('o)T
p(o)Cp(o)Tp(o)Gp(o)Ap(0)A
p(o)-Ap(o)Ap(o)Tp(o)Gp(o)
Production of oligodeoxyribonucleotide (hereinafter referred to as DO-20) represented by G (in the sequence, p (o) represents that the atom bonded to the phosphorus atom of the internucleotide moiety by a double bond is an oxygen atom) ( 1) Capping agent (bis(+, 1.1, 3.8.3
Synthesis of phosphorus trichloride (871, IM) with stirring at -20'C under a nitrogen atmosphere
) was added dropwise over about 1 hour and 30 minutes. After the dropwise addition, the mixture was gradually warmed to room temperature and stirred for about 2 hours, then transferred to a separating funnel and left overnight. Then distilled under reduced pressure to obtain a boiling point of 40-42℃/
22vi+l1g (40"C/ 20mmml1g) fraction was collected and diluted with isoprobyl phosphorodichloride) (
20.1g, 0.125M, 12.5%).

This isoprobyl phosphorodichloridite (20.1g
, 0.125M) was dissolved in anhydrous ether (1
Anhydrous triethylamine (0.35 M, 48.85 ml) dissolved in anhydrous ether (60 ml) was added dropwise with stirring at -20"C, then 1,1 dissolved in anhydrous ether (Bowl) was added dropwise with stirring. .1゜3.
3+3-heki f7/Iz o D-2-fo ha) -ru (0,
5M, 52.8ml) was added dropwise. The mixture was gradually warmed to room temperature and stirred for about 12 hours. Thereafter, the hydrochloride was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was distilled under reduced pressure to obtain a boiling point of 46-4.
Target bis(1,1,1°3.3.3-hexafluoro-2-propyl)-2-propyl + Sphite (43.4g, 0.10
2M, yield 82%).

”P-NOR schedule (CDCI2): 13
9.9ftppm(2) CPG into which oligomer-20mer synthetic nucleoside was introduced (28mg, 0.5
μM, 17.9 μM/g) was placed in a reaction tube, and dedimethoxytritylation was performed with a 3% trichloroacetic acid methylene chloride solution (1+ml×2).
) and then acetonitrile. after that,
After drying CPG under reduced pressure, phosphite Ninit (300μm, 0.5M) was dissolved in acetonitrile.
/l) and N-methylimidazole (
60 μM, 750 μM) was placed in a reaction tube and reacted for 300 minutes, followed by washing with acetonitrile (2 sl×5).

Next, O,1M N-methylimidazole solution (TIIF
/ 1120 = 38/2 (in v/v)) for 2 minutes and washed with acetonitrile (21 x 5). Next, acetonitrile (75 μm) and bis(1,1,
I, 3,3.3-hexafluoro-2-propyl-2-
Propyl phosphite (37,5μH111,4μl)
and N-methylimidazole (18
After adding 7.5 μM (15 μl) and carrying out a capping reaction for 15 minutes, the mixture was washed with acetonitrile (2 ml×5). Finally, 0.1M N-methylimidazole solution (Tr
rF/It20-98/2 (v/v)) and 2
After reacting for a minute, wash with acetonitrile (2 ml x 5) and methylene chloride (2 ml x 5) and return to dedimethoxytritylation.

This series of operations was repeated 19 times.

TIIP/Py/II with 0.1M iodine in a 20-mer obtained by manual synthesis. 0-44/3/3 (v/v
) Solution (21) was added, and the mixture was allowed to react at room temperature for 1 hour to perform oxidation. Then pyridine/acetonitrile-1/1
(v/v) and acetonitrile to synthesize an oligomer-20mer in which all atoms bonded to the phosphorus atom of the internucleotide portion by double bonds were oxygen atoms.

(3) Deprotection, purification, and structural confirmation of oligomer-20mer. Excision from CPG was performed by adding 28% ammonia water (21) and treating at room temperature for 1 hour. Thereafter, CPG and insoluble matter were removed by filtration using a membrane filter, and the filtrate was concentrated under reduced pressure. This was purified by reverse phase It PLC. When the 1-1 compound was eluted using a linear gradient of O, IN-TE^^ buffer-acetonitrile polarity (10 to 50%) as an eluent, a 20-mer having a dimethoxytrityl group at the 5°-terminus was converted. After concentrating this, 80% acetic acid aqueous solution (21) was added and dedimethoxytritylation was performed at room temperature for 15 minutes, followed by washing with ether. Thereafter, the target 20-mer (DO-20) was obtained by elution using reverse phase II r'LC using 0, IM) ethyl ammonium acetate buffer (5-40% acetonitrile). Reverse phase I after purification
It was analyzed using an I PLC column (see Figure 2). The structure was confirmed to be a 20-mer by electrophoresis on a 20% polyacrylamino gel containing 7M urea (see Figure 3) and enzymatic degradation with snake venom phosphodiesterase and alkaline phosphatase (see Figure 4). In FIG. 3, lane 1 represents the 20-mer as a chain length standard, and lane 2 represents the 20-mer obtained in Example 1. After treatment with snakevenom phosphodiesterase (37°C, 2 hours) and alkaline phosphatase (32°C, 1 hour), it was inactivated (90°C, 30 minutes) and reversed phase 11PI,C (C-
18) Te0. IN) Analyzed using IJ ethylammonium acetate (0.8% acetonitrile) and confirmed the sequence (5dC: 3dG: 4dT: 8d^-
4,87 pieces 3.00:4.25+7.81).

Example 2 Base sequence: d-Ap(s)Cp(s)Ap(o)Op(o)Cp(
o)Cp(o)Ap(o)-Ap(o)Tp(o)Tp
(o)Cp(o)Tp(o)Gp(o)Ap(o)Ap
(o)-Ap(o)Ap(o)Tp(s)Gp(s)G
(In the sequence, p(s) represents that the atom bonded to the phosphorus atom of the internucleotide part by a double bond is a sulfur atom, and p(o) is the same as above.) , DSO3-20
The same procedure as in Example 1 was carried out except for the oxidation reaction. The oxidation reaction was carried out as follows.

After capping the dimer, trimer, 19fi form, and 20°C form, carbon disulfide containing 5% by weight of sulfur: pyridine-1 (v/v) was added to the reaction tube and further mixed. Triethylamine was added to the solution in an amount of 5% by volume, and sulfur oxidation was performed for 15 minutes. Note that, before the sulfur oxidation (2 times at 21) and after (4 times at 21), sufficient washing was performed with carbon disulfide:pyridine-1:l (v/v) to remove residual sulfur. The other internucleotide parts are 0.1H +2-TII
F: Viridine: 1120-44: 3:3 (v/v)
was added and treated for 1 minute to oxidize the phosphite to phosphate.

Erareta Purpose Tossru Diomer (Dsos-20) Rough M
This was confirmed by 20% polyacrylamide gel electrophoresis containing urea (see Figure 5). In FIG. 5, lane 1 represents the 20-mer obtained in Example 2, and lane 3 represents the 20-mer as a chain length standard. Also, 0.025M I
□-Desulfurization was performed using pyridine solution, and 0DS-11
PLc(0,IN)! Purification with J-T-thiL7 ammonium acetate (10-30% acetonitrile) yielded the normal diomer. As in Example 1, after enzymatic decomposition with snake venom pospogesterase and alkaline phosphatase, the sequence was confirmed by analysis by II PLC on TSK gel oligo-needle blade ram (
5dC: 3dG: 4dT: 8dA-4,52:
2.18: 4.21: 7.50) was performed (
(See Figure 6).

Example 3 Base sequence: d-Ap(s)Cp(s)Ap(s)Cp(s)Cp(
s)Cp(s)Ap(s)-Ap(s)Tp(s)Tp
(s)Cp(s)Tp(s)Gp(s)Ap(s)Ap
(s)-Ap(s)Ap(s)Tp(s)Gp(s)G
(In the sequence, p(s) is the same as above.) Production of oligodeoxyribonucleotide (hereinafter referred to as DS-20) The same procedure as in Example 1 was carried out except for the oxidation reaction. The oxidation reaction was carried out as follows.

After capping each step, carbon disulfide:pyridine (volume ratio 1:1) containing 5% by weight of sulfur is added;
Further, 5% by volume of triethylamine was added to the mixed solution to carry out sulfur oxidation for 15 minutes.

The obtained target diomer (DS-20) was prepared in Example 2.
This was confirmed by gel electrophoresis in the same manner as in (see Figure 5). In FIG. 5, lane 2 is 20
Lane 3 represents the 20-mer as a chain length standard. In addition, the sequence was confirmed in the same manner as in Example 2.
: 3dG: 4dT: 8d^-5, 41: 2.09
: 5.02 : 7.59) (see Figure 7).

Test Example 1 Anti-111v Activity Test Regarding the obtained synthetic probe, we asked Mr. Naoki Yamamoto and Mr. Osamu Yoshida, professors of Yamaguchi University School of Medicine, to conduct an anti-1i 1V activity test. 111■ (101-
0,01) was infected (37°C, 1 hour) and prepared (20x
10' cells/ml). The oligomers obtained in Examples 1 to 3 were added to RPMI-1640 (RO3WIELL PA) supplemented with 10% FBS (fetal bovine serum).
RK MEMORIAI.

Made by lN5TITUTrE, G.E. Moore (G, E
, Moore) et al., j,^1M,^,肚,591 (
1957)) and dissolved in 0.45. After sterilization by filtration, the mixture was serially diluted 2 times to obtain various concentrations. In order to examine the effect of the oligomer itself on cells, the same procedure was performed on uninfected cells and an activity test was conducted. On the fourth day of infection, the inhibitory effect was determined by Trivan blue staining method and indirect fluorescent antibody method. It obtained in Examples 1.2 and 3.
-20, DSO8-20 and DS-20 are shown in Figures 8, 9 and 10, respectively. In the bar graphs in Figures 8 to 1O, the hatched cells represent old V-infected cells, and the unhatched cells represent 111■
Represents uninfected cells.

Based on the results, oligomers of the present invention 〇〇-20 and D
It was found that SO8-20 showed activity when the concentration was 25 μX or more. Furthermore, the oligomer DS-20 of the present invention
The activity was observed at 5 μH or more, and it was revealed that it showed particularly excellent activity.

The method for administering the III IV activity inhibitor of the present invention includes:
Oral, enteral or parenteral administration methods can be selected. Specific formulation forms include, for example, tablets, capsules, fine granules, syrups, suppositories, ointments, and injections.

The carrier for the formulation of the old V activity inhibitor of the present invention includes oral,
Organic or inorganic solid or liquid, usually inert, pharmaceutical carrier materials suitable for enteral or other parenteral administration are employed.

Specifically, for example, crystalline cellulose, gelatin, lactose, starch, and magnesium stearate.

Mention may be made of talc, vegetable and animal fats and oils, gums, polyalkylene glycols, and the like.

The compound of the present invention can be included in the formulation from 0.2 to 100%.

The It l V activity inhibitors of the present invention are generally administered at dosages that achieve the desired effect without side effects.

[Effects of the Invention] The stable antisense oligodeoxyribonucleotide of the present invention can effectively inhibit the activity of IIV (which causes A103 onset).

[Brief explanation of the drawing]

Diagram m1 is a drawing showing the tat gene site (splicing site) of IIIVRN^. Figure 2 shows Example 1
Reverse phase II of the obtained DO-20 at 254ns+
It is a chromatogram by PLC. FIG. 3 is a drawing showing the electrophoresis pattern of Do-20 obtained in Example 1 by 20% polyacrylamide gel electrophoresis. Figure 4 shows 254 ns of the product obtained after enzymatically decomposing the DO-20 obtained in Example 1 with snake venom phosphodiesterase and alkaline phosphatase.
This is a chromatogram obtained by II PLC. FIG. 5 is a drawing showing electrophoresis patterns of DSO3-20 obtained in Example 2 and O3-20 obtained in Example 3 by 20% polyacrylamide gel electrophoresis. Figure 6 shows the product 254 obtained after enzymatically decomposing DSO3-20 obtained in Example 2 with snakevenom phosphodiesterase and alkaline phosphatase.
Chromatogram by II PLC in rv. Figure 7 shows the product 254 obtained by enzymatically decomposing O8-20 obtained in Example 3 using snake venom phosphodiesterase and alkaline phosphatase.
This is a chromatogram based on n+g+kore (old) 1, C. Figures 8 to 10 are DO-20 and DSO3, respectively.
-20, is a bar graph showing anti-III IV activity for DS-20. Figure 22 Time (minutes) Figure 3 Figure 4 Time (minutes) Figure 5 Figure 26 Time (minutes) Off figure time (minutes) Figure O8 Concentration of H-V infected cells (LIM) Figure 9 RoH Engineering ■ Infected cell concentration (JJM) Age 1o Figure ZH Engineering V infected cell concentration (JJM)

Claims (1)

[Claims] 1 Base sequence: d-Ap(s)Cp(s)Ap(o)Cp(o)Cp(
o)Cp(o)Ap(o)-Ap(o)Tp(o)Tp
(o)Cp(o)Tp(o)Gp(o)Ap(o)-A
p(o)Ap(o)Ap(o)Tp(s)Gp(s)G
(In the sequence, p(s) represents that the atom bonded to the phosphorus atom of the internucleotide portion by a double bond is a sulfur atom, and p(o) represents the atom bonded to the phosphorus atom of the internucleotide portion by a double bond. An antisense oligodeoxyribonucleotide (representing that the atom is an oxygen atom). 2 Base sequence: d-Ap(x)Cp(x)Ap(x)Cp(x)Cp(
x) Cp(x)Ap(x)-Ap(x)Tp(x)Tp
(x)Cp(x)Tp(x)Cp(x)Ap(x)-A
p(x)Ap(x)Ap(x)Tp(x)Gp(x)G
(In the sequence, p(x) is independently p(s) or p(o), and p(s) represents that the atom bonded to the phosphorus atom of the internucleotide part by a double bond is a sulfur atom. , p(o) represents that the atom bonded by a double bond to the phosphorus atom of the internucleotide moiety is an oxygen atom), which contains an antisense oligodeoxyribonucleotide as an active ingredient. 3 The base sequence of the antisense oligodeoxyribonucleotide is d-Ap(o)Cp(o)Ap(o)Cp(o)Cp(
o)Cp(o)Ap(o)-Ap(o)Tp(o)Tp
(o)Cp(o)Tp(o)Gp(o)Ap(o)-A
p(o)Ap(o)Ap(o)Tp(o)Gp(o)G
Claim 2 (in the arrangement, p(o) is the same as above)
The HIV activity inhibitor described. 4 The base sequence of the antisense oligodeoxyribonucleotide is d-Ap(s)Cp(s)Ap(o)Cp(o)Cp(
o)Cp(o)Ap(o)-Ap(o)Tp(o)Tp
(o)Cp(o)Tp(o)Gp(o)Ap(o)-A
p(o)Ap(o)Ap(o)Tp(s)Gp(s)G
(In the sequence, p(s) and p(o) are the same as above.) The HIV activity inhibitor according to claim 2. 5 The base sequence of the antisense oligodeoxyribonucleotide is d-Ap(s)Cp(s)Ap(s)Cp(s)Cp(
s)Cp(s)Ap(s)-Ap(s)Tp(s)Tp
(s)Cp(s)Tp(s)Gp(s)Ap(s)-A
p(s)Ap(s)Ap(s)Tp(s)Gp(s)G
Claim 2 (in the arrangement, p(s) is the same as above)
The HIV activity inhibitor described.