JPH0578386A - New anti-hiv agent and its production - Google Patents

Abstract

PURPOSE:To obtain a new anti-human immunodeficiency virus (anti-HIV) agent excellent in antiviral action with hardly any toxicity. CONSTITUTION:An anti-HIV agent is composed of a compound expressed by formula I [(m) is 1-50; B denotes B2 to Bm+1 (B2, B3...Bm. and Bm+1) and B1 to Bm+2 are hypoxanthin-9-yl, etc., except when all B1 to Bm+2 are the same]. The above-mentioned compound expressed by formula I is obtained by subjecting a long-alkylamino controlled pore glass(CPG) having thymidine introduced thereinto to detritylation treatment with trichloroacetic acid, adding a phosphoramidite unit such as d-DMTrA<bz>p (DMTr is dimethoxytrityl group; bz is benzoyl group) suited to the objective sequence and 1H-tetrazole to the resultant long-alkylamino CPG, carrying out condensation, introducing sulfur into the phosphoric acid ester part and repeating the above-mentioned operation (n-1) times. For example, a compound expressed by formula II [(d) has a sequence unit structure of deoxynucleoside unit; T is thymidine; C is 2'- deoxycytidine; G is 2'-deoxyguanosine; A is 2'-deoxyadenosine; ps is phosphoric acid ester bond, expressed by formula III and connecting OH at the respective 5'-and 3'-positions of T, C, G and A to phosphorothioic acid].

Description

Detailed Description of the Invention

[0001]

The present invention relates to HIV (Human Immuno
deficiency Virus; human acquired immunodeficiency virus; AIDS virus) sequences encoding major proteins in genes (eg gag, pol, vif, vpr, tat, rev, env, nef, LT)
R) and the like or an oligonucleotide complementary to all or a part of it (that is, antisense DNA) is prepared, and its phosphoric acid ester portion is chemically modified to phosphorothioate, which is used for the HIV gene represented by the following general formula [I]. The present invention relates to antisense oligodeoxyribonucleoside phosphorothioates, and a method for producing the same,
The present invention also relates to an anti-AIDS virus agent containing, as an active ingredient, antisense oligodeoxyribonucleoside phosphorothioates against the HIV gene synthesized by this production method.

[0002]

PRIOR ART As an anti-AIDS drug, AZT (azidothymidine) [Proc. Natl. Acad. Sci. USA 82 7096 (1985)] which is currently the only approved drug and ddI (dideoxyinosine) [Proc] which is a clinical drug. . Nat
l. Acad. Sci. USA 82 1911 (1986)] and the like are well known. Also, dCS ₂₈ (oligophosphorothioate 28mer of cytidine) [Proc. Natl. Acad. Sci. USA 84 7706 (1987)] is known to have an anti-HIV effect in vitro. Furthermore, there is a report that a part of the phosphorothioate derivative of the oligoribonucleotide derivative has anti-AIDS virus activity (Japanese Patent Laid-Open No. 3-128391).

[0003]

AZT and ddI are nucleic acid derivatives belonging to the group of compounds called dideoxynucleosides. The mechanism of action of dideoxynucleosides is
It acts directly on the retrovirus reverse transcriptase and exerts an antiviral effect by its competitive inhibition effect and chain termination effect. Therefore, it can be said that these compounds are reverse transcriptase inhibitors. On the other hand, in the AIDS virus (HIV) replication cycle, there are many virus-specific proteins and their associated steps. And it can be said that these stages of the replication cycle and virus-specific proteins are excellent targets from the side of developing antiviral agents.

The virus-specific process (stage) is, for example, 1. Adsorption of virus to target cells 1. Penetration into cells 3. 3. Uncoating of virus particles Reverse transcription of viral RNA into DNA (Reverse T
ranscription) 5. Incorporation of viral DNA on cellular genes (Int
egration) 6. Replication of viral RNA (Replication) 7. Translation of viral RNA into viral proteins (Trans
lation) 8. Activation by viral regulatory proteins (Transactivati
on) 9. Modification of viral RNA by viral proteins (Assemb
ly) 10. Budding of virus particles can be mentioned.

Examples of proteins specific to AIDS virus include, for example, a. Glycoprotein (gp-120) a. Reverse transcriptase (RT; reverse transcriptase, and RNase H; ribo)
nuclease H) C. A protein that activates HIV gene expression (tat; trans-acting transcriptional activator) d. Regulator of expression of virion protei (rev;
n) E. Examples include protein-cleaving enzyme protease (pro; protease).

The above-mentioned AZT and ddI are antiviral agents which inhibit the reverse transcription step of 4 as a step and the reverse transcriptase of b as a protein. However, as seen above, there are still many points that may be able to overcome the virus, and it is very difficult to develop a drug that targets the stage or enzyme where the dideoxynucleosides such as AZT and ddI cannot exert an effect. Is significant and important. If a drug with a different action, which is not seen in conventional drugs, can be developed in this way, it is possible to reduce the dosage of each other by combination therapy with, for example, AZT, or to expect a synergistic effect not seen by single administration. .. AZT is not only in vitro but also in vitro.
Although it is an excellent anti-AIDS drug that exerts its effect in vo and clinical settings, recently AZT's side effects (bone marrow toxicity, etc.) have become a problem, and from this point of view, it has a different action point from AZT. The development of medicines to carry is urgent and important.

An object of the present invention is to provide a novel antiviral agent having new efficacy and a method for synthesizing the same.

[0008]

The present invention relates to HIV (Huma).
n Immunodeficiency Virus; Human acquired immunodeficiency virus
Code for the major protein in the gene
Sequence (eg gag, pol, vif, vpr, tat, rev, en
v, nef, LTR, etc.)
Produces gonucleotides (ie antisense DNA)
The phosphoric acid ester moiety is chemically modified to
Antisense oligo against HIV gene
It is based on the discovery that deoxyribonucleoside phosphorothioates have anti-HIV activity.

The present invention provides the HI described in the underlined section above.
The present invention relates to a novel anti-HIV agent containing at least one kind of antisense oligodeoxyribonucleoside phosphorothioates for V gene as an active ingredient and a method for producing the same.

The present invention also provides oligodeoxyribonucleoside phosphorothioates represented by the following general formula [I]: [In the formula, m represents an integer of 1 to 50, B represents any _one of B ₂ to B _{m + 1} , that is, B = B ₂ , B ₃ , B ₄
……… B _m , B _{m + 1} ) B _{1 to} B _{m + 2} are the same or different from each other, and are hypoxanthine-9-yl, guanine-9-yl, adenine-9-yl, cytosine-, respectively. 1- Ill,
Indicates either uracil-1-yl or thymine-1-yl. However, although each of B _{1 to} B _{m + 2} may be partially the same, it is not always the same. That is, the following six cases in general formula [I] are excluded. A. When B _{1 to} B _{m + 2} are all hypoxanthyl-9-yl a. When B _{1 to} B _{m + 2} are all guanine-9-yl c. When B _{1 to} B _{m + 2} are all adenine-9-yl d. When B _{1 to} B _{m + 2} are all cytosine-1-yl e. When B _{1 to} B _{m + 2} are all uracil-1-yl. B _{1 to} B _{m + 2} are all thymin-1-yl] and an anti-HIV agent containing the same, and thymidine (or 2′-deoxyadenosine, 2 ′) that has been detritylated with trichloroacetic acid. -Deoxyguanosine, 2'-deoxycytidine, 2'-deoxyuridine,
2'-deoxyinosine) -introduced long alkylamino CPG (controlled pore glass) has a phosphoramidite unit (eg, d-DMTrA ^bz p, d-DMTrC ^an p,
d-DMTrG ^ibu p, d-DMTrTp, d-DMT
rIp, d-DMTrUp, etc.) at 30 to 50 equivalents and 1H-
A series of operations consisting of adding 150 equivalents of tetrazole to cause a condensation reaction, and then performing an oxidation reaction with sulfur to introduce sulfur into the phosphoric ester portion is performed n-1 times (however, n
Represents the target chain length) A method of synthesizing antisense oligodeoxyribonucleoside phosphorothioates by using the phosphoramidite method [wherein the above phosphoramidite unit has the structure shown below, for example] A compound or its derivative. (Here, bz is a benzoyl group, an is an anisoyl group,
DMTr is dimethoxytrityl group, NCEt relates cyanoethyl group, i-Pr ₂ N represents a diisopropylamino group, respectively).

In the present invention, the novel anti-HIV of the present invention
The reagents used in synthesizing antisense oligodeoxyribonucleoside phosphorothioates against the HIV gene, which is an agent, are all commercially available reagents, but if necessary, easily available compounds are well known. However, it is safe to use it after guiding by a simple method.

[0012]

The action of the antisense oligodeoxyribonucleoside phosphorothioates against the HIV gene of the present invention will be described below.

The anti-HIV effect of the antisense oligodeoxyribonucleoside phosphorothioates on the HIV gene of the present invention shows that when the replication cycle of AIDS virus (HIV) is classified as follows, replication of viral RNA of 6 is performed. It can be said that it mainly inhibits. 1. Adsorption of virus to target cells 1. Penetration into cells 3. 3. Uncoating of virus particles Reverse transcription of viral RNA into DNA (Reverse T
ranscription) 5. Incorporation of viral DNA on cellular genes (Int
egration) 6. Replication of viral RNA (Replication) 7. Translation of viral RNA into viral proteins (Trans
lation) 8. Activation by viral regulatory proteins (Transactivati
on) 9. Modification of viral RNA by viral proteins (Assemb
ly) 10. Budding of virus particles

However, since the above steps 1 to 10 are a series of cycles, as a result, as a result, 6
Replication The following stages (ie Replication, Translat
ion, Transactivation, Assembly, Budding) is inhibited.

From a different point of view, the antisense oligodeoxyribonucleoside phosphorothioates for the HIV gene of the present invention are anti-HIV agents capable of suppressing the growth of HIV after it has been incorporated as a provirus into the gene of human cells. Is. Therefore, the antisense oligodeoxyribonucleoside phosphorothioates against the HIV gene of the present invention can be said to be a new type of anti-HIV agent that is effective not only for the prevention of HIV infection but also for patients after infection. This is
Clearly, dideoxynucleosides such as AZT are anti-HIV agents that block the step before HIV is incorporated as a provirus (the step of reverse transcription).

The fact that the stages in which the effects are exerted are different allows the simultaneous administration while suppressing side effects, and
It opens the way for more flexible response to various symptoms caused by.

By the way, sequences encoding various viral proteins are found on the HIV gene. These are, for example, gag, pol, vif, vpr, tat, rev, env, nef, LTR. Each of these sequences is the HIV of the present invention.
Antisense oligodeoxyribonucleoside phosphorothioates against genes can be targeted. However, in the above various sequences, tat and r
Antisense oligodeoxyribonucleoside phosphorothioates of genes encoding regulatory proteins such as ev are less susceptible to gene mutation due to viral replication and are expected to have higher anti-HIV effects.

The length of the antisense oligodeoxyribonucleoside phosphorothioates for the HIV gene of the present invention can be freely selected according to the purpose of use, but it is easy to synthesize and adaptable to mutation.
(Following property) , Penetration into cells, Ease of purification , Drug
Cost puffs resulting from the correlation between stability , length and efficacy
When combined with other factors, nucleoside-5'-1-
Phosphoric acid can be used as a unit up to its 50-mer (that is, within 50 mer counted as a nucleoside), but among them, up to the 20-mer (20mer) is more suitable.

The antisense oligodeoxyribonucleoside phosphorothioates for the HIV gene of the present invention have a phosphorothioate bond as a partial structure. The phosphorothioate bond is a phosphoric acid bond found in ordinary RNA or DNA that is chemically modified to introduce a sulfur functional group, and has a structure that is difficult to be cleaved by various enzymes in the living body. Therefore,
It can be transported to a desired place for expressing the activity without being hindered by decomposition and the like.

On the other hand, the usual antisense strand [Zame
cnic et al. Proc. Natl. Acad. Sci. USA, 75 280 (1978)]
Since it is a phosphate bond, it is susceptible to degradation in the body.

As already mentioned many times, the present invention is the first antisense oligodeoxyribonucleoside phosphorothioates for the HIV gene.

However, as a compound having a structurally similar skeleton, dCS ₂₈ (28mer of deoxycytidine phosphorothioate) reported by Matsukura et al. Is known [Pr.
oc. Natl. Acad. Sci. USA 84 , 7706 (1987)] Matsukura et al.'s dCS ₂₈ is a ₂₈ -mer in which deoxycytidine (2'-deoxycytidine) is linked by a phosphorothioate bond, so to speak, oligodeoxycytidine phosphorothioate 28mer.
It is a compound that should be called.

The antisense oligodeoxyribonucleoside phosphorothioates for the HIV gene of the present invention are similar to Matsukura et al.'S dCS ₂₈ in that they have a phosphorothioate bond in the molecule. However, it is completely different from the stage of the idea / idea to the effect / point of action.

The biggest difference is the overall structure (ie sequence). The antisense oligodeoxyribonucleoside phosphorothioates for the HIV gene of the present invention can be used for HIV (Human Immunodeficiency).
Virus; human acquired immunodeficiency virus; AIDS virus) sequences encoding major proteins in genes (eg gag, pol, vif, vpr, tat, rev, env, nef, LTR, etc.)
An oligonucleotide (that is, an unsense DNA) complementary to all or part of HI was prepared, and its phosphoric acid ester moiety was chemically modified to form phosphorothioate.
Antisense oligodeoxyribonucleoside phosphorothioates for V gene. Therefore, the antisense oligodeoxyribonucleoside phosphorothioates against the HIV gene of the present invention are completely “complementary” to the sequence encoding the main protein of HIV. Therefore, it binds "specifically" and "regioselectively" to the sequence encoding the main protein of HIV, and inhibits the synthesis of viral proteins, and consequently suppresses the growth of HIV.

To give an example, tat of HIV genes
An antisense oligodeoxyribonucleoside phosphorothioate for the compound of the present invention represented by the following sequence: [However, T; 2'-deoxythymidine (or simply thymidine), C; 2'-deoxycytidine, G; 2'-
Deoxyguanosine, A; 2'-deoxyadenosine, p _s is phosphorothioic aci
d) represents the phosphate bond. ] Is H
5348 to 5367 of the 10 kb gene of IV
CCAU, which is the th gene code (part of the sequence encoding the regulatory protein tat)
It is completely "complementary" to the structure UUUCAGAAUUGGGGUGU, and specifically binds to this portion to form a double strand, suppressing the expression of the HIV gene.

That is, this CCAUUUCAGAAUU
The structure GGGUGU is unique to a gene fragment encoding a special protein called tat in the HIV gene, and therefore the same CCAUUUUCAGAAU is used.
It does not bring any interaction with the human gene, which is expected to have the possibility of having the structure UGGGUGU in the gene. That is, it does not bind to the human genome. Therefore, it can be said that it is an ideal drug with few side effects and very high anti-HIV selectivity.

On the other hand, since the above-mentioned dCS ₂₈ is a repeating unit of deoxycytidine, it is expected to have high selectivity for the HIV gene as seen in the antisense oligodeoxyribonucleoside phosphorothioates for the HIV gene of the present invention. Can not. This is d
Not only expected avidity and selectivity of weakness against HIV genes CS _28, tells us that the hidden danger that binds randomly to the human gene. Therefore,
Naturally, the side effects also increase, and the therapeutic index as a drug (dCS ₂₈
Is reduced (compared to antisense oligodeoxyribonucleoside phosphorothioates for the HIV gene of the present invention).

Further, according to the disclosure of Shibahara et al. (Japanese Patent Laid-Open No. 3-128391), a part of the phosphorothioate derivative of the oligoribonucleotide derivative shows anti-HIV activity. It has a nucleoside as a unit partial structure and is essentially completely different from the antisense oligodeoxyribonucleoside phosphorothioates of the present invention having a 2'-deoxyribonucleoside as a partial unit structure. Also, the action is very different. For example, according to the disclosure of Shibahara et al. (Japanese Patent Laid-Open No. 3-128391), their compounds have an anti-H concentration of 1 to 3 μM.
Although it is said that there is an IV effect, the antisense oligodeoxyribonucleoside phosphorothioate form against the HIV gene of the present invention already shows a remarkable effect at a concentration of 1.0 to 5.0 × 10 ⁻³ μM. Furthermore, no toxicity is observed even at a concentration of 5-6 μM or more. This indicates that the action mechanism, antiviral effect, and drug potential of each compound are completely different.

[0029] As described above, the antisense oligodeoxyribonucleoside phosphorothioates for the HIV gene of the present invention can be used for HIV (Human Immuno-d
eficiency Virus; human acquired immunodeficiency virus; AIDS virus) sequences encoding major proteins in genes (eg gag, pol, vif, vpr, tat, rev, env, nef, LTR)
Is a new type of anti-HIV agent that has high selectivity and low toxicity not found in conventional anti-HIV agents because it is an oligodeoxyribonucleoside phosphorothioate complementary to all or part of is there.

The antisense oligodeoxyribonucleoside phosphorothioates against the HIV gene of the present invention are HIV (Human Immunodeficiency Virus;
A sequence encoding a major protein in the gene of human acquired immunodeficiency virus (AIDS virus) (for example, ga
g, pol, vif, vpr, tat, rev, env, nef, LTR, etc.), which may be an antisense oligodeoxyribonucleoside phosphorothioate
Although it exhibits activity, in particular, a gene encoding a regulatory protein such as tat or rev is less likely to be mutated, and therefore antisense oligodeoxyribonucleoside phosphorothioate against a gene encoding a regulatory protein such as tat or rev has a higher anti-resistance. It is suitable for expressing HIV activity.

Among these, the following compounds show particularly high anti-HIV activity. That is, the compound is a) a compound represented by the following sequence among antisense oligodeoxyribonucleoside phosphorothioates for the rev region of HIV gene. [However, d indicates that the unit structure of this sequence consists of deoxynucleoside units, and T; 2
′ -Deoxythymidine (or simply thymidine), C;
2′-deoxycytidine, G; 2′-deoxyguanosine, A; 2′-deoxyadenosine, and p _s is phosphorothio of the respective 5′- and 3′-position hydroxyl groups of T, C, G, and A. Phosphate bond by acid ie Is represented. ]

B) Among antisense oligodeoxyribonucleoside phosphorothioates for the tat region of HIV gene, compounds represented by the following sequences. [However, d indicates that the unit structure of this sequence consists of deoxynucleoside units, and T; 2
′ -Deoxythymidine (or simply thymidine), C;
2′-deoxycytidine, G; 2′-deoxyguanosine, A; 2′-deoxyadenosine, and p _s is phosphorothio of the respective 5′- and 3′-position hydroxyl groups of T, C, G, and A. Phosphate bond by acid ie Is represented. ] Etc. are mentioned.

The antisense oligodeoxyribonucleoside phosphorothioates for the HIV gene according to the present invention can be easily synthesized using commercially available chemical reagents.

The HIV of the present invention used in the present invention below
The synthetic method of antisense oligodeoxyribonucleoside phosphorothioates for genes is shown (Table 1 and flowchart).

[0035]

[Table 1] Synthetic cycle step of oligodeoxyribonucleoside phosphorothioate Reagent or solvent amount (ml) Time (min) Number of operations 1 3% TCA in CH ₂ Cl ₂ 1 × 2 2 CH ₂ Cl ₂ 1 × 3 3 CH ₃ CN 2 × 3 4 vacuum 10 5 Phosphoramidite unit (30eq.) 6 vacuum, N ₂ atmosphere 10 7 1H-tetrazole (150eq.) In 0.45 20 CH ₃ CN 8 CH ₃ CN 2 × 3 9 Ac ₂ O / Lutidine / THF = 1 / 1/8 0.25 7% DMAP in THF 0.25 2 10 CH 3 CN 2 11 CS 2 / Pyridine = 1/1 2 × 2 12 5% S 8 in CS 2/15 Pyridine / Et 3 N = 12/12/1 13 CS ₂ / Pyridine = 1/1 2 × 4 14 CS ₃ CN / Pyridine = 1/1 2.5 × 3 15 CH ₃ CN 2 × 3 16 CH ₂ Cl ₂ 1 × 3 17 Step 1 until the nucleotide sequence is complete. Repeat ~ 16.

[0036] Synthesis cycle flow chart

That is, thymidine (or 2'-deoxyadenosine, 2'-deoxyinosine, 2'-deoxycytidine, 2'is placed in a glass reaction tube equipped with a glass filter.
-Deoxyuridine, 2'-deoxyguanosine) -introduced long alkylamino CPG (controlled pore glass, manufactured by Etrectronic Nucleonic Co., Ltd. in the UK) (3
μmol, supported amount 18 μmol / g) is added, and after detritylation reaction with 3% trichloroacetic acid-methylene chloride solution, methylene chloride and acetonitrile are thoroughly washed in this order. And C
After the PG was dried under reduced pressure, the phosphoramidite unit (d-DMTrA) having a base suitable for the target sequence was prepared.
^bz p, d-DMTrC ^an p, d-DMTrG ^ibu p, d
-DMTrTp, d-DMTrUp, d-DMTrI
p) (30 to 45 eq, manufactured by ABI, USA) and 0.45 ml of a 1H-tetrazole (150 eq) acetonitrile solution were added, and a condensation reaction was carried out for 20 minutes. After washing the reaction tube with acetonitrile, acetic anhydride: 2,6-lutidine: anhydrous TH was added to the reaction tube.
F = 1: 1: 8 (v / v), DMAP: anhydrous THF = 7:
93 (v / v) was added to cap the unreacted terminal 5'-hydroxyl group (acetylate the 5'-terminal hydroxyl group of the unreacted compound,
Follow the procedure below to make it easier to remove. It was washed with fluff and acetonitrile and then subjected to an oxidation reaction with sulfur in order to bring it into a basic and stable state in vivo as a pentavalent phosphoric acid derivative.

In order to perform sulfur oxidation treatment on all phosphoric acid groups, carbon disulfide containing 5% sulfur in the reaction tube after completion of each condensation reaction / capping operation: anhydrous pyridine: anhydrous triethylamine = 12: 12: 1 (v / v ) Was added and a sulfur oxidation operation was performed for 15 minutes. However, carbon disulfide: anhydrous pyridine = 1: 1 (v / v) before and after the sulfur oxidation so that the atmosphere is adjusted so that the sulfur oxidation proceeds sufficiently and does not affect the next condensation reaction. Therefore, it is necessary to perform sufficient cleaning to remove residual sulfur. Through the above sulfur oxidation treatment, the phosphite form was converted into a phosphorothioate form. Next, anhydrous acetonitrile: anhydrous pyridine = 1: 1
Wash thoroughly with (v / v), anhydrous acetonitrile, and anhydrous methylene chloride in this order. This series of operations is performed n-1 times (however, n
Represents the target chain length) and the target probe was synthesized by repeating it (Table 1). For cleaning,
After being dried by compressed air, the next step was carried out.

Deprotection of the obtained target chain length (n-mer)
When the product is treated with 28% ammonia water at 60 ° C. for 6 hours in an oil bath, the product is desorbed from CPG. At the same time, under these conditions, deprotection of the inter nucleotide site and the base site is performed. Next, TEA containing 20-50% acetonitrile to separate only those with DMTr groups attached.
This DMTr group-containing oligomer is treated with an 80% acetic acid aqueous solution for 15 minutes at room temperature to perform detritylation with an A (triethylamine ammonium acetate) elution solvent. The reaction product was subjected to ion exchange HPLC (TSK gel DEAE-2SW, manufactured by Tosoh Corporation) using a gradient solvent of an ammonium formate aqueous solution (pH 6.8, 0.38 to 0.36 M) containing 20% acetonitrile, and the reaction product was separated according to the dissociation number of the phosphoric acid moiety. Refined,
The desired antisense oligodeoxyphosphorothioate was synthesized.

The structure of the antisense oligodeoxyphosphorothioates thus obtained can be confirmed as follows. That is, an iodine-pyridine solution was added to an aqueous solution of each synthetic oligomer, and the mixture was allowed to stand at room temperature for 60 minutes to replace the phosphorothioate bond with a phosphate body, and then this oligomer was treated with snake venom phosphodiesterase and alkaline phosphotase (both sigma (Manufactured by the company). Since the degradation production ratio (dC: dG: T: dA) was the same as the degradation product of the target gene fragment in the HIV gene, it was judged that the target sequence was obtained.

As described above, according to the synthetic method of the present invention, a novel anti-HIV agent according to the present invention, that is, a novel anti-HIV agent according to the present invention, is prepared by a simple method using a commercially available reagent.
Sequences encoding major proteins in the gene of HIV (Human Immunodeficiency Virus; human acquired immunodeficiency virus; AIDS virus) (eg gag, pol, vif, vp
Antisense oligodeoxyribonucleoside phosphorothioates against the HIV gene, which are complementary to all or part of r, tat, rev, env, nef, LTR, etc.) can be synthesized.

[0042]

The present invention will be described in more detail with reference to the following examples. However, the following examples are shown only for the purpose of explanation, and are not intended to limit the scope of the present invention in any case.

(1) Manufacturing Example Manufacturing Example 1. HIV (Human Immunodeficiency Virus)
Synthesis of 20-mer antisense oligomer of rev region

From the viral genome of HIV, from 5550
The sequence up to 5569, that is, the base sequence is AUGGC AGGAA GAAGC GGAGA
Antisense oligodeoxyribonucleoside phosphorothioate [However, T, C, G, and A are thymidine, deoxycytidine, deoxyguanosine, and deoxyadenosine, respectively, and p _s represents a phosphate bond by phosphorothioic acid. ] Was synthesized as follows.

(I) Synthesis of Antisense Oligomer 20-mer Having Protective Groups at Base and Internucleotide Sites

[Procedure I] A long alkylamino CPG (controlled pore glass, manufactured by Nucleonic Co., Ltd. in England) (83 mg, 3 μmol) into which thymidine had been introduced was put into a glass reaction tube having an inner diameter of 20 mm equipped with a glass filter, and 3% Detritylation is carried out with a solution of trichloroacetic acid in methylene chloride (1 ml x 2) and washed with anhydrous methylene chloride (1 ml x 3). Further wash with acetonitrile (2 ml x 3).

[Operation II] After the CPG was dried under reduced pressure, a phosphor having a base suitable for the target sequence was used.
Place the midite unit (90 μmol, 30 eq) in the reaction tube and dry again under reduced pressure. 0.45 ml of 1H-tetrazole (450 μmol, 150 eq) in anhydrous acetonitrile is added thereto, and the condensation reaction is performed for 20 minutes.

[Procedure III] Anhydrous acetonitrile (2 ml
After washing with × 3), a mixed solvent consisting of acetic anhydride, 2,6-lutidine and anhydrous THF in the reaction tube [acetic anhydride: 2,6-lutidine: anhydrous THF = 1: 1: 8 (v / v) ] To 0.3 ml,
0.3 ml of a mixed solvent of dimethylaminopyridine (DMAP) and anhydrous THF [DMAP: anhydrous THF = 7: 93 (v / v)] was added to capping (acetylate 5′-terminal hydroxyl group of unreacted compound, The operation for making it easier to remove by performing the washing operation in 1). It was washed again with anhydrous acetonitrile (2 ml × 3).

[Operation IV] Subsequently, an oxidation reaction was carried out with sulfur. That is, after each condensation reaction and capping operation of the HIV antisense strand, 5% is left in the reaction tube.
Add a mixed solvent consisting of carbon disulfide solution containing sulfur, anhydrous pyridine and anhydrous triethylamine [carbon disulfide solution: anhydrous pyridine: anhydrous triethylamine = 12: 12: 1 (v / v)] and perform a sulfur oxidation operation for 15 minutes. went. However, a mixed solvent of carbon disulfide and pyridine before and after sulfur oxidation [carbon disulfide: pyridine = 1: 1
(V / v)] (2 ml × 4) to sufficiently wash the atmosphere to remove residual sulfur.

[Operation V] Further, the mixture was washed with a mixed solvent of anhydrous acetonitrile and anhydrous pyridine [anhydrous acetonitrile: anhydrous pyridine = 1: 1 (v / v)] (2.5 ml × 3),
Then, it is thoroughly washed with anhydrous acetonitrile (2 ml × 3) and anhydrous methylene chloride (2 ml × 3) in this order.

The series of [Operation I] to [Operation V] described above is repeated n-1 times (where n represents the target chain length, and therefore the target compound is a 20-mer, n =
20) By repeating, a target 20-mer (although it is still a protected oligomer at this stage) was synthesized. However, in the underlined part in [Operation II] above ,
-A phosphoramidite unit with a base suitable for
DOO phrase is, d-DMTrA ^bz p shown below, d-D
MTrC ^an p, d-DMTrG ^ibu p, d-DMTrT
represents either p.

[0052]

A series of operations [operation I] to [operation V]
Per one time, d-DMTrA ^bz p, d-DMTrC
^{an p, d-DMTrG ibu p} , only one of the d-DMTrTp is introduced in the operation III]. Phosphoramidite unit to be charged (ie, d-DMT
rA ^bz p, d-DMTrC ^an p, d-DMTrG
^ibu p or d-DMTrTp), in this case, the target HIV nucleotide sequence (ie, AUGGC).
AGAGA GAGAC GGAGA) must be added sequentially. Therefore, the phosphoramidite unit is replaced by ([A]; d-DMTr
A ^bz p, [C]; d-DMTrC ^an p, [G]; d-D
MTrG ^ibu p, [T]; abbreviated as d-DMTrTp) <T>, [C], [T], [C], [C], [G],
[C], [T], [T], [C], [T], [T],
[C], [C], [T], [G], [C], [C],
[A] and [T] were added in this order and reacted. Here, the first <T> has been input in [Operation I].

(Ii) Deprotection of antisense oligomer 20-mer Antisense oligomer 20 synthesized in experimental section (i)
The oligomer (however, still a protected oligomer at this stage) was taken out of the reaction tube, transferred to a vacuum vial, filled with 28% ammonia water, sealed, and treated at 60 ° C for 6 hours using an oil bath. Then, it is eliminated from CPG, and at the same time, the internucleotide site and the base part are deprotected. The reaction solution is filtered with a membrane filter and concentrated under reduced pressure.

A TEAA solution was added to the obtained residue to dissolve it, and reverse phase HPLC (ODS-80 TM CTR, manufactured by Tosoh Corporation) was performed using a TEAA elution solvent containing 20 to 50% CH ₃ CN.
Then, the colored portion of the DMTr group is collected. This is concentrated under reduced pressure, 80% aqueous acetic acid solution is added to the residue, and the mixture is treated for 15 minutes. The solution is concentrated under reduced pressure and the concentrate is dissolved in distilled water. The aqueous solution is washed with a small amount of ethyl acetate, and the aqueous layer is concentrated under reduced pressure. Furthermore, this concentrate was subjected to ion exchange HPLC (TS gel) using a gradient solvent of an ammonium formate aqueous solution (pH 6.8, 0.38 to 1.36 M) containing 20% acetonitrile.
Isolation and purification with DEAE2SW) and the target product, HIV (Human
Immunodeficiency Virus rev region antisense oligomer 20-mer (deprotected form) was obtained. Total yield of Production Example 1 42%.

The compound thus obtained was subjected to electrophoresis using a 20% polyacrylamide gel to confirm a single spot between the bromophenol blue (BPB) and the xylene cyanol (XC) label. There is.

Production Example 2. HIV (Human Immunodeficien
cy virus) tat region antisense oligomer 20-mer synthesis.

Of the HIV viral genome, from 5548
The sequence up to 5567, that is, the base sequence is CCAUU UUCAG AAUUG GGUGU
Antisense oligodeoxyribonucleoside phosphorothioate [However, T, C, G, and A are thymidine, deoxycytidine, deoxyguanosine, and deoxyadenosine, respectively, and p _s represents the phosphate bond by the phosphorothioic acid. ] Was synthesized as follows.

That is, {1} Production Example 1. In [Operation I] of (i) therein, a long alkyl CPG having adenine introduced is used instead of the long alkyl CPG having introduced thymidine, {2} and [Operation II]
The order of phosphoramidite units to be added in
Target HIV nucleotide sequence, CCAUU UUCAG
<A> in order to be complementary to AAUUGGGGUGU,
[C], [A], [C], [C], [C], [A],
[A], [T], [T], [C], [T], [G],
[A], [A], [A], [A], [T], [G],
[G] (here, the first <A> has already been input in [Operation I]. Also, [A], [C], [G],
[T] is a phosphoramidite unit d-
DMTrA ^bz p, d-DMTrC ^an p, d-DMTrG
^ibu p, d-DMTrTp (which is an abbreviation corresponding to d-DMTrTp) except that the above two points {1} and {2} are changed as described above. , An antisense oligomer of the target tat region of HIV,
Obtained in a yield of 45%.

(2) Structure and Purity Confirmation The purity and structure confirmation of the antisense oligodeoxynucleoside phosphorothioates against the HIV gene, which were synthesized according to the above-mentioned production examples, were carried out by the methods shown below, and all were intended compounds. It was confirmed.

(I) Gel electrophoresis 40% polyacrylamide solution (25 ml) in urea (21 g),
After adding TBE buffer (5 ml) to completely dissolve urea and degassing with an aspirator, 1.2% ammonium persulfate solution (4.15 ml) and N, N, N ', N'-tetramethylethylenediamine (TEMED) (25 μl) Then, quickly pour air into the glass plate with a gap of 0.5 mm so that air does not enter, and insert the slot maker into the upper part. The gel is left to stand overnight, the slot maker is removed, the buffer is washed off with a running buffer (TBE buffer diluted 10 times), and then 400 V pre-run is performed with a new running wave for 30 minutes. After completion of the pre-electrophoresis, the sample (60 μl of a mixture of the 0.3% to 0.5 OD oligomer aqueous solution and the same amount of 80% formaldehyde buffer) and the dye marker (BPB, XC) (10 μl) were placed in the slots.
After migrating at 50 V for 50 minutes and confirming the migration of the oligomer to the gel, perform main migration at 400 V. After the electrophoresis is completed, the gel is wrapped in a plastic wrap and directly printed on a photographic paper with a UV lamp (2537A) to take a photograph.

For both compounds, a single spot was obtained between the BPB and XC labels.

(Ii) Dissociation of phosphorothioate bond (Synthesis of oligodeoxyribonucleoside phosphate) The oligodeoxyribonucleoside phosphorothioate (1A ₂₆₀ unit) obtained in Production Example was dissolved in distilled water (50 μl), and then dissolved.
Add 026 M iodine-pyridine solution (150 μl),
The mixture was allowed to stand at room temperature for 1 hour to dissociate the phosphorothioate bond, and the phosphate body was substituted. Distilled water (1.5 ml) was added to it, and then diethyl ether (2 ml x 6)
After washing with, the aqueous layer is dried under reduced pressure. Dissolve the residue in distilled water and add 0.15M containing 8.75 to 20% acetonitrile.
Isolated and purified by reverse phase HPLC (Inertsil PREP-ODS) using a gradient solvent of -TEAA (pH 7.0) (33-45%).

(Iii) Enzymatic degradation The oligodeoxyribonucleoside phosphate (0.33A ₂₆₀ unit) obtained in (ii) above was dissolved in 0.01M-Trisacetate buffer (pH 8.8, 500 μM) and treated with snake venom phosphodiesterase (4.6 μg). Incubation was carried out at 37 ° C for 1 hour. This solution was deactivated by heat treatment at 90 ° C. for 1 hour, and then centrifuged. The supernatant was analyzed by reverse phase HPLC (Inertsil PREP-ODS). The composition ratio of the oligomers is the peak area at 260 nm and the molar extinction coefficient of nucleoside (e: dC7300, dG 11700, dT8800, dA 15400).
Quantitative calculation based on

The composition ratios of the oligomers of all the compounds were in good agreement with the theoretically calculated values, and the structures could be confirmed.

(3) Antiviral Test The antiviral action of the compound represented by the general formula [I] synthesized in the present invention, that is, the antisense oligodeoxyphosphorothioates against the HIV gene, is the test method of Pauwels et al., MTT assay. (J. Virol. Methods 20
309 (1988)).

The results of antiviral tests of representative compounds are shown in FIGS. Viruses used HIV cells used MT-4, that is, ODS19 (rev. 20mer) of the present invention is shown in FIG. 1, ODS'19 (tat, 20mer) is also shown in FIG. 2, and the activity of these compounds of the present invention is relatively high. The test results for AZT in a similar manner are shown in FIG.

As is apparent in FIGS. 1-3, AZT
Shows an anti-HIV effect in the range of 0.001 μM to 0.2 μM, while the compound of the present invention ODS19 (rev, 20me
r) and ODS'19 (tat, 20mer) are both 0.0
The anti-HIV effect is shown in the range of 002 μM to 0.6 μM or more. That is, the compound of the present invention ODS19 (re
v, 20mer) and ODS'19 (tat, 20mer) both show effects at concentrations lower than AZT, and their range is wide. Furthermore, ODS19 (rev, 20mer) and ODS
´19 (tat, 20mer) are all 500 × 10 ^-3 μ
Since it has no cytotoxic effect even at the concentration of M, it can be said to be an excellent drug having high effect and low cytotoxicity.

However, in the figure, ODS19 (rev, 20me
r) corresponds to the compound that can be synthesized according to Production Example 1 of the present invention, and ODS'19 (tat, 20mer) corresponds to the compound that is synthesized according to Production Example 2 of the present invention. To do.

The horizontal axis (Concentration) in the figure represents the concentration of the drug, and the vertical axis (Viability) represents the ratio of the number of viable MT-4 cells [the number of virus-infected cells when the drug concentration is 0]. The relative ratio (%) when 100%] is shown. Further, in the figure, the black filled columns indicate cells infected with the virus (HIV), and the shaded columns indicate cells not infected with the virus (HIV).

[0071]

INDUSTRIAL APPLICABILITY As described above, the present invention provides a compound having low toxicity and excellent antiviral activity. In particular, it is extremely effective as a therapeutic agent for AIDS.

[Brief description of drawings]

FIG. 1 is a test result diagram showing the anti-HIV effect and cytotoxicity of ODS19 (rev, 20mer).

FIG. 2 is a test result diagram showing the anti-HIV effect and cytotoxicity of ODS′19 (tat, 20mer).

FIG. 3 is a test result diagram showing the anti-HIV effect and cytotoxicity of AZT [comparative example].

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] October 29, 1991

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0028

[Correction method] Change

[Correction content]

Furthermore, the disclosure of Shibahara et al.
391), a part of the phosphorothioate derivative of the oligoribonucleoside derivative exhibits anti-HIV activity. However, this compound has a ribonucleoside as a unit partial structure, and the compound of 2 ' -Essentially quite different from the antisense oligodeoxyribonucleoside phosphorothioates which have deoxyribonucleosides as the partial unit structure. Also, the action is very different. For example, the disclosure of Shibahara et al.
128391), their compounds are 1-3μ.
From the concentration of M, it is said that it has an anti-HIV effect. However, the antisense oligodeoxyribonucleoside phosphorothioate compound against the HIV gene of the present invention has
At a concentration of 5.0 × 10 ⁻³ μM, a remarkable effect is already seen. Furthermore, no toxicity is observed even at a concentration of 5-6 μM or more. This indicates that the action mechanism, antiviral effect, and drug potential of each compound are completely different.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0058

[Correction method] Change

[Correction content]

Of the HIV viral genome, 5348
From the sequence from No. to 5367, that is, the base sequence, CCAUU UUCAG AAUUGGGU
Antisense oligodeoxyribonucleoside phosphorothioate for the sequence GU [However, T, C, G, and A are thymidine, deoxycytidine, deoxyguanosine, and deoxyadenosine, respectively, and p _S represents a phosphoric ester bond by the phosphorothioic acid. ] Was synthesized as follows.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C07H 21/04 ZNA B 7822-4C // C12N 15/00 8828-4B (72) Inventor Naoki Yamamoto Haramachi House, 3-11-17, Ebisu Minami, Shibuya-ku, Tokyo (72) Inventor Hideki Nakajima 4-3 Musamurayama City Gakuen, Tokyo 10-405 (72) Inventor Hiroshi Takaku 4-5 Shintakane, Funabashi City, Chiba Prefecture -9

Claims (22)

[Claims] 1. An oligodeoxyribonucleoside phosphorothioate represented by the following general formula [I]. [In the formula, m represents an integer of 1 to 50, B represents any _one of B ₂ to B _{m + 1} , and (B = B ₂ , B ₃ , B ₄ ... B
_m , B _{m + 1} ) B _{1 to} B _{m + 2} are the same or different from each other, and are hypoxanthine-9-yl and guanine-9-, respectively.
Ill, adenine-9-yl, cytosine-1-yl, uracil
-1-yl or thymine-1-yl. However, the case where all of B _{1 to} B _{m + 2} are the same is excluded. ] 2. HIV (Human Immunodeficiency Viru)
s; human acquired immunodeficiency virus; AIDS virus) gag, pol, vif, vpr, t, which encodes the major protein in the gene
Oligonucleotides complementary to all or part of the sequence such as at, rev, env, nef, LTR (antisense
Antisense oligodeoxyribonucleoside phosphorothioates for the HIV gene in which the phosphoric acid ester moiety of DNA) is chemically modified to form phosphorothioate. 3. The antisense oligodeoxyribonucleoside phosphorothioates for the HIV gene according to claim 1, wherein m is 1-18. 4. The antisense oligodeoxyribonucleoside phosphorothioates for the HIV gene according to claim 1, wherein the HIV gene is the rev region of the HIV gene. 5. The antisense oligodeoxyribonucleoside phosphorothioates for the HIV gene according to claim 1, wherein the HIV gene is the tat region of the HIV gene. 6. The HIV gene re according to claims 1 to 4.
A compound represented by the following sequence, which is an antisense oligodeoxyribonucleoside phosphorothioate for the v region. [However, d indicates that the unit structure of this sequence consists of deoxynucleoside units, and T; 2
′ -Deoxythymidine (or simply thymidine), C;
2′-deoxycytidine, G; 2′-deoxyguanosine, A; 2′-deoxyadenosine, and p _s is phosphorothio of the respective 5′- and 3′-position hydroxyl groups of T, C, G, and A. Phosphate bond linked by acid Is represented. ] 7. The HIV gene ta according to claims 1 to 5.
A compound represented by the following sequence, which is an antisense oligodeoxyribonucleoside phosphorothioate for the t region. [However, d indicates that the unit structure of this sequence consists of deoxynucleoside units, and T; 2
′ -Deoxythymidine (or simply thymidine), C;
2′-deoxycytidine, G; 2′-deoxyguanosine, A; 2′-deoxyadenosine, and p _s is phosphorothio of the respective 5′- and 3′-position hydroxyl groups of T, C, G, and A. Phosphate bond linked by acid Is represented. ] 8. HIV (Human Immunodeficiency Viru)
s; human acquired immunodeficiency virus; AIDS virus) gag, pol, vif, vpr, t, which encodes the major protein in the gene
Oligonucleotides complementary to all or part of the sequence such as at, rev, env, nef, LTR (antisense
The phosphoramidite method is used in the synthesis of antisense oligodeoxyribonucleoside phosphorothioates for the HIV gene, in which the phosphoric acid ester moiety of DNA) is chemically modified to phosphorothioate, and the general formula [I] ] The synthetic method of the antisense oligodeoxy ribonucleoside phosphorothioate shown by these. 9. Thymidine (or 2'deoxyadenosine, which has been detritylated with trichloroacetic acid)
2'deoxyguanosine, 2'deoxycytidine, 2 '
-Deoxyuridine, 2'-deoxyinosine) -introduced long alkylamino CPG (controlled pore glass) has d-D having a base suitable for the target sequence.
MTrA ^bz p, d-DMTrC ^an p, d-DMTrG
^ibu p, d-DMTrTp, d-DMTrIp, d-D
30 to 50 phosphoramidite units such as MTrUp
Equivalent amount and 1H-tetrazole 150 equivalent are added to cause a condensation reaction, followed by an oxidation reaction with sulfur to introduce sulfur into the phosphate ester part, a series of operations n-1 times (where n is the target chain). The method for synthesizing the antisense oligodeoxyribonucleoside phosphorothioates according to claims 1 to 7, wherein the synthetic method is repeated. 10. In the method for synthesizing antisense oligodeoxyribonucleoside phosphorothioates for the HIV gene shown in claim 9, sufficient washing is performed with a mixed solvent of carbon disulfide and anhydrous pyridine before and after sulfur oxidation, A method for synthesizing antisense oligodeoxyribonucleoside phosphorothioates, which comprises adjusting an atmosphere and removing residual sulfur. 11. An antiviral agent containing at least one of the antisense oligodeoxyribonucleoside phosphorothioates according to claim 1 as an active ingredient. 12. An antiretroviral agent comprising at least one of the antisense oligodeoxyribonucleoside phosphorothioates according to claim 1 as an active ingredient. 13. An anti-AIDS virus agent containing at least one of the antisense oligodeoxyribonucleoside phosphorothioates according to claim 1 as an active ingredient. 14. An experimental reagent and an experimental drug used in genetic engineering, which comprises at least one of the antisense oligodeoxyribonucleoside phosphorothioates according to claim 1 as an active ingredient. 15. An antiviral agent comprising the compound or derivative thereof according to claim 6 as an active ingredient. 16. An antiretroviral agent comprising the compound or derivative thereof according to claim 6 as an active ingredient. 17. An anti-AIDS virus agent containing the compound or its derivative according to claim 6 as an active ingredient. 18. An experimental reagent and an experimental drug used in genetic engineering and immunology, which comprise the compound or derivative thereof according to claim 6 as an active ingredient. 19. An antiviral agent comprising the compound or derivative thereof according to claim 7 as an active ingredient. 20. An antiretroviral agent comprising the compound or its derivative according to claim 7 as an active ingredient. 21. An anti-AIDS virus agent containing the compound or its derivative according to claim 7 as an active ingredient. 22. An experimental reagent and an experimental drug used in genetic engineering and immunology, which comprise the compound or derivative thereof according to claim 7 as an active ingredient.