JPH04506074A - α-anomeric oligonucleotide compounds that inhibit retroviral replication - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] The present invention provides for nucleotides having a non-natural α-anomeric configuration as opposed to the natural β-anomeric configuration. A compound consisting of an oligonucleotide or an oligodeoxynucleotide containing a deoxynucleotide sequence Regarding compounds.

Such compounds are disclosed in patent applications, in particular in French application No. 87 04 in the name of the applicant. No. 339 (PCT WO38104301) and Specification No. 88 12264 They form the country of origin, but especially if you find descriptions about their manufacturing methods there. Meso's application should be referred to.

More specifically, the present invention describes such species useful for inhibiting retrovirus replication. Oligonucleotide compounds of the same class, especially those that inhibit the replication of the HIV virus. Concerning oligonucleotides useful in.

According to their essential characteristics, the compounds according to the invention have a non-natural α-anomeric configuration. consisting of oligoribonucleotide or oligodeoxyribonucleotide sequences; The sequence consists of the binding site of the tRNA primer involved in viral RNA replication. The so-called TBS or PBS (tRNA binding site or primer binding site) Complementary to sequences contained in the sequence or in the viral RNA sequence upstream from the TBS sequence It is true.

Among these sequences upstream from the TBS or PBS sequence, the CAP sequence is unique. It is mentioned in

The α-oligonucleotide compounds according to the invention optionally contain intercalating agents or reactive agents. or an effector agent such as a photoactive chemical group.

In one embodiment of the compound according to the invention, the latter has the following formula: The groups B may be the same or different, each optionally modified to form a non-natural α represents a nucleobase attached to a glycosidic ring according to the anomeric configuration: Group B is a sequence marker contained in the TBS sequence or sequences upstream from the proviral RNA. considered sequential but complementary to the oligonucleotide bases; The groups X may be the same or different, and each So anion 01 Thio anion S 1 Alkyl group, alkoxy or aryl group Represents an xy group, an aminoalkyl group, an aminoalkoxy group or a thioalkyl group; R and R' may be the same or different, and each represents a hydrogen atom or a group -Y-Z. represent; Y is a linear or branched alkylene group -alk- or E -C-alk, C-NK-alk, -P-U-alk, -P-alkoo　o　 .

-alk-C-N-alk, -P-U-alk-N-C-alk and -P -Ll-a　I　k-C-N-a　l　kll　ro　II　I　MI OHOHO00H (U-0, N or S) or alternatively a group -Y'-0-Y-(Y 'or Y' has the meaning indicated for Y): E has the same meaning as X: J represents a hydrogen atom or a hydroxyl group; Z is a group corresponding to an effector agent; ; n is an integer including O; L represents an oxygen atom, a sulfur atom or an -NH- group.

In this formula 1, the following abbreviations for nucleosides are used, but two , where the (3°) and (5°) ends are shown.

Formula I represents a sequence of nucleotides that may be the same or different, where n is a molecule It simply indicates the number of nucleotides contained therein, where n is preferably 1 to 30, more preferably or a number between 1 and 20, depending on the size of the retroviral RNA target sequence. It should be noted that

Generally, for example, a TBS sequence has about 20 nucleotides.

Effector groups are compatible with effector agents, which are compounds known in the nucleic acid art. I guess. They are, for example, compounds that can be inserted into DNA or RNA structures. .

These intercalators are in particular acridine, flocoramarin daunomycin, 1.10 -phenanthroline, phenanthridinium, profilins, dipyride [1, 2-a:3-. 2--d] imidazole derivative, eributicin or eributidine consists of polycyclic compounds that grow in a planar arrangement, such as um and their derivatives.

These effector agents may be reactive chemical groups, such as chemically cleavable groups, and These groups can then react directly or indirectly to cleave the nucleotide chain.

Preferably, these reactive chemical groups may be activated, e.g. chemically or photochemically. Yes.

Activable reactive cleavage groups are e.g. -Ethylenediaminetetraacetic acid monodiethylenetriaminepentaacetic acid -Porphyrins -1,10-phenanthroline, busoralen and absorbs light in the near UV and visible region derivatives of compounds such as other aromatic groups.

These groups, which can be chemically activated in the presence of metal ions, oxygen and reducing agents, cleavage is induced in the nucleic acid sequences located adjacent to each other.

Group B consists of a nucleobase attached to the sugar moiety of the nucleotide by an α-anomeric configuration. Ru. These include thymine, adenine, cytosine, guanine or uracil. deer However, modified, especially I\logenated or azide, nucleobases, e.g. -bromouracil or 8-azidoadenine; amino acids such as 2-aminoadenine and aminoalkylene groups or azidophenylalkyl groups at N6, for example. derivatives thereof substituted with a lene group; in Oe, for example (ω-alkylene)-9- Group substituted with acridine group or 8-(ω-aminoalkyl)aminoadenine Using anine and its derivatives substituted with an acridine group in ω-NH2 Both are possible.

According to the invention, it is possible to introduce normal bases and modified bases. phase "Effector" bases capable of covalently bonding to the complementary β-strand may also be introduced. For this reason, 4th place Functionalization of C or T with aziridine groups in the two groups on G and A, respectively. This results in the formation of a covalent CH-CH2 bridge between complementary strands.

Thus, more specifically, group B is thymine, adenine, cytosine, guanine, 4 -azidocytosine, 4-azidothymine, 8-azidoadenine, uracil and 5- selected from bromouracil.

The group X preferably represents an oxo anion, but can also have other meanings. When group X represents an alkyl group, the latter is preferably a C-C7 lower alkyl group, For example, an ethyl, methyl or propyl group: when the group X represents an alkoxy group The latter is preferably a C-C lower alkoxy group, such as a methoxy or ethoxy group. When X represents an aminoalkyl or aminoalkoxy group, the latter may be monosubstituted. or a disubstituted aminoalkyl group or alternatively an amino group in the form of a quaternary ammonium salt. be. Under these conditions, the substituents are preferably lower alkyl as defined above. group, and with respect to the alkyl or alkoxy chain linking the amino group to the phosphorus, it is Preferably it is a straight chain or branched chain having 1 to 10 carbon atoms. alkyl or If the alkoxy group is substituted with a nitrogen-containing heterocycle, the latter is especially quaternized. It is a saturated 5- to 6-membered ring containing one optional nitrogen atom. Finally, group X is thioalkyl the latter is a lower thioalkyl group, i.e. a group containing 1 to 7 carbon atoms. It is preferable that

The group -alk- may be a straight-chain or branched alkylene group having 1 to 10 carbon atoms. is preferable.

In particular, starting from the 3- or 5'' terminal alcohol functionality of the alpha oligomer, The vector is attached to a functionalized Z of another nature in the glycoside portion of the oligomer. (CH2). It may also be introduced by a chain.

The following formula: or -〇-Z'-(C)I)-effector (Z)n 5゜ Starting from, depending on what is represented by Z', for example - a phosphate of the formula or methylphosphonate U-0, N or S −Ether of the following general formula 3゛ Or -MCH←CH2i Z 5゛ One ester of the following general formula or instead Carbamate of the following general formula -O-+GON))-+CH2→]-Z.

is obtained.

According to the invention, it is possible to obtain Compounds in racemic form or in the form of R or S optical isomers are also included.

According to the invention, the above-mentioned compounds of the D or L series are also included.

In an aspect of the invention, α-oligodeoxynucleotide e, is used. The latter is the case where X-0, R and R''-H, B are A, CST and and G: A compound in which L is an oxygen atom.

The subject of the invention is antiviral pharmaceutical compositions, especially antiviral pharmaceutical compositions containing said compounds as active substances. Treatment of viral symptoms caused by RNA viruses such as the AIDS virus HIVI It is also a composition useful for medical treatment.

Finally, the subject matter of the present invention relates to the preparation of antiviral pharmaceutical compositions particularly useful in the treatment of AIDS. 1. Uses of compounds according to the invention in relation to.

As mentioned above, the application of α-oligonucleotide compounds according to the present invention is retroviral. virus replication, especially the replication of the AIDS human immunodeficiency virus HIVI.

HTLV-111 virus (HTLV-human T leukemia virus) or AIDS-related Retrovirus involved in lymphadenopathy (LAV), also known as ARV (ARV). Rovirus was more recently named HIVI and is now considered the pathogen involved in AIDS. It is virtually recognized that

RNA viruses, especially the AIDS virus, also more commonly known as reverse transcriptase In fact, the main propagation mechanism is the RNA-dependent DNA polymerase enzyme ing. This enzyme transforms the viral RNA into a single-stranded DNA (cD Copy to NA). To do this, the 3-end of the RNA template to be copied must be We must find an oligonucleotide primer to pair with.

Reverse transcriptase is responsible for establishing the proviral state, initiating viral replication and It is necessary for both transformations.

In retroviruses, the primer is a cell-sourced tR present inside the pillion. It is NA. Especially when a virus infects a cell, reverse transcriptase (RT) The lysine 3-tRNA serves as an initiator for the DNA being synthesized. stand. The synthesized strand is guided by the primer groove. Then, it was synthesized in this way The DNA strand is in single-stranded form as a result of RNase activity coupled to RT, and complementary The target DNA strand is synthesized by the DNA-dependent polymerase activity of RT.

The structure of a retrovirus before and after integration into host cell DNA is as follows: Abbreviated as: a) Integration of the retrovirus into the DNA of the host cell □ Viral RN A reverse transcriptase 1 genome RNA 5' R-05-011-TBS-NO-code region-NC-Pur-AA-U 3-R3゜(RNA) Retroviral RNA contains sequences whose coding regions are essential for viral replication and expression. Single-stranded RNA flanked by columns. These sequences extend from the 5' to the 3-terminus. consisting of the following -R (short chain “repeat”): Open sequence that contains the CAP sequence necessary for translation to occur. A short sequence at the 5′ end that includes the beginning (similar nucleotides repeated at the 3′ end) ) -U: Unique sequence at the 5-end (consisting of about 80 nucleotides) -UU: two uracil nucleotides -TBS (“tRNA binding site 0)”: This acts as a primer during replication This is the site where possible tRNA binding (through its 3' end) occurs. Complementary? Two antiparallel bonds contain approximately 20 pb.

-NC: non-coding sequence One coding region: This is centrally located and it contains only a few genes. be.

these are: “gag” (relating to “group-specific antigen”), i.e. nucleoid when cleaved Concerning group antigens that code for polyproteins that give rise to proteins in the body. genes. One of these internal proteins has the antigenicity of the group.

“po1” (for “polymerase °”), which codes for reverse transcriptase; “env” codes for envelope glycoprotein (“envelope” ). One of these has the antigenicity of each virus serotype.

In the coding region, retrovirus-specific viral proteins (or proteins) There can also be genes encoding for protein segments (e.g. The onc gene is found in cancer retroviruses. This is a cancer retrovirus It's not about the AIDS virus; it doesn't make cells cancerous, but it doesn't. and destroy them).

-NC: non-coding area -Purnibulin base-rich sequences -AA: two adenine nucleotides -U: Unique sequence at the 3″ end (approximately 300 nucleotides) -R: D at the 3-end (short chain “repeat 1)” after action of 1 reverse transcriptase NA :Non-integrated viral DNA 3°TT-U -R-U5-AA-TBS-NC-code region-NCP y 　r　-T　T　-U　3-R-υ5-＾A　5' After the action of reverse transcriptase, the transcribed double-stranded DNA molecule becomes the corresponding genomic DNA. longer than In fact, Knee U3 sequence with 2 sequences added at the 2nd end is added at the 5' end - an assembler where the Us sequence is added at the 3' end Bully U-R-U5 is called “LTR” (long chain terminal repeat). There is one LTR for each.

1.Integrated viral DNA (provirus) double-stranded linear DNA becomes closed circular DNA. It is then opened again and integrated into the host cell's DNA. Built-in Wi rus DNA is called a "provirus." During integration, the transcribed DNA (and dinucleotides (TT,, AA) located at the ends of is removed.

Conjugation at the integration site requires proviral Direct repeat sequence DR in conjunction with DR.

All DRs are 4-6 bp in length. Their arrangement is different. exactly the same pro For viruses, different DRs should also be found in the host cell's DNA.

Therefore, the integration site of the virus is not specific, and the virus has several different integration sites. can be integrated into the host's genome at any point.

- Regarding viral DNA, there are two inverted repeat sequences IR at the ends of retroviruses. A sequence is called an “inverted repeat” when one is both opposite and complementary to the other. It is called.

Antisense oligonucleotides, i.e. proteins synthesized by liposomes It is complementary to the part of the virus' messenger RNA that directly codes for N. Attempts have been made to combat the replication of the A upper segment AIDS virus.

α-oligonucleotides are heterozygous with their complementary β-oligonucleotides. The fact that the latter is not a substrate for RNase H enzymes ・Inhibition of protein expression by ribonucleotides is caused by liposomes inhibiting mRNA. It is recognized that this could probably be done by preventing the virus from being translated into proteins. Ta.

However, this understood experiment was inconclusive.

Conversely, the initiation sequence of viral RNA, especially the primer tRN to proviral RNA, This book aims to use oligonucleotides complementary to the A-bond initiation site. The inventive approach proved effective.

For this reason, the subject of the present invention is that the sequence of the PB of the proviral RNA of the HIV virus is It is an α-oligonucleotide that is complementary to positions 182-199 of the S sequence. Immediately α-oligodeoxynucleotide d (ACCGCGGGCTTGTCCCTG)” or more generally the formula% ( For ribodeoxyribonucleotides, XT or α-oligoribonucleotides. case X-υ) or complementary sequences by interchanging X and A1 on the one hand and C and G on the other It is.

Other features and advantages of the invention will become apparent from the examples below.

Figure 1 shows α-oligonucleotide activity by examining reverse transcriptase activity (pl). Do d (ACCGCGGGCTTGTCCCTG) 3° (α-5° Figure 2 shows the inhibition of virus production in MT4 cells with oligo-PBS''.

Example 1: Cytotoxicity of α-oligonucleotides Typical of oligonucleotides used Regardless of the type, these oligonucleotides end up being more or less rapidly separated by cellular enzymes. be understood. α-oligonucleotides and their α-nucleosides and α-nucleosides It is shown below that cleotide catabolites are not cytotoxic.

Therefore, a priori, it is possible to Nonsensical oligonucleotide a-d (CCTCTCGTTCTTTAC) , a-oligo Tat has a CD5o of 250 ag/at or more. Similarly, α− Oligothymidylate, α-(dT) (2≦n≦12) was also used for similar cell lines. It was found to be non-cytotoxic.

Finally, the α-nucleases that can result from slow hydrolysis of oligonucleotides Osides and α-nucleotides are also present in various cell lines (He1a, Vero, It has no cytotoxicity to MT-4).

Cytotoxicity of α-oligodeoxyribonucleotides and components (nucleoside and nucleotides) α-dT5-phosphate (α-dTMP)) 1250α-d (CCπmAG)> 250 α-d(TT))100 α-d('m1')>100 a - d (TrTTTr) > 100 α-diπ Nukie TT) > to.

α−d (π汀汀TETE汀)　＞100 1. 2 details required to produce microscopically detectable changes in normal cell morphology. The evaluation required to cause 50% cell death is M after infection with the HIV virus. Based on studies of the cytopathic effects of the HIVI virus on T4 cell lines, The formation of fused cells was observed 4-6 days after infection, followed by the production of virus particles, This is followed by cell death.

Destruction of 14928 cells, which are essential for immune defense, is the main cause of immunodeficiency characteristic of HIV infection. This is the key cause. This virus is known to kill cells by multiplying there. and where it is extracted from them, it damages cell membranes. HI V is also probably indirectly transmitted through the gp120 protein present on the membrane of infected cells. He must have killed 14,928 pitches. In fact, 14928 spheres contain gp120 protein The healthy 14928 sphere has a surface molecule CD4 receptor that binds to the protein. It binds to protein and fuses with infected cells. The resulting cell aggregate is a non-viable “fusion” "cell" and all the healthy cells it is composed of die along with the infected cells.H IV can also induce a normal immune response against infected lymphocytes. of antibodies With or without help, cytotoxic defense cells have viral proteins on their surface Destroy infected cells. Finally, the gp120 protein is present in solution in the blood of infected individuals. Free and sometimes circulating, it binds to the CD4 receptor on healthy cells and induces sensitization. promotes infection and induces a destructive response in uninfected cells.

A fused cell is a structure consisting of several cell nuclei surrounded by a single cell membrane. , they are a sign of infection by HIV in cell culture, and the fused cells are Infected cells that synthesize the 22120 molecule and transport it on their surface carry the CD4 molecule. Occurs when fused with healthy cells that have

b) Compound to be tested α-d” ACCGCGGGCTTGTCCCTG O, 54U sterile double distilled water Stock solution containing IB/sl in stock solution stored at -80°C.

c) MT4 exam Combine MT4 cells (in 96-well microplates) 2 days after final passage. Serial dilution of compound at 3X10 cells/100 μg for 1 hour at 37°C. Brain incubate.

Infection was performed by adding 100 μg of a 10-4 dilution of HIV virus. performed with Rhunil (the dilution of the virus is such that it induces the formation of fused cells over a period of 4 days). ).

After 1 hour incubation at 37°C, infected MT4 cells were incubated with different selected dilutions. at a concentration of 3 x 105 cells/Akebono in 24-well microplates in the presence of Wash 5 times before maintaining culture.

Every 3 or 4 days, cells were diluted 3 or 4 times and further incubated with R in the presence of α-oligo PBS. PMI medium, 10% FCS.

Adjust to a concentration of 3 x 105 cells 11 with 1% PSN, 1% Gluta. .

Every 2 or 3 days, the appearance of fused cells is observed in culture.

d) Reverse transcriptase assay Virus production in MT4 cells was monitored by reverse transcriptase activity ( This is done by examining Figure 1, RT). Briefly, enzyme activity is determined by synthetic primers and Tested in vitro using substrates radiolabeled with 3H and 3H. The amount of radioactive isotope labeled substance displayed by count/win is the amount of reverse transcriptase. activity, which itself is proportional to the amount of virus produced in infected MT4 cells. I'm giving an example.

2. Results D3 D4 D5 D7 DIODll D12 014 018100μg/ it −−−−−−−−−−−(+) −(−) (+)++ → 25μg/ if (+) - ('') - (+) - ('') (+) (+) → + ← + → + +++KKlOμg/ml −(+) (”) (”) + ← ＋＋＋＋ ←+ ＋→→＋＋＋−＋→＋＋KK5μg7111　(+)(+)　＋＋＋ ++　10＋＋＋　→ ＋＋＋＋＋＋→ ＋＋ ＋＋＋＋＋＋＋＋1μg/ ml (+) (+) (+) (+) + (+) ” ++ → ++ ++ ++ ++ ← ++ ++ → ÷+100μg/ml (+) (+) (+ )” + + + + + + + + + + + + + + + + + + + K 10+50μg/ml (+) (+) + ++ + ++ + ++ ++ + + ++ ++ ++ ++ ++++ → K111vl (+) (+) + ＋ ＋＋＋＋ ＋＋＋＋ ＋＋ 10＋ ＋＋＋＋ ＋＋＋＋ 4＋4−＋K K annotation d - No fused cells +)・Fusion cell formation +: Appearance of fused cells →・Fused cells K・Cell death 3. Conclusion Inhibition of the cytopathogenic effect of HIV virus in the MT4 system by α-oligo PBS In toxicity evaluation, the delay for formation of fused cells is 10-100 μg/sl. a concentration of 100 μg/ml extended this delay at the end of the 12th day postinfection. can be passed.

At the same time, in monitoring virus production by infected MT4 cells, the α- No virus production was obtained in the presence of HIV virus regardless of the dose of ``RigoPBS''. This indicates that the peak virus production peak is also delayed. (Figure 1).

In summary, these results indicate that α-oligo PBS has antiviral properties against the HIV1 virus. Infected with HIVI BRU, the presence of oligonucleotides was shown to have a viral effect. The production of virus particles by CEM cells cultured in the presence of cps).

Oligonucleotide “α-Tat” is α-oligonuclease 5゛ Corresponds to leotide d GTAAAAGTCTTAACCCAC3°. That's E It is complementary to the sequence of the Tat gene of the virus.

Oligonucleotide "α-random ° is any α-, ? Reconucle, t + )’d”’ACTGACTGACT corresponds to “ACTGAC” Nemo.

Reverse transcriptase results are lower when compared to random control in count/win As shown below.

Oligonucleotide “α-PBS” was used at concentrations of 100 μg/ml and 50 μg/ml. degree of inhibition of virus production.

At a concentration of 6.25 μg intestine 1, the oligonucleotide “α-Tat” was released for 11 days. No evidence of virus production. The same applies to random in 3.1.

These results can be summarized as follows: 100 μg/g of oligonucleotide “α-PBS” ■ Antiviral effect against HIV1 virus at a concentration of 1 and 50μgeml However, oligonucleotide “α-Tat” and oligonucleotide “α -Random” has no antiviral effect at all.

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Claims (10)

[Claims] 1. An oligonucleotide compound useful in inhibiting retrovirus replication. So, Oligoribonucleotides or oligodeoxyribonucleotides with non-natural α-anomeric configuration Consisting of an octide sequence, the sequence is the TBS sequence or the sequence upstream from the viral RNA. , specifically complementary to the start sequence of the viral RNA contained in the CAP sequence. Featured oligonucleotide compounds. 2. The oligonucleotide of claim 1, further covalently linked to an effector agent. Otide compound. 3. The compound has the following formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [I] [In the above formula: The groups B may be the same or different, each optionally modified to form a non-natural α represents a nucleobase linked to a glycosidic ring according to the anomeric configuration; Group B is the target origin of sequences in the TBS sequence or sequences upstream from the viral RNA. considered sequential but complementary to the nucleotide bases; The groups X may be the same or different, each containing an oxo anion O, a thio anion OnS■, alkyl group, alkoxy or aryloxy group, aminoalkyl represents a group, an aminoalkoxy group or a thioalkyl group; R and R' are the same or may be different, each representing a hydrogen atom or a group -Y-Z; Y is a straight chain or branched alkylene group -alk- or ▲a numerical formula, chemical formula, table, etc. ▼、▲There are mathematical formulas, chemical formulas, tables, etc.▼、▲There are mathematical formulas, chemical formulas, tables, etc. ▼、▲There are mathematical formulas, chemical formulas, tables, etc.▼▲There are mathematical formulas, chemical formulas, tables, etc.▼▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ and ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (U =O, N or S) or alternatively a group -Y''-O-Y'(Y'' or Y′ has the meaning indicated for Y; E has the same meaning as X; J represents a hydrogen atom or a hydroxyl group; Z is a group corresponding to an effector agent. ; n is an integer including 0; according to claim 1 or 2, wherein L represents an oxygen atom, a sulfur atom, or an -NH- group. oligonucleotide compounds. 4. Useful for inhibiting the replication of the AIDS virus HIV and whose sequence is similar to that of the viral protein. Complementary to sites 182-199 of the viral RNA, i.e. the α-oligonucleo ACCGCGGGGCXXGXCCCXG (X=T or U) or on the other hand A, the complementary sequence thereof with interchanging C and G on the other hand, claims 1- 3. The compound according to any one of 3. 5. In the compound of formula I, X=O; J, R, R'=H; B is A, C, T and G. selected from; L is an oxygen atom α-oligodeoxynucleotide; 5. A compound according to any one of claims 1 to 4. 6. Contains a compound according to any one of claims 1 to 5 as active substance An antiviral pharmaceutical composition characterized by: 7. A pharmaceutical composition useful in the treatment of AIDS, comprising as the active substance the alpha anomer configuration d5'(ACCGCGGGCTTGTCCCTG)3' oligonucleotide A pharmaceutical composition comprising: 8. Useful for the treatment of viral infections caused by RNA viruses, the active substance and Preparation of a pharmaceutical composition containing the compound according to any one of claims 1 to 5. 6. Use of a compound according to any one of claims 1 to 5 for the preparation of a compound. 9. 9. The use according to claim 8, for the manufacture of a pharmaceutical composition useful in the treatment of AIDS. 10. α-Oligonucleotide for the production of a pharmaceutical composition useful for the treatment of AIDS Usage of d5'(ACCGCGGGCTTGTCCCTG)3'.