WO1996019240A1

WO1996019240A1 - Oligonucleotide-dendrimer conjugates

Info

Publication number: WO1996019240A1
Application number: PCT/EP1995/004933
Authority: WO
Inventors: Robert Häner; Konstantinos Skobridis
Original assignee: Novartis Ag
Priority date: 1994-12-21
Filing date: 1995-12-13
Publication date: 1996-06-27
Also published as: EP0790837A1; IL116456A0; JPH10510822A; CA2206915A1; FI972548A; NO972868L; NO972868D0; HUT77521A; AU4344696A; FI972548A0

Abstract

Oligonucleotide-dendrimer conjugates, wherein dendrimer is the monovalent residue of a dendrimer of the first to tenth generation and oligonucleotide is a natural, modified or synthetic sequence which is composed of natural, modified or synthetic deoxynucleosides or peptide nucleic acid building blocks which are linked via internucleotide bridges and which encompasses a region which is complementary, preferably completely complementary, to a target nucleic acid (target RNA or target DNA), with the dendrimer being bonded to an internucleotide bridge, a nucleic acid base or a sugar of the oligonucleotide, and the salts thereof.

Description

Oligonucleotide-dendrimer conjugates

The present invention relates to oligonucleotide-dendrimer conjugates, to a process for preparing these conjugates, to the use of these conjugates and to pharmaceutical preparations which comprise these conjugates.

Oligonucleotides have attracted widespread interest as antiviral active ingredients or as a result of their ability to interact with nucleic acids (antisense oligonucleotides) and the biological activity associated therewith. A substantial problem in this context is that they are only taken up in small quantities by cells. Hitherto, efforts have been made to increase the cellular uptake of antisense oligonucleotides by covalently linking the oligonucleotides to, or substituting them by, various chemical groups. Examples are conjugates with cationic compounds such as poly-(L)-lysine, poly-(L)-ornithine or aminoalkanes, conjugates with lipophilic compounds such as cholesterol, alkanes, phospholipids or aromatic substances, or conjugates with other groups such as polyethylene glycol. These groups are attached at any position in the oligonucleotide, for example at the 3'- or 5' end, at any base, at a sugar or at another site in the backbone. In addition, liposome and nanoparticle formulations have been used to increase the cellular uptake of oligonucleotides. A further possibility is to admix cationic lipids with the oligonucleotides.

It has now been found that oligonucleotide-dendrimer conjugates have an improved cellular uptake, a high resistance to nucleases and advantageous pharmacokinetics. This is of value for antisense oligonucleotide or antigen oligonucleotide applications, in the

transfection of cells with foreign hereditary material and in medical diagnosis. By means of linking the oligonucleotides to dendrimers, groups having an extremely high degree of lipophilia or an ionic character can be introduced in a simple manner. The influence of the dendrimer moiety on the conjugate as a whole can readily be controlled by the size or number of the end groups.

The invention relates to oligonucleotide-dendrimer conjugates, where dendrimer is the monovalent residue of a dendrimer of the first to tenth generation and oligonucleotide is a natural, modified or synthetic sequence which is composed of natural, modified or synthetic deoxynucleosides or peptide nucleic acid building blocks which are linked via internucleotide bridges and which encompasses a region which is complementary, preferably completely complementary, to a target nucleic acid (target RNA or target DNA), with the dendrimer being directly bonded, or bonded via a bridging group B, to an internucleotide bridge, a nucleic acid base or a sugar of the oligonucleotide, and the physiologically tolerated salts thereof.

Preferably, the target nucleic acid is a target ribonucleic acid (target RNA). Accordingly, polyribonucleic acids (RNA) can be present. These are preferably mRNA (messenger RNA), pre-mRNA (precursor mRNA) and viral RNA. The RNA has sufficient building blocks to ensure that a complex (double strand) can be formed with the oligonucleotide.

The oligonucleotide can be partially or completely constructed of natural DNA building blocks which are complementary to the target RNA or completely constructed of unnatural, synthetic nucleotides which are likewise complementary to the target RNA, with partially denoting that natural DNA building blocks which are complementary to the target RNA are replaced in the oligonucleotide sequence by unnatural, synthetic nucleotides which are likewise complementary. Synthetic building blocks comprise the modifications of natural building blocks in the nucleic acid base, the furanose ring and/or the bridging groups of the oligonucleotides. In general, synthetic building blocks are employed in order to strengthen complex binding in duplex structures and/or to increase the stability of the oligonucleotides towards degradation which is caused, for example, by nucleases. A wide variety of modified nucleosides have become known which can be used, within the sphere of "antisense technology", for synthesizing or modifying complementary oligonucleotides and such nucleotides will not, therefore, be dealt with in more detail here (cf., for example,

E. Uhlmann et al., Chemical Reviews, Volume 90, Number 4, pages 543 to 584 (1990)).

Possible modifications are modifications in the nucleic acid base moiety (for example substitutions or omission of substituents), in the nucleotide-bridging group (for example modification of the phosphoric ester group or its replacement by other bridging groups) and in the furanose ring (for example substitutions on the 2'-hydroxyl group, replacement of the furanose O atom, replacement of the furanose ring by monocarbocyclic or bicarbocyclic rings, or replacement of the furanose ring by open-chain structures). The choice and the order of the building blocks in the sequence of the oligonucleotide is determined by the necessity of forming a duplex with a target RNA. The nature and the site of linkage to the dendrimer can also affect the choice and the order of the building blocks.

The number of building blocks in the oligonucleotide is designed so that hybridization is achieved with the target RNA. The oligonucleotides can, for example, contain from 5 to 100, preferably from 5 to 50, particularly preferably from 8 to 30 and, very particularly, from 10 to 25, building blocks. The nucleotide building blocks which pair with the target RNA are preferably arranged in the central sequences of the oligonucleotide, for example between the fourth building blocks from each end of the sequence, or between the third from each end, or between the second from each end or between the last building blocks at each end of the sequence. For example, in an oligonucleotide having 20 building blocks, building blocks which pair are preferably located in the region from the fourth to the seventeenth building block.

The oligonucleotides are preferably constructed from nucleosides of the purine series and the pyrimidine series. They are particularly preferably constructed from 2'-deoxy-2-amino-adenosine, 2'-deoxy-5-methylcytidine, 2'-deoxyadenosine, 2'-deoxycytidine, 2'-deoxyguanosine and thymidine. Very particular preference is given to 2'-deoxyadenosine (A), 2'-deoxycytidine (C), 2'-deoxyguanosine (G) and thymidine (T). Modified building blocks are preferably derived from natural nucleosides of the purine series and the pyrimidine series, particularly preferably from adenosine, cytidine, guanosine, 2-aminoadenosine, 5-methylcytosine, uridine and the previously mentioned deoxy derivatives. The nucleosides can also be 2'-modified ribonucleosides.

In a very particularly preferred embodiment of the invention, the oligonucleotide which is complementary to a target RNA is constructed from natural deoxynucleosides, particularly preferably from the group 2'-deoxyadenosine (A), 2'-deoxycytidine (C), 2'-deoxyguanosine (G), and 2'-thymidine (T), or from complementary, unnatural synthetic building blocks.

Within the scope of the invention, those modified nucleosides are particularly preferred which increase the stability of the oligonucleotide towards nucleases.

The oligonucleotide can also consist of sequences of peptide nucleic acids (PNA), with the dendrimer preferably being bonded to the amino end or the carboxyl end. The same preferences apply to the structure of the PNA sequence as to that of the oligonucleotides. Examples of PNA's can be found in Science 254:1497-1500 (1991).

Within the scope of the present invention, the dendrimer contains an initiator core having at least three valencies, with one valency being used for the bond to the oligonucleotide, and at least two monovalent branches which are bonded to the initiator core, with each branch consisting of at least one branching point having at least three valencies. The dendrimer itself, and also its building blocks, are physiologically tolerated or harmless.

The initiator core and the branching point can, independently of each other, be a single atom, a cyclic or heterocyclic, saturated or unsaturated aliphatic radical having from three to twelve, preferably from five to eight, ring members, a bicyclic or heterobicyclic aliphatic radical having from five to twelve ring members or a mononuclear or polynuclear aromatic or heteroaromatic radical having from six to eighteen, preferably from six to fourteen, in particular from six to twelve, ring members, where the ring members are carbon atoms which are, where appropriate, interrupted by from one to three heteroatoms which are selected from the group consisting of nitrogen, oxygen and sulfur. A preferred embodiment of the present invention is represented by those compounds in which the initiator core and the branching point are, independently of each other, a single atom, a cyclic or heterocyclic, saturated or unsaturated aliphatic radical or a mononuclear or polynuclear aromatic or heteroaromatic radical. Compounds are particularly preferred in which the initiator core and the branching point are, independently of each other, a cyclic or heterocyclic, saturated or unsaturated aliphatic radical or a mononuclear or polynuclear aromatic or heteroaromatic radical.

All atoms having at least three valencies are possible single atoms for the initiator core or the branching point; those which are preferred are carbon, nitrogen, silicon or phosphorus, in particular carbon.

In one embodiment of the present invention, the cyclic or heterocyclic aliphatic radical as the meaning of the initiator core and of the branching point is derived from compounds which are selected from the group consisting of cycloalkanes and cycloalkenes which preferably have from 5 to 7 ring carbon atoms. In another embodiment of the present invention, the bicyclic or heterobicyclic aliphatic radical as the meaning of the initiator core and of the branching point is derived from compounds which are selected from the group consisting of bicycloalkanes and

bicycloalkenes which preferably have from 5 to 7 ring carbon atoms.

In a further embodiment of the present invention, the aromatic or heteroaromatic radical as the meaning of the initiator core and the branching point is derived from compounds which are selected from the group consisting of benzene, naphthalene, anthracene,

phenanthrene, naphthacene, indene, fluorene, indacene, biphenylene, triphenylene, pyrrole, indole, carbazole, furan, benzofuran, dibenzofuran, thiophene, benzothiophene, dibenzothiophene, pyridine, quinoline, isoquinoline, acridine, phenanthridine, pyridazine, cinnoline, phthalazine, pyrimidine, quinazoline, pyrazine, quinoxaline, phenazine, pteridine, purine, pyrazole, indazole, imidazole, benzimidazole, isoxazole, oxazole, furazan, thianthrene, xanthene, triazine, phenanthroline, benzoxazole and benzothiazole.

Preference is given to compounds which are selected from the group consisting of benzene, naphthalene, fluorene, biphenylene, pyrrole, carbazole, furan, dibenzofuran, thiophene, dibenzothiophene, pyridine, acridine, pyrazine, phenazine, furazan, thianthrene and xanthene, with particular preference being given to compounds which are selected from the group consisting of benzene, pyrrole, furan, thiophene, pyridine, pyrazine and furazan, with benzene being in particular preferred.

The valencies of the initiator core are occupied by the bond to the oligonucleotide and those to the branches. The valencies of the first generation branching point are occupied by the bond to the initiator core and those to the second generation branching point. The valencies of the branching points of later generations are occupied by the bond to the branching point of the preceding generation and those to the branching points of the subsequent generation. If, in all these cases, valencies are still free, these free valencies are then occupied, independently of each other, by hydrogen or a substituent selected from the group consisting of halogen, C₁-C₆alkyl, C₁-C₆hydroxyalkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, C₆-C₁₂aryl, C₆-C₁₂Ar-C₁-C₆alkyl, -CN and -NO₂ .

One of the valencies of the branching points at the periphery of the branch is occupied by the bond to the branching point of the preceding generation. The free valencies are, independently of each other, occupied by monovalent end groups. In one embodiment of the present invention, end groups are understood to mean groups having a high degree of lipophilia or an ionic character.

It has been found to be advantageous to select an end group from the group consisting of hydrogen, C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, C₆-C₁₀aryl, C₇-C₁₇aralkyl, hydroxyl, amino, nitro and an organic radical which is derived from a carboxylic acid derivative. Preference is given to an end group which is selected from the group consisting of hydrogen and C₁-C₆alkoxy. End groups which are in particular preferred are hydrogen and -OCH₃.

The invention also relates to oligonucleotide-dendrimer conjugates in which the initiator core in the dendrimer residue is linked to the first generation branching point via a bivalent bridging group Z.

The present invention also relates to oligonucleotide-dendrimer conjugates in which, in the dendrimer residue, the branching points of consecutive generations are linked via a bivalent bridging group Z.

Within the scope of the present invention, the bivalent bridging group Z is advantageously selected from the group consisting of C₁-C₄alkylene; C₁-C₄alkylene which is interrupted once or more than once by a representative selected from the group consisting of oxygen atom, sulfur atom, nitrogen atom, carbonyl radical, thio radical, sulfoxide radical and a radical of the formula -Si(OR')(OR")-O-, in which R' and R" are, independently of each other, hydrogen or C₁-C₆alkyl; C₂-C₄alkenylene; C₂-C₄alkenylene which is interrupted once or more than once by a representative selected from the group consisting of oxygen atom, sulfur atom, nitrogen atom, carbonyl radical, thio radical, sulfoxide radical, and a radical of the formula -Si(OR')(OR")-O-, in which R' and R" are, independently of each other, hydrogen or C_1-C₆alkyl; C₂-C₄alkynylene; C₂-C₄alkynylene which is interrupted once or more than once by a representative selected from the group consisting of oxygen atom, sulfur atom, nitrogen atom, carbonyl radical, thio radical, sulfoxide radical, and a radical of the formula -Si(OR')(OR")-O-, in which R' and R" are, independently of each other, hydrogen or C_1-C₆alkyl; aryl; aralkyl and alkyloxy. Particular preference is given to the bivalent bridging group Z which is selected from the group consisting of C₁-C₄alkylene and C₁-C₄alkylene which is interrupted once or more than once by a representative selected from the group consisting of oxygen atom, sulfur atom, nitrogen atom, carbonyl radical, thio radical, sulfoxide radical, and a radical of the formula -Si(OR')(OR")-O-, in which R' and R" are, independently of each other, hydrogen or C₁-C₆alkyl. Preference is in particular given to the bivalent bridging group Z being C₁-C₄alkylene or C₁-C₄alkylene which is interrupted once by an oxygen atom. The most preferred bivalent bridging group Z is -OCH₂-.

Within the scope of the present invention, the number of the generations indicates the number of consecutive branching points. Those compounds are preferred in which dendrimer denotes the monovalent residue of a dendrimer of the first to seventh, particularly preferably of the first to fifth, in particular of the first to fourth, very particularly preferably of the first to third, generation.

The novel residue of a dendrimer can be constructed in accordance with the formula I,

in which x₁ is the initiator core, (I₁) and (l₂) are each a bridging group Z, x₂ and x₃ are each a branching point and E is an end group. As already indicated above, the initiator core and the branching points may have more than three valencies, the initiator core may or may not be linked to the first generation branching point via a bivalent bridging group Z, the branching points of consecutive generations may or may not be linked via a bivalent bridging group Z, and the generation number determines how often branching points succeed each other and, correspondingly, how frequently bridging groups Z may be present. According to the invention, the residue of the dendrimer of the formula (I) may be branched to a greater extent than indicated in formula (I), i.e. the radical E in formula (I) represents additional bifurcations which are constructed from additional initiator cores x₄, Xs etc., which, where appropriate, are bridged via additional radicals (l₃), (l₄) etc., and which end in an end group, for example H or -OCH₃ .

Those dendrimer residues of the formula I are preferred in which x₁, x₂ and x₃ is benzene, (I₁) and (l₂) are -O-CH₂-, and E is H or -OCH₃ .

The dendrimer is, for example, bonded to N, S or O atoms in the 3' or 5' end groups of the oligonucleotide sequence. However, it can also be bonded to C, N or O atoms of nucleic acid bases in or at the end of the sequence, to 2' positions in the furanose ring, to O, S or N atoms in or at the end of the sequence, or to O, S or N atoms of the nucleotide-bridging group in the sequence. The nature of the bond depends on the dendrimer and on the nature of its functional groups. The bond to the oligonucleotide can be ionic or, preferably, covalent. The dendrimer can also be bonded to the 6' carbon atom of a carbocyclic nucleotide analogue.

It has been found to be particularly advantageous for the dendrimer to be bonded via a bridging group B. Within the scope of the present invention, the bridging group B is a group of the formula II

-Xp-[A-X]_n-A'_m- (II) in which

X and X' are, independently of each other, a radical which is unsubstituted or is substituted by C₁-C₁₀alkoxy, preferably C₁-C₆alkoxy. F, Cl, Br, -CN, C₁-C₁₀alkyl, preferably C₁-C₆alkyl, aryl, hydroxy-C₁-C₁₀alkyl, preferably hydroxy-C₁-C₆alkyl, amino-C₁-C₁₀alkyl, preferably amino-C₁-C₆alkyl, OH, NR₁₂or -NO₂ and which is selected from the group consisting of C₁-C₂₀alkylene, C₂-C₁₂alkenylene, C₂-C₁₂alkynylene, C₃-C₈cycloalkylene, C₆-C₁₂arylene and C₆-C₁₂aralkylene,

A and A' are, independently of each other, -O-, -S-, -S-S-, -NR₁₂-CO-NR₁₂-, -NR₁₂-CS-NR₁₂-,

-NR₁₂-, -NR₁₂-C(O)-O-, -C(O)O-, -C(O)S-, -C(O)NR₁₂-, -C(S)S-, -C(S)O-, -C(S)NR₁₂-,

-SO₂NR₁₂-, -SO₂-, -P(O)(OH)O-, -OP(O)(OH)O-, -P(S)(SH)O-, -OP(S)(SH)O-, -P(S)(OH)O-, -OP(S)(OH)O-, -P(O)(OH)-NR₁₂-, -OP(O)(OH)-NR₁₂-, -P(S)(SH)-NR₁₂-, -OP(S)(SH)-NR₁₂-,

-P(S)(OH)-NR₁₂- or -OP(S)(OH)-NR₁₂-,

R₁₂ is H or C₁-C₁₀alkyl, preferably H or C₁-C₆alkyl;

n is a number from 1 to 50, preferably from 1 to 20, particularly preferably from 1 to 5, in particular from 1 to 3, where, when more than one (A-X') unit is present, the meanings of A and X' in the individual units are identical or different, and

m and p are, independently of each other, 0 or 1 .

Some examples of possible meanings of X and X' are methylene, ethylene, 1 ,2- or 1 ,3-propylene, 1 ,2-, 1 ,3- or 1 ,4-butylene, 1 ,2-, 1 ,3-, 1 ,4- or 1 ,5-pentylene, 1 ,2-, 1 ,3-, 1 ,4-, 1 ,5- or 1 ,6-hexylene, 1 ,2-, 1 ,3-, 1 ,4-, 1 ,5-, 1 ,6- or 1 ,7-heptylene, 1 ,2-, 1 ,3-, 1 ,4-, 1 ,5-, 1 ,6-, 1 ,7- or 1 ,8-octylene, and the isomers of nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecylene, octadecylene, nonadecylene and eicosylene; cyclopentylene, cyclohexylene; naphthylene, and particularly, phenylene; benzylene and phenylethylene.

In one embodiment of the present invention, X is a radical which is unsubstituted or substituted by C₁-C₁₀alkoxy, preferably C₁-C₆alkoxy, F, Cl, Br, -CN, C₁-C₁₀alkyl, preferably C₁-C₆alkyl, aryl, hydroxy-C₁-C₁₀alkyl, preferably hydroxy-C₁-C₆alkyl, amino-C₁-C₁₀alkyl, preferably amino-C₁-C₆alkyl, OH, NR₁₂ or -NO₂ and which is selected from the group consisting of C₁-C₂₀alkylene, C₃-C₈cycloalkylene, C₆-C₁₂arylene and C₇-C₁₂aralkylene.

In a preferred embodiment, X is C₁-C₂₀alkylene, particularly preferably C₁-C₁₀alkylene, in particular C₁-C₅alkylene. The radical -CH₂- has been found to be particularly advantageous.

Advantageously, the novel compounds contain X, that is p is preferably 1.

In a further embodiment of the present invention, X' is a radical which is unsubstituted or substituted by C₁-C₁₀alkoxy, preferably C₁-C₆alkoxy, F, Cl, Br, -CN, C₁-C₁₀alkyl, preferably C₁-C₆alkyl, aryl, hydroxy-C₁-C₁₀alkyl, preferably hydroxy-C₁-C₆alkyl, amino-C₁-C₁₀alkyl, preferably amino-C₁-C₆alkyl, OH, NR₁₂ or -NO₂ and which is selected from the group consisting of C₁-C₂₀alkylene, C₃-C₈cycloalkylene, C₆-C₁₂arylene and C₇-C₁₂aralkylene.

In a preferred embodiment, X' is a radical which is unsubstituted or substituted by hydroxy - C₁-C₁₀alkyl, preferably hydroxy-C₁-C₆alkyl, amino-C₁-C₁₀alkyl, preferably amino-C₁-C₆alkyl or OH and which is selected from the group consisting of C₁-C₂₀alkylene, C₃-C₈cycloalkylene, C₆-C₁₂arylene and C₇-C₁₂aralkylene. Particularly preferably, X' is a radical which is unsubstituted or substituted by hydroxy-C₁-C₂alkyl or OH and which is selected from the group consisting of C₁-C₁₀alkylene and C₁-C₁₀aralkylene. It is preferred, in particular, that X' is selected from the group consisting of -(CH₂)₂-, -(CH₂)₆-, -(CH₂)₁₀-, -CH₂CH(OH)CH₂-,

In one embodiment of the present invention, A is -O-, -NR₁₂-CO-NR₁₂-, -NR₁₂-, -NR₁₂-C(O)-O-, -C(O)O-, -C(O)NR₁₂-, -P(O)(OH)O-, -OP(O)(OH)O-, -P(O)(OH)-NR₁₂- or -OP(O)(OH)-NR₁₂-, particularly preferably, A is -O-, -NR₁₂-CO-NR₁₂-, -NR₁₂-, -NR₁₂-C(O)-O-, -C(O)O- or -C(O)NR₁₂-, and, in particular preferably, -O-, -C(O)O- oder -C(O)NH-.

Within the scope of the present invention, those compounds are preferred in which A' is not present, that is m is 0, or is -P(O)(OH)O- or -P(S)(OH)-.

The bridging groups of the formula IIIa, IIIb or IIIc

have been found to be particularly suitable as bridging groups B.

The bridging groups of the formula IIId or IIIe

are likewise particularly suitable.

The present invention furthermore relates to intermediates in the preparation of the novel compounds. These are the compounds of the formula IV dendrimer-X_p-[A-X']_n-R₁ (IV) where dendrimer, X, p, A and X' have the abovementioned meanings, n' is a number from 0 to 49, and R₁ is a monovalent functional group.

Within the scope of the present invention, the monovalent functional group is preferably selected from the group consisting of -OR₁₀, -SR₁₀, -NCO, -NCS, -NHR₁₁, -C(O)OR₁₁, -C(O)SH, -C(O)CI, -C(S)SR₁₁, -C(S)OR₁₁, -SO₃R₁₁, -SO₂CI, -OP(O)(OR)(OH), -OP(S)(OR)(OH), -OP(O)(SR)(SH), -OP(O)(OH), -OP(O)(SH), -OP(OCH₃)N[CH(CH₃)₂]₂, -OP(OCH₂CH₂CN)N[CH(CH₃)₂]₂ and P(OCH₂CH₂CN)N[CH(CH₃)₂]₂, where R is a phosphate protecting group, for example β-cyanoethyl, 2,6-dichlorobenzyl, 2-chlorophenyl, 4-chlorophenyl or S-phenyl, R₁₀ is H, -C(O)NH₂, -C(S)NH₂, -C₁-C₆alkyl, -C_xH_2x-NHz, -C_xH_2x-SH or -(C_xH_2xO)_y-H and R₁₁ is H, -C₁-C₆alkyl, -C_xH_2x-NH₂, -C_xH_2x-SH or -(C_xH_2xO)_y-H, and x is a number from 2 to 6, and y is a number from 1 to 20. The functional group is particularly preferably selected from the group consisting of -OR₁₀, -SR₁₀, -NCO, -NCS, -NHR₁₁, -C(O)OR₁₁ and -P(O)(OH)₂, in particular selected from the group consisting of -NCS, -C(O)OR₁₁ and -P(O)(OH)₂.

The present invention furthermore relates to a process for preparing the novel compounds, which comprises reacting a compound of the formula IV with a compound of the formula Va

R₁-[A-X']_n-A'_m-oligonucleotide (Va) in which R_1' and n" each have one of the meanings mentioned above for R₁ and n', and A, X', A', m and oligonucleotide have the abovementioned meanings.

The process can, for example, be carried out such that the compounds of the formulae IV and Va are dissolved in a solvent, preferably in equivalent quantities, and then reacted with each other at elevated temperatures. Expediently, condensation catalysts, for example concentrated mineral acids, in particular hydrochloric acid, or acidic ion exchangers, are used concomitantly. It can be expedient to add water-binding agents or to remove the water of reaction from the reaction mixture.

The reaction temperature can, for example, be from 40 to 220°C, preferably from 50 to 150°C.

Examples of suitable solvents are water and polar protic solvents which advantageously are miscible with water, and also polar aprotic and non-polar solvents. Examples of such solvents are alcohols (methanol, ethanol, n- or i-propanol, butanol, ethylene glycol, propylene glycol, ethylene glycol monomethyl ether, diethylene glycol, and diethylene glycol monomethyl ether), ethers (diethyl ether, dibutyl ether, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol diethyl ether and triethylene glycol dimethyl ether), halogenated hydrocarbons (methylene chloride, chloroform, 1 ,2-dichloroethane, 1 ,1 ,1-trichloroethane, 1,1,2,2-tetrachloroethane and chlorobenzene), carboxylic esters and lactones (ethyl acetate, methyl propionate, ethyl benzoate, 2-methoxyethylacetate, γ-butyrolactone, δ-valerolactone and pivalolactone), N-alkylated carboxamides and lactams (N,N-dimethylformamide, N,N-diethylformamide, N,N-dimethylacetamide, tetramethylurea, hexamethylphosphoric triamide, N-methyl-γ-butyrolactam, N-methyl-∈-caprolactam and N-methylpyrrolidone), sulfoxides (dimethyl sulfoxide and tetramethylene sulfoxide), sulfones (dimethyl sulfone, diethyl sulfone, trimethylene sulfone and tetramethylene sulfone), tertiary amines (trimethylamine, triethylamine, N-methylpiperidine, N-methylmorpholine and pyridine), substituted benzenes (chlorobenzene, o-dichlorobenzene, 1 ,2,4-trichlorobenzene, nitrobenzene, toluene and xylene) and nitriles (acetonitrile, propionitrile, benzonitrile and phenylacetonitrile).

The novel process for preparing the oligonucleotide conjugates can, for example, be carried out such that an oligonucleotide which is or is not functionalized is dissolved in a solvent or solvent mixture and the dendrimer carrying a suitable functional group is then added, and the reaction mixture is subsequently allowed to react, if desired while stirring. The conjugate which is formed can then be purified in a manner known per se and isolated, if desired.

The reaction temperature can, for example, be from 0 to 120°C, preferably from 20 to 80°C. Particularly preferably, the reaction is carried out at room temperature.

If the linking is an esterification, transesterification or amidation reaction, corresponding carboxylic acid groups can be activated in advance in a known manner, for example by reaction with carbodiimides and N-hydroxysuccinimide.

The reactants are expediently employed in molar ratios. However, an excess of the catalyst or the oligonucleotide can be used.

The customary methods, advantageously, for example, dialysis, electrophoresis and chromatographic methods such as high pressure liquid chromatography (HPLC), reverse-phase HPLC, affinity chromatography, ion exchange chromatography and gel

chromatography, can be used for the purification. The oligonucleotides which are to be used and which are or are not functionalized can be prepared in a manner known per se using automated synthesizers which are commercially available. Nucleosides for their synthesis are known and can either be obtained

commercially or prepared using analogous methods. The phosphorotriester method, the phosphite triester method or the H-phosphonate method, with which the person skilled in the art is familiar, can, for example, be used in the case of the bridging group -P(O)O--. In the phosphite triester method, the approach can, for example, be to react the nucleosides with a protecting group reagent, for example 4,4'-dimethoxytriphenylmethyl chloride, and to use a linker, for example succinic anhydride, to bind the resulting compound to a solid support material, for example to control pore glass (CPG) which contains long-chain alkylamino groups. In a separate procedure, a hydroxyl group of such compounds is derivatized, for example to form a phosphoramidite using R'OP[N(i-propyl)₂)]₂.

After the protecting group, for example the DMT group, of the material bound to the support has been eliminated, coupling is carried out while eliminating -N(i-C₃H₇)₂, any free hydroxyl groups which are present are blocked (capped), and the phosphite which has been formed is then oxidized to the phosphate. Following deprotection of the dimer, the reaction cycle is repeated using another protected compound until an oligomer having the desired number of monomer units has been synthesized, after which the product is released from the support material. In this manner, oligonucleotides can be prepared having any monomer units in any sequence, depending on the use of synthetic, natural and novel nucleoside building blocks in the individual reaction cycles.

The novel compounds have anti-viral and anti-proliferative properties and can consequently be used as pharmaceuticals. In addition, the novel oligonucleotides exhibit a high degree of stability towards degradation by nucleases. Their unexpectedly high cellular uptake is particularly surprising. In addition, they pair in an outstanding manner with complementary nucleic acid strands, especially of the RNA type. The novel oligonucleotides are therefore particularly suitable for antisense technology, that is for inhibiting the expression of undesirable protein products by means of binding to suitable, complementary mRNA nucleotide sequences (EP 266, 099, WO87/07300 and WO89/08146). They may be employed for treating infections and diseases, for example by means of blocking the expression of bioactive proteins at the stage of the nucleic acids (for example oncogenes). More than 30 families of such oncogenes are known which are thought to be involved in the formation of tumours in humans. An example of such a family is the raf gene family which comprises three highly conserved genes which are designated A-raf, B-raf and c-raf (also termed raf-1). The raf genes encode protein kinases which are assumed to play an important role in cellular signal transduction which regulates cell proliferation. There are indications that abnormal expression of the c-raf protein, in particular, is associated with abnormal cell profileration. (Review: U. Rapp et al., "The Oncogene Handbook", E.P. Reddy et al., eds., Elsevier Science Publishers, New York, 1988, pp. 213-253.)

Surprisingly, it has been observed, within the scope of the present invention, that oligonucleotide-dendrimer conjugates whose olignucleotide sequence is complementary to a segment of the 3'-non-translated region of human c-raf mRNA and has, in particular, the sequence 5'-TCCCGCCTGTGACATGCATT-3' (nucleosides linked via -P(S)O-, SEQ. ID. NO. 5) possess outstanding properties as regards decreasing the expression of c-raf, determined, for example, in cell cultures, and decreasing tumour growth in vivo.

Consequently, the invention furthermore relates to novel oligonucleotide-dendrimer conjugates in which the oligonucleotide moiety has the sequence

5'-TCCCGCCTGTGACATGCATT-3' (nucleosides linked via -P(O)S-- ("PS"), SEQ. ID. NO. 5) and the dendrimer moiety is as defined above. Preference is given to oligonucleotide- dendrimer conjugates of the formulae (VI) and (VII)

in which Q' is , r is 0 or 1 , and s is 1 , 2 or 3. Preferably, r is 1 and

s is 2.

The protein kinase C (PKC) family, which comprises several isoforms (isozymes) such as PKC α, β, γ, δ, ε, ξ and η, forms another class of proteins which play an important role in signal transduction and abnormal cell proliferation (cf., for example, Gescher et al., Anti Cancer Drug Design 4 (1989), pp. 93-105; Nishizuka, Nature 334 (1988), pp. 661 -665).

Within the scope of the present invention, it has been observed, surprisingly, that oligonucleotide-dendrimer conjugates which have an oligonucleotide sequence

5'-GTTCTCGCTGGTGAGTTTCA-3' (nucleosides linked via -P(S)O-, SEQ. ID. NO. 6), which is complementary to human PKC α mRNA, are outstandingly suitable for decreasing the expression of PKC α, for example in cell cultures, and for reducing tumour growth in vivo.

The invention furthermore relates to novel oligonucleotide-dendrimer conjugates in which the oligonucleotide moiety has the sequence 5'-GTTCTCGCTGGTGAGTTTCA-3'

(nucleosides linked via -P(O)S-- ("PS"), SEQ. ID. NO. 6) and the dendrimer moiety is defined as above. Preference is given to oligonucleotide-dendrimer conjugates of the formulae (VIII) and (IX)

in which Q' is , r is 0 or 1 , and s is 1 , 2 or 3. Preferably, r is 1 and

s is 2.

The novel oligonucleotide-dendrimer conjugates are also suitable for use as diagnostic agents and can be used as gene probes for detecting viral infections or genetically determined diseases by means of selective interaction at the stage of single-stranded or double-stranded nucleic acids (gene probes). In particular - as a result of the increased stability towards nucleases - it is possible to use the conjugates for diagnostic applications in vivo (for example tissue samples, blood plasma and blood serum) as well as in vitro. Such possible uses are described, for example, in WO 91/06556.

The invention furthermore relates to the use of the novel compounds as diagnostic agents for detecting viral infections or genetically determined diseases.

The invention also relates to the novel compounds for use in a therapeutic process for treating diseases in homoiothermic animals, including man, by means of inactivating nucleotide sequences in the body. When administering to homoiothermic animals of about 70 kg body weight, the dose can, for example, be from 0.01 to 1000 mg per day.

Administration is preferably effected parenterally, for example intraveneously or

intraperitoneally, in the form of pharmaceutical preparations.

The invention also relates to a pharmaceutical preparation which comprises an effective quantity of a novel compound, either alone or together with other active compounds, a pharmaceutical excipient, preferably in a significant quantity, and auxiliary substances, if desired.

The pharmacologically active novel compounds can be used in the form of preparations which can be administered parenterally or of infusion solutions. Such solutions are, preferably, isotonic aqueous solutions or suspensions, with it being possible for the latter, for example in the case of lyophilized preparations which comprise the active substance alone or together with an excipient, for example mannitol, to be prepared prior to use. The pharmaceutical preparations can be sterilized and/or comprise auxiliary substances, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating the osmotic pressure and/or buffers. The pharmaceutical preparations, which, if desired, can comprise additional pharmacologically active substances, for example antibiotics, are produced in a manner which is known per se, for example by means of conventional dissolution or lyophilization methods, and comprise from about 0.1% to 90%, in particular from about 0.5% to about 30%, for example from 1% to 5%, of active compound(s).

The following examples illustrate the invention in more detail.

In the examples, the dendrimers which conform to the following scheme are described, with the clarifications given above in association with the compounds of the formula (I) applying in an analogous manner:

Preparation of the starting compounds

Example A1 : Preparation of [G-2]-CH₂-OH (4) a) 15.1 g of methyl 3,5-dihydroxybenzoate are dissolved in 400 ml of acetone, 4.75 g of 18-crown-6, 26.65 ml of benzyl bromide and 31.0 g K₂CO₃ (anhydrous) are added and the whole is heated under reflux for 40 h. The precipitate is filtered off and washed with acetone and the filtrate is concentrated in vacuo. H₂O and CH₂Cl₂ are added to the residue and the organic phase is separated off and dried and the solvent is removed; the residue is recrystallized from diethyl ether/hexane (1 :1). The compound (1) [G-1]-CO₂-CH₃ (1) is obtained. b) 19.0 g of compound (1) are dissolved in 300 ml of diethyl ether, the solution is cooled and 3.8 g of LiAlH₄ are added in several portions. The mixture is subsequently heated under reflux for a total of 18 h. The reaction mixture is hydrolysed dropwise with H₂O and filtered, the solvent is removed and the residue is then dried overnight under high vacuum. The compound (2)

[G-1]-CH₂-OH (2) is obtained. c) 14.0 g of compound (2) are dissolved in 200 ml of tetrahydrofuran (argon), and 13.7 g of triphenylphosphine and 17.4 g of CBr₄ are added at room temperature. The mixture is stirred at room temperature for 1.5 h, the precipitate is filtered off and the filtrate is concentrated in vacuo. CH₂Cl₂ and H₂O are added to the residue, the organic phase is removed and dried (Na₂SO₄) and the solvent is removed. Purification is effected by means of flash chromatography. Eluent: CH₂Cl₂:hexane (1 :1). The compound (3) .

[G-1]-CH₂Br (3) is obtained. d) 14.5 g of compound (3) and 2.4 g of 3,5-dihydroxybenzyl alcohol are dissolved in acetone (argon atmosphere), 5.5 g of (K₂CO₃) (anhydrous) and 0.9 g of 18-crown-6 are added and the whole is heated under reflux for 18 hours. The mixture is filtered and the filtrate is concentrated in vacuo. CH₂Cl₂ and H₂O are added to the residue, and the organic phase is separated off and dried and the solvent is removed; the residue is recrystallized from toluene:hexane (3:1). The title compound, compound (4), is obtained.

Example A2: Preparation of [G-3]-CH₂-OH (6) a) 6.0 g of compound (4) are dissolved in 100 ml of tetrahydrofuran (argon), and 2.6 g of triphenylphosphine and 3.3 g of CBr₄ are added at room temperature. The mixture is stirred at room temperature for 1.5 h, the precipitate is filtered off and the filtrate is concentrated in vacuo. CH₂Cl₂ and H₂O are added to the residue, and the organic phase is removed and dried (Na₂SO₄) and the solvent is removed. Purification is effected by means of flash chromatography. Eluent: CH₂Cl₂:hexane (3:1). The compound (5)

[G-2]-CH₂-Br (5) is obtained. b) 5.0 g of compound (5) and 5.0 g of 3,5-dihydroxybenzyl alcohol are dissolved in 50 ml of acetone (argon atmosphere), 1.03 g of K₂CO₃ (anhydrous) and 158 mg of 18-crown-6 are added and the whole is heated under reflux for 18 hours. The mixture is filtered and the filtrate is concentrated in vacuo. CH₂Cl₂ and H₂O are added to the residue, and the organic phase is separated off and dried and the solvent is removed. Purification is effected by means of flash chromatography. Eluent: CH₂Cl₂. The title compound, compound (6) is obtained.

Example A3: Preparation of [G-2]-CH₂-O-(CH₂)₁₀-OH (7)

0.5 g of 1 ,10-decanediol are dissolved in 15 ml of tetrahydrofuran, 672 mg of NaH are added and the whole is heated under reflux for 15 min. The mixture is brought to room temperature and a solution of 0.3 g of compound (5) in tetrahydrofuran is added dropwise. After this addition, the mixture is refluxed overnight and then cooled; the excess NaH is hydrolysed by adding a few drops of H₂O, and H₂O/CH₂Cl₂ is added to the mixture. The organic phase is separated off and dried (Na₂SO₄) and the product is purified by flash chromatography. Eluent: CH₂Cl₂. The title compound, compound (7), is obtained.

Example A4: Preparation of [G-3]-CH₂-O-(CH₂)₁₀-OH (9)

250 mg of 1 ,10-decanediol are dissolved in 10 ml of tetrahydrofuran, 50 mg of NaH are added and the whole is heated under reflux for 15 min. The mixture is brought to room temperature and a solution of 0.3 g of compound (8)

[G-3]-CH₂-Br (8) in tetrahydrofuran is added dropwise. After this addition, the mixture is refluxed overnight and then cooled; the excess NaH is hydrolysed by adding a few drops of H₂O, and

H₂O/CH₂Cl₂ is added to the mixture. The organic phase is separated off and dried (Na₂SO₄) and the product is purified by flash chromatography. Eluent: CH₂Cl₂:acetone (19:1). The title compound, compound (9) is obtained.

Example A5: Preparation of [G-4]-CH₂OH (10)

1 .4 g of compound (8) and 56 mg of 3,5-dihydroxybenzyl alcohol are dissolved in 40 ml of acetone (argon atmosphere), 140 mg of K₂CO₃ (anhydrous) and 53 mg of 18-crown-6 are added and the whole is heated under reflux for 18 hours. The mixture is filtered and the filtrate is concentrated in vacuo. CH₂Cl₂and H₂O are added to the residue, and the organic phase is separated off and dried and the solvent is removed. Purification is effected by means of flash chromatography. Eluent: CH₂Cl₂/acetone (50:1). The title compound, compound (10), is obtained.

Example A6: Preparation of [M-1]-CH₂OH (11)

4.0 g of 3,5-dimethoxybenzyl bromide and 1.12 g of 3,5-dihydroxybenzyl alcohol are dissolved in 100 ml of acetone (argon atmosphere), 2.75 g of K₂CO₃ (anhydrous) and 0.45 g of 18-crown-6 are added and the whole is heated under reflux (argon) for 24 hours. The mixture is filtered and the filtrate is concentrated in vacuo. CH₂Cl₂ and H₂O are added to the residue, and the organic phase is separated off, dried (Na₂SO₄) and concentrated, and the residue is purified by flash chromatography. Eluent: CH₂Cl₂/acetone (9:1). The title compound, compound (11), is obtained.

Example A7: Preparation of [M-2]-CH₂-OH (13)

10 g of 3,5-dimethoxybenzyl bromide and 3.53 g of methyl 3,5-dihydroxybenzoate are added together to 200 ml of acetone, after which 1.11 g of 18-crown-6 and 6.2 g of K₂CO₃ are added, with the mixture subsequently being heated under reflux (argon) for about 20 h. The residue is filtered off and the filtrate is concentrated and CH₂Cl₂/Η₂O is added. The organic phase is separated off, dried (Na₂SO₄) and concentrated, and the solid is recrystallized from toluene/hexane. The compound (12)

[M-1]-COOCH₃ (12) is obtained. b) 1.2 g of LAH are added to a solution of 8 g of compound (12) in 150 ml of

tetrahydrofuran. The suspension is stirred at room temperature under argon for 0.5 h and then at 40ºC for 16 hours. While being cooled, the reaction mixture is carefully hydrolysed with H₂O, acidified with dilute HCl and filtered. CH₂Cl₂/Η₂O is added to the filtrate, and the organic phase is separated off and dried (Na₂SO₄). The solvent is removed and the residue is dried under high vacuum, treated with hexane, filtered off and dried. The compound (11) is obtained. c) 1.5 g of compound (11), 1.05 g of triphenylphosphine and 1.32 g of CBr₄ are stirred, at room temperature for 1 h, in 50 ml of THF. The precipitate is filtered off and the filtrate is concentrated to dryness. CH₂Cl₂/Η₂O is added to the residue, and the organic phase is separated off and dried (Na₂SO₄) and the solvent is removed. Purification is effected by means of flash chromatography. Eluent: CH₂Cl₂. The compound (46)

[M-1]-CH₂Br (46) is obtained. d) 0.8 g of compound (46) and 105 mg of 3,5-dihydroxybenzyl alcohol are dissolved in 50 ml of acetone (argon atmosphere), and 276 mg of K₂CO₃ (anhydrous) and 42.3 mg of 18-crown-6 are added and the whole is heated under reflux (argon) for 24 hours. The mixture is filtered and the filtrate is concentrated in vacuo. CH₂Cl₂ and H₂O are added to the residue, and the organic phase is separated off, dried (Na₂SO₄) and concentrated; the residue is purified by flash chromatography. Eluent: CH₂Cl₂/acetone (19:1). The title compound, compound (13), is obtained.

a) 0.2 g of compound (4) is added to a suspension of 18 mg of NaH in 10 ml of absolute tetrahydrofuran and the mixture is heated under reflux for 15 min. It is then cooled down to room temperature, 65 mg of α-bromo-p-toluic acid are added and the whole is heated under reflux overnight. The reaction mixture is allowed to cool down to room temperature, after which it is carefully treated with a few drops of H₂O and acidified with dilute HCl.

CH₂Cl₂/Η₂O is added to the solution, and the organic phase is separated off and dried (Na₂SO₄) and the solvent is removed. The residue is subsequently dried under high vacuum. The compound (14)

[G-2]-Q-COOH (14) is obtained. b) 100 mg of compound (14) and 13.8 mg of N-hydroxysuccinimide are dissolved in 5 ml of absolute tetrahydrofuran, the solution is cooled down to 0°C, and a solution of 25.8 ml of DCC in 5 ml of tetrahydrofuran is added. The mixture is stirred at room temperature overnight, after which the solvent is removed in vacuo and the residue is purified by flash chromatography. The title compound, compound (15), is obtained.

a) 1.0 g of compound (6) is added to a suspension of 48 mg of NaH in 20 ml of absolute tetrahydrofuran, and the mixture is heated under reflux for 15 min. It is then cooled down to room temperature, 0.15 g of α-bromo-p-toluic acid is added and the whole is heated under reflux overnight. The reaction mixture is allowed to cool down to room temperature, after which it is carefully treated with a few drops of H₂O and acidified with dilute HCl. CH₂Cl₂/H₂O is added to the solution, and the organic phase is separated off and dried (Na₂SO₄) and the solvent is removed. Purification is effected by means of flash

chromatography. Eluent: CH₂Cl₂:acetone (9:1 ). The compound (16)

[G-3]-Q-COOH (16) is obtained. b) 200 mg of compound (16) and 14.0 mg of N-hydroxysuccinimide are dissolved in 5 ml of absolute tetrahydrofuran, the solution is cooled down to 0°C and a solution of 27 mg of DCC in 5 ml of tetrahydrofuran is added. The mixture is stirred at room temperature overnight, after which the solvent is removed in vacuo and the residue is purified by flash chromatography. Eluent: CH₂Cl₂. The title compound, compound (17) is obtained.

a) 0.32 g of compound (6) and 16 mg of 4-(dimethylamino)pyridine are dissolved in 2 ml of pyridine, after which 25 mg of succinic anhydride are added. The mixture is stirred for 16 hours and then treated with CH₂Cl₂ and extracted twice with an ice-cold 10% solution of citric acid on each occasion. The organic phase is separated off and dried (Na₂SO₄) and the solvent removed; the residue is dried under high vacuum. The compound (18).

[G-3]-W-COOH (18) is obtained. b) 125 mg of compound (18) and 11.5 mg of N-hydroxysuccinimide are dissolved in 10 ml of absolute tetrahydrofuran, the solution is cooled down to 0°C and 25 ml of DCC are added. The mixture is allowed to stand overnight, after which the solvent is removed in vacuo and the residue is purified by flash chromatography. Eluent: CH₂Cl₂:acetone (19:1). The title comopund, compound (19), is obtained.

a) 8.5 mg of 4-(dimethylamino)pyridine and 50 ml of triethylamine are added to 120 mg of compound (7) and the reaction mixture is then stirred at room temperature overnight. It is then treated with 3.0 ml of CH₂Cl₂ and extracted twice with ice-cold 10% citric acid on each occasion. The organic phase is subsequently washed with H₂O and dried (Na₂SO₄) and the solvent removed; the residue is dried under high vacuum. The compound (20)

[G-2]-W-COOH (20) which is used without further purification, is obtained. b) 130 mg of compound (20) are dissolved in 5 ml of absolute tetrahydrofuran, the solution is cooled down to °C and 35 mg of N-hydroxysuccinimide and 17.5 mg of DCC are then added. In order to ensure complete reaction, the reaction mixture is stirred at room temperature overnight. The precipitate is filtered off, the filtrate is concentrated and the residue is purified by flash chromatography. Eluent: CH₂Cl₂:acetone (19:1). The title compound, compound (21), is obtained.

O

a) 60 mg of 4-N,N-dimethylaminopyridine and 50 μl of triethylamine are added to 140 ml of compound (9) and the reaction mixture is then stirred at room temperature overnight. It is treated with 3.0 ml of CH₂Cl₂ and extracted twice with ice-cold 10% citric acid on each occasion. The organic phase is subsequently washed with H₂O and dried (Na₂SO₄) and the solvent removed; the residue is dried under high vacuum. The compound (22)

[G-3]-W-COOH (22) which is used without further purification, is obtained. b) 140 mg of compound (22) are dissolved in 5 ml of absolute tetrahydrofuran, the solution is cooled down to 0°C and 11 mg of N-hydroxysuccinimide and 20.5 mg of DCC are then added. In order to ensure complete reaction, the reaction mixture is stirred at room temperature overnight. The precipitate is filtered off, the filtrate is concentrated and the residue is purified by flash chromatography. Eluent: CH₂Cl₂:acetone (19:1). The title compound, compound (23), is obtained.

a) 1.0 g of compound (10) is added to a suspension of 12 mg of NaH in 10 ml of absolute tetrahydrofuran and the mixture is heated under reflux for 15 min. It is then cooled down to room temperature, 36.5 mg of α-bromo-p-toluic acid are added and the whole is heated under reflux overnight. The reaction mixture is allowed to cool down to room temperature after which it is carefully treated with a few drops of H₂O and acidified with dilute HCl.

CH₂Cl₂/H₂O is added to the solution, and the organic phase is separated off and dried (Na₂SO₄) and the solvent removed. Purification is effected by means of flash

chromatography. Eluent: CH₂Cl₂:acetone (19:1 ). The compound (24)

[G-4]-Q-COOH (24) is obtained. b) 100 mg of compound (24) and 4.5 mg N-hydroxysuccinimide are dissolved in 5 ml of absolute tetrahydrofuran, the solution is cooled down to 0°C and a solution of 10 mg of DCC in 5 ml of tetrahydrofuran is added. The mixture is stirred at room temperature overnight, after which the solvent is removed in vacuo and the residue is purified by flash chromatography. Eluent: CH₂Cl₂/acetone (50:1). The title compound, compound (25), is obtained.

a) 5.0 g of compound (2) are reacted with 1.2 g of sodium hydride and 3.44 g of α-bromo-p- toluic acid in 200 ml of absolute tetrahydrofuran in analogy with method B1a). The compound (B7.1)

[G1]-Q-COOH (B7.1) is obtained. b) 3.0 g of compound (B7.1) are reacted with 0.83 g of N-hydroxysuccinimide and 1.55 g of N,N-dicyclohexylcarbodiimide in 50 ml of absolute tetrahydrofuran in analogy with method B1 b). The title compound, compound (B7.2) is obtained.

a) 545 mg of 3-amino-1 ,2-propanediol are added, while stirring vigorously, to 3.1 g of compound (15) in 40 ml of tetrahydrofuran. The reaction mixture is stirred at room temperature for a total of 3 h and at 50°C for 15 min. It is then concentrated and the residue purified by column chromatography (flash chromatography). Eluent: MeOH:CH₂Cl₂ (1:9). The product is subsequently dried under high vacuum. The compound (26)

[G-2]-Q-C(O)-NH-CH₂-CH(OH)-CH₂-OH (26) is obtained.

b) 2.6 g of compound (26) are dissolved in 20 ml of absolute pyridine, the solution is cooled down to 0°C and 1.01 g of dimethoxytrityl chloride (DMTrCl) are added in several portions at this temperature. After that, the mixture is stirred at 0°C for 1 h, slowly brought to room temperature and stirred overnight. 2 ml of methanol are added to the reaction solution and the solvent (pyridine + methanol) is removed. 100 ml of CH₂Cl₂ are added and the mixture is washed with a 5% solution of NaHCO₃ and with H₂O. The organic phase is dried, the solvent is removed and the residue is purified by flash chromatography. Eluent:

CH₂Cl₂:acetone (9:1) + 0.5 % triethylamine. The compound (27)

[G-2]-Q-C(O)-NH-CH₂-CH(OH)-CH₂-O-DMTr (27) is obtained. c) 1.25 g of compound (27) are dissolved in 5.0 ml of CH₂Cl₂, and 0.15 g of succinic anhydride are added. 61 mg of 4-(dimethylamino)pyridine and 140 μl of triethylamine are then added to the reaction mixture and the whole is stirred at room temperature overnight. The solvent is removed in vacuo and 20 ml of CH₂Cl₂ are added to the residue, and this solution is washed with an ice-cold 10% solution of citric acid and with H₂O. The organic phase is separated off and dried (Na₂SO₄) and the solvent is removed in vacuo. The residue is dissolved by adding 5 ml of CH₂Cl₂ , and the product is precipitated out by adding approximately 100 ml of hexane while at the same time stirring vigorously. The solvent is decanted off and the residue is dried under high vacuum. The compound (28)

is obtained. d) 258 mg of DCC are added to a reaction mixture consisting of 0.68 g of compound (28), 70 mg of p-nitrophenol, 100 μl of pyridine and 2 ml of dioxane and the whole is stirred at room temperature. After a total reaction time of 3 h, the mixture is filtered. The compound (29)

is obtained. e) The resulting compound (29) is added to a suspension of 3 g of dried (HV pump over P₂O₅) long chain alkylamine controlled pore glass (LCAA-CPG) in 7 ml of

dimethylformamide and the whole is stirred overnight. The mixture is filtered, a further 3 g of LCAA-CPG is added to the filtrate, with the whole then being stirred overnight and subsequently filtered. 1 ml of acetic anhydride, 50 mg of 4-(dimethylamino)pyridine and 10 ml of pyridine are in each case added to the solid support (2x 3 g in each case) and the mixture is stirred for 1 h. The support is filtered off, washed with dimethylformamide, methanol and diethyl ether (100 ml in each case) and dried in a desiccator. The title compound, compound (30) is obtained.

a) 1.4 g of compound (17) and 136 mg of serinol are added together to 20 ml of tetrahydrofuran and the reaction mixture is stirred at room temperature. CH₂Cl₂ and H₂O are added to it, and the organic phase is separated off and dried and the solvent is removed. The product is purified by flash chromatography. Eluent: CH₂Cl₂:MeOH (19:1). The compound (31)

[G-3]-Q-C(O)-NH-CH(CH₂OH)₂ (31) is obtained. b) 0.7 g of compound (31) is dissolved in 15 ml of absolute pyridine, the solution is cooled down to 0°C and 140 mg of dimethoxytrityl chloride (DMTrCI) are added. The mixture is stirred at 0°C for 1 h, brought to room temperature and stirred overnight. Working-up is effected in analogy with Example C1 b). The compound (32)

[G-3]-Q-C(O)-NH-CH(CH₂OH)-CH₂-O-DMTr (32) is obtained. c) 0.4 g of compound (32) are dissolved in 5.0 ml of CH₂Cl₂ , and 30 mg of succinic anhydride are added. 12.5 mg of 4-N,N-dimethylaminopyridine and 30 μl of triethylamine are subsequently added to the reaction mixture, which is then stirred at room temperature overnight. Working-up is effected in analogy with Example C1c). The compound (33)

is obtained. d) 82.5 mg of DCC are added to a reaction mixture consisting of 0.35 g of compound (33), 28 mg of p-nitrophenol, 50 μl of pyridine and 4 ml of dioxane and the whole is stirred at room temperature. After a total reaction time of 3 h, the mixture is filtered. The compound (34)

is obtained. e) The title compound, compound (35), is obtained after reacting the resulting compound (34) with 3 g of LCAA/CPG (dried over P₂O₅ under high vacuum).

a) 1.5 g of compound (B7.2)) are reacted with 0.32 g of serinol in 30 ml of absolute tetrahydrofuran at room temperature for 3 hrs and then at 50°C for 1 hr, in analogy with method C2a). The compound (C3.1)

[G1]-Q-C(O)-NH-CH(CH₂OH)₂ (C3.1) is obtained. b) 1.4 g of the compound (C3.1) are reacted with 0.95 g of 4,4'-dimethoxytrityl chloride (DMTrCI) in 30 ml of absolute pyridine in analogy with method C2b). The compound (C3.2)

[G1]-Q-C(O)-NH-CH(CH₂OH)-CH2-O-DMTr (C3.2) is obtained. c) 0.60 g of the compound (C3.2) is reacted with 0.10 g of succinic anhydride, 49 mg of 4-N,N-dimethylaminopyridine and 110 μl of triethylamine in 5 ml of methylene chloride, in analogy with method C2c). The compound (C3.3)

is obtained. d) 0.60 g of the compound (C3.3) is reacted with 91 mg of p-nitrophenol, 310 mg of N,N-dicyclohexylcarbodiimide and 150 μl of pyridine in 5 ml of dioxane, in analogy with method C2d). The compound (C3.4)

is obtained. e) The title compound, compound (C3.5), is obtained by reacting the resulting compound (C3.4) with 6 g of LCAA/CPG, in analogy with method C1.

0.75 g of compound (4) is dissolved in 30 ml of hot acetonitrile, the solution is then cooled down to room temperature and 85.5 mg of diisopropylammonium tetrazolide are added. 0.33 ml of [bis(diisopropylamino)-2-cyanoethoxy]phosphine is added dropwise, under an argon atmosphere, and the mixture is stirred at room temperature for 1 h and at 30°C for 2 h. The title compound, compound (36), is obtained.

Synthesis and purification of oligonucleotides

The oligonucleotides are synthesized on an Applied Biosystems 392 DNA-RNA synthesizer (synthesis scale, 0.1 - 10 μmol) or on a Millipore 8800 large scale DNA synthesizer (synthesis scale, 10 - 100 μmol) using the common cyanoethyl phosphoramidite method and employing 4-tert.-butylphenoxyacetyl-protected building blocks on a solid phase support.

If the dendrimer moiety is to be attached to the 3' end of the conjugate (cf. also Examples E6 to E20), the oligonucleotide synthesis is then carried out using a dendrimer-modified solid phase support, e.g. compounds 30, 35 or C3.5. After the synthesis, the crude polynucleotides are detached from the solid phase support, and deprotected, by being treated, at room temperature for 16 hrs, with a cone, aqueous solution of ammonia. The solution is concentrated and the crude oligonucleotide is purified by reverse phase high pressure chromatography (Waters HPLC system) using a Nucleosil C 18 column (gradient: from 85% 0.05 M triethylammonium acetate and 15% acetonitrile to 100% acetonitrile over 65 min). The oligonucleotide-containing fractions are collected and lyophilized. Molecular weights are determined on an LD1 1700 (Linear Scientific Inc., Reno, USA).

Introduction of the dendrimer at the 5' end (cf. also Examples E1 to E5) is carried out, as described, by using a suitable dendrimer derivative and an appropriately amino-substituted oligonucleotide. Alternatively, the dendrimer can be introduced directly during the oligonucleotide synthesis by using a dendrimer-phosphoramidite.

Preparation of oligonucleotide conjugates The preparation of oligonucleotide-dendrimer conjugates is described below.

Oligonucleotides having the following sequences are used:

PO: Linkage of the nucleosides via -P(O)O-- PS: Linkage of the nucleosides via -P(O)S--

at room temperature for 20 h, in the presence of 160 μl of solvent

(dimethylformamide:dioxane:H₂O = 1 :1 :4) and 5 μl of N,N-diisopropylethylamine. The title compound, compound (38), is obtained.

The title compound, compound (39), is obtained by reacting compound (17) in analogy with Example (E1).

120 μl of a mixture of dimethylformamide and H₂O (5:1) and 2 μl of diisopropylethylamine are added to 2.0 mg of compound (40)

together with compound (37), and the mixture is stirred at 40°C for 16 h. The title compound, compound (41), is obtained.

The title compound, compound (42), is obtained by reacting 2.0 mg of compound (21 ) and 4 OD of compound (37) in analogy with Example (E3).

The title compound, compound (43), is obtained by reacting 2 mg of compound (23) and 4 OD of compound 37 in analogy with Example (E4)

Examples E6 to E20:

The following 3'-oligonucleotide-dendrimer conjugates are synthesized by using dendrimers which are bound to solid phase supports::

F: Fluorescein

PO: Linking of the nucleosides via -P(O)O--

PS: Linking of the nucleosides via -P(O)S-- Biological activity

In vivo anti tumor activity against human lung carcinoma A549 of oligonucleotide dendrimer conjugates

Determination of anti tumor activities are carried out in male Balb/c nude mice bearing serially passaged (minimum of three consecutive transplantations) human lung carcinoma A549 (CCL185, American Type culture collection ATCC; Rockville, Maryland, USA) The cells are cultured as recommended by ATCC. Tumor fragments of approximately 25 mg are transplanted into the left flank of each animal (n = 6 per group). Treatments with oligonucleotide dendrimer conjugates according to the present invention are started when the tumors reach a mean tumor volume of 150 - 200 mm³. Tumor growth is monitored twice weekly by measuring perpendicular diameters. Tumor volumes are determined as described in T. Meyer et al., Int. J. Cancer 43 (1989), pp. 851 -856. Treatment schedule used in these experiments is once daily i.v (tail vein) starting day 10 after tumor

transplantation.

Example F1:

The novel oligonucleotide-dendrimer conjugate from Example E15 is employed for the treatment in accordance with the above protocol.

Example F2:

The novel oligonucleotide-dendrimer conjugate from Example E16 is employed in analogy with Example F1.

Example F3:

The novel oligonucleotide-dendrimer conjugate from Example E17 is employed in analogy with Example F1. Example F4:

The novel oligonucleotide-dendrimer conjugate from Example E18 is employed in analogy with Example F1.

Example F5:

The novel oligonucleotide-dendrimer conjugate from Example E19 is employed in analogy with Example F1.

Example F6:

The novel oligonucleotide-dendrimer conjugate from Example E20 is employed in analogy with Example F1.

Claims

WHAT IS CLAIMED IS:

1. An oligonucleotide-dendrimer conjugate, wherein dendrimer is the monovalent

residue of a dendrimer of the first to tenth generation and oligonuleotide is a natural, modified or synthetic sequence which is composed of natural, modified or synthetic deoxynucleosides or peptide nucleic acid building blocks which are linked via internucleotide bridges, and which encompasses a region which is complementary to a target nucleic acid, with the dendrimer being bonded directly or via a bridging group B to an internucleotide bridge, a nucleic acid base or a sugar of the

oligonucleotide, and the physiologically tolerated salts thereof.

2. An oligonucleotide-dendrimer conjugate according to claim 1 , wherein the dendrimer contains an initiator core having at least three valencies, with one valency being used for the bond to the oligonucleotide, and at least two monovalent branches which are bonded to the initiator core, with each branch consisting of at least one branching point having at least three valencies.

3. An oligonucleotide-dendrimer conjugate according to claim 2, wherein the initiator core and the branching point are, independently of each other, a single atom, a cyclic or heterocyclic, saturated or unsaturated aliphatic radical having from three to twelve ring members, a bicyclic or heterobicyclic aliphatic radical having from five to twelve ring members, or a mononuclear or polynuclear aromatic or heteroaromatic radical having from six to eighteen ring members, where the ring members are carbon atoms which are, where appropriate, interrupted by from one to three heteroatoms which are selected from the group consisting of nitrogen, oxygen and sulfur.

4. An oligonucleotide-dendrimer conjugate according to claim 3, wherein the initiator core and the branching point are, independently of each other, a single atom, a cyclic or heterocyclic, saturated or unsaturated aliphatic radical, or a mononuclear or polynuclear aromatic or heteroaromatic radical.

5. An oligonucleotide-dendrimer conjugate according to claim 4, wherein the initiator core and the branching point are, independently of each other, a cyclic or

heterocyclic, saturated or unsaturated aliphatic radical or a mononuclear or polynuclear aromatic or heteroaromatic radical.

6. An oligonucleotide-dendrimer conjugate according to claim 3, wherein the atom is carbon, nitrogen, silicon or phosphorus.

7. An oligonucleotide-dendrimer conjugate according to claim 6, wherein the atom is carbon.

8. An oligonucleotide-dendrimer conjugate according to claim 3, wherein the cyclic or heterocyclic aliphatic radical is derived from compounds which are selected from the group consisting of cycloalkanes and cycloalkenes having from five to seven ring carbon atoms.

9. An oligonucleotide-dendrimer conjugate according to claim 3, wherein the bicyclic or heterobicyclic aliphatic radical is derived from compounds which are selected from the group consisting of bicycloalkanes and bicycloalkenes having from five to seven ring carbon atoms.

10. An oligonucleotide-dendrimer conjugate according to claim 3, wherein the aromatic or heteroaromatic radical is derived from compounds which are selected from the group consisting of benzene, naphthalene, anthracene, phenanthrene,

naphthacene, indene, fluorene, indacene, biphenylene, triphenylene, pyrrole, indole, carbazole, furan, benzofuran, dibenzofuran, thiophene, benzothiophene,

dibenzothiophene, pyridine, quinoline, isoquinoline, acridine, phenanthridine, pyridazine, cinnoline, phthalazine, pyrimidine, quinazoline, pyrazine, quinoxaline, phenazine, pteridine, purine, pyrazole, indazole, imidazole, benzimidazole, isoxazole, oxazole, furazan, thianthrene, xanthene, triazine, phenanthroline, benzoxazole and benzothiazole.

11. An oligonucleotide-dendrimer conjugate according to claim 10, wherein the aromatic or heteroaromatic radical is derived from compounds which are selected from the group consisting of benzene, naphthalene, fluorene, biphenylene, pyrrole, carbazole, furan, dibenzofuran, thiophene, dibenzothiophene, pyridine, acridine, pyrazine, phenazine, furazan, thianthrene and xanthene.

12. An oligonucleotide-dendrimer conjugate according to claim 11 , wherein the aromatic or heteroaromatic radical is derived from compounds which are selected from the group consisting of benzene, pyrrole, furan, thiophene, pyridine, pyrazine and furazan.

13. An oligonucleotide-dendrimer conjugate according to claim 12, wherein the aromatic radical is derived from benzene.

14. An oligonucleotide-dendrimer conjugate according to claim 2, wherein free valencies of the initiator core and of branching points within the branch are occupied, independently of each other, by hydrogen or a substituent selected from the group consisting of halogen, C₁-C₆alkyl, C₁-C₆hydroxyalkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, -CN and -NO₂ .

15. An oligonucleotide-dendrimer conjugate according to claim 2, wherein free valencies of the branching points at the periphery of the branch are occupied, independently of each other, by a monovalent end group.

16. An oligonucleotide-dendrimer conjugate according to claim 15, wherein the end group is selected from the group consisting of hydrogen, C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₆alkylthio, C₆-C₁₀aryl, C₇-C₁₇aralkyl, hydroxyl, amino, nitro and an organic radical which is derived from a carboxylic acid derivative.

17. An oligonucleotide-dendrimer conjugate according to claim 16, wherein the end group is hydrogen or C₁-C₆alkoxy.

18. An oligonucleotide-dendrimer conjugate according to claim 17, wherein the end

group is hydrogen or -OCH₃ .

19. An oligonucleotide-dendrimer conjugate according to claim 2, wherein bivalent

bridging groups Z, independently of each other, link the initiator core to the branching point of the first generation and/or the branching points of consecutive generations.

20. An oligonucleotide-dendrimer conjugate according to claim 19, wherein the bridging group Z is selected from the group consisting of C₁-C₄alkylene; C₁-C₄alkylene which is interrupted once or more than once by a representative selected from the group consisting of oxygen atom, sulfur atom, nitrogen atom, carbonyl radical, thio radical, sulfoxide radical and a radical of the formula -Si(OR')(OR")-O-, in which R' and R" are, independently of each other, hydrogen or C₁-C₆alkyl; C₂-C₄alkenylene;

C₂-C₄alkenylene which is interrupted once or more than once by a representative selected from the group consisting of oxygen atom, sulfur atom, nitrogen atom, carbonyl radical, thio radical, sulfoxide radical, and a radical of the formula

-Si(OR')(OR")-O-, in which R' and R" are, independently of each other, hydrogen or C₁-C₆alkyl; C₂-C₄alkynylene; C₂-C₄alkynylene which is interrupted once or more than once by a representative selected from the group consisting of oxygen atom, sulfur atom, nitrogen atom, carbonyl radical, thio radical, sulfoxide radical, and a radical of the formula -Si(OR')(OR")-O-, in which R' and R" are, independently of each other, hydrogen or C_1-C₆alkyl; aryl and aralkyl.

21. An oligonucleotide-dendrimer conjugate according to claim 20, wherein the bridging group Z is selected from the group consisting of C₁-C₄alkylene and C₁-C₄alkylene, which is interrupted once or more than once by a representative selected from the group consisting of oxygen atom, sulfur atom, nitrogen atom, carbonyl radical, thio radical, sulfoxide radical and a radical of the formula -Si(OR')(OR")-O-, in which R' and R" are, independently of each other, hydrogen or C₁-C₆alkyl.

22. An oligonucleotide-dendrimer conjugate according to claim 21 , wherein the bivalent bridging group Z is C₁-C₄alkylene or a C₁-C₄alkylene which is interrupted once by an oxygen atom.

23. An oligonucleotide-dendrimer conjugate according to claim 22, wherein the bivalent bridging group Z is -OCH₂- .

24. An oligonucleotide-dendrimer conjugate according to claim 1 , wherein dendrimer is the monovalent residue of a dendrimer of the first to seventh generation.

25. An oligonucleotide-dendrimer conjugate according to claim 24, wherein dendrimer is the monovalent residue of a dendrimer of the first to fifth generation.

26. An oligonucleotide-dendrimer conjugate according to claim 25, wherein dendrimer is the monovalent residue of a dendrimer of the first to fourth generation.

27. An oligonucleotide-dendrimer conjugate according to claim 26, wherein dendrimer is the monovalent residue of a dendrimer of the first to third generation.

28. An oligonucleotide-dendrimer conjugate according to claim 1 , wherein the dendrimer conforms to the formula I

in which x₁ is the initiator core, (I₁) and (I₂) are each a bridging group Z, x₂ and x₃ are each a branching point and E is an end group.

29. An oligonucleotide-dendrimer conjugate according to claim 28, wherein x₁, x₂ and x₃ are benzene, (I₁) and (I₂) are -O-CH₂- and E is H or -OCH₃ .

30. An oligonucleotide-dendrimer conjugate according to claim 1 , wherein the dendrimer is bonded to N, S or O atoms in the 3' or 5' end groups of the oligonucleotide sequence, to C, N or O atoms of nucleic acid bases in or at the end of the sequence, to 2' positions in the furanose ring, to O, S or N atoms in or at the end of the sequence, or to O, S or N atoms of the nucleotide bridging group in the sequence, or to the 6' carbon atom of a carbocyclic nucleotide analogue.

31. An oligonucleotide-dendrimer conjugate according to claim 30, wherein the

dendrimer is bonded to the oligonucleotide sequence via a bridging group B.

32. An oligonucleotide-dendrimer conjugate according to claim 31 , wherein the bridging group B conforms to a group of the formula II

-X_p-[A-X']_n-A'_m- (II) in which X and X' are, independently of each other, a radical which is unsubstituted or is substituted by C₁-C₁₀alkoxy, F, Cl, Br, -CN, C₁-C₁₀alkyl, aryl, hydroxy-C₁- C₁₀-alkyl, amino-C₁-C₁₀alkyl, OH, NR₁₂ or -NO₂ and which is selected from the group consisting of C₁-C₂₀alkylene, C₂-C₁₂alkenylene, C₂-C₁₂alkynylene, C₃- C₈cycloalkylene, C₆-C₁₂arylene and C₇-C₁₂-aralkylene, A and A' are, independently of each other, -O-, -S-, -S-S-, -NR₁₂-CO-NR₁₂-, -NR₁₂-CS-NR₁₂-, -NR₁₂-, -NR₁₂-C(O)-O-, -C(O)O-, -C(O)S-, -C(O)NR₁₂-, -C(S)S-, -C(S)O-, -C(S)NR₁₂-, -SO₂NR₁₂-, -SO₂-, - P(O)(OH)O-, -OP(O)(OH)O-, -P(S)(SH)O-, -OP(S)(SH)O-, -P(S)(OH)O-,

-OP(S)(OH)O-, -P(O)(OH)-NR₁₂-, -OP(O)(OH)-NR₁₂-, -P(S)(SH)-NR₁₂-, -OP(S)(SH)- NR₁₂-, -P(S)(OH)-NR₁₂- or -OP(S)(OH)-NR₁₂-, R₁₂ is H or C₁-C₁₀alkyl, n is a number from 1 to 50, when more than one (A-X') unit is present, the meanings of A and X' in the individual units are identical or different, and m and p are, independently of each other, 0 or 1.

33. An oligonucleotide-dendrimer conjugate according to claim 32, wherein X is a radical which is unsubstituted or is substituted by C₁-C₁₀alkoxy, F, Cl, Br, -CN, C₁-C₁₀alkyl, aryl, hydroxy-C₁-C₁₀alkyl, amino-C₁-C₁₀alkyl, OH, NR₁₂ or -NO₂ and which is selected from the group consisting of C₁-C₂₀alkylene, C₃-C₈cycloalkylene, C₆-C₁₂arylene and C₇-C₁₂aralkylene.

34. An oligonucleotide-dendrimer conjugate according to claim 33, wherein X is a radical which is unsubstituted or is substituted by C₁-C₆alkoxy, F, Cl, Br, -CN, C₁-C₆alkyl, aryl, hydroxy-C₁-C₆alkyl, amino-C₁-C₆alkyl, OH, NR₁₂ or -NO₂ and which is selected from the group consisting of C₁-C₂₀alkylene, C₃-C₈cycloalkylene, C₆-C₁₂arylene and C_7-C₁₂aralkylene.

35. An oligonucleotide-dendrimer conjugate according to claim 33, wherein X is

C₁-C₂₀alkylene.

36. An oligonucleotide-dendrimer conjugate according to claim 35, wherein X is

C₁-C₁₀alkylene.

37. An oligonucleotide-dendrimer conjugate according to claim 36, wherein X is

C₁-C₃alkylene.

38. An oligonucleotide-dendrimer conjugate according to claim 36, wherein X is -CH₂-.

39. An oligonucleotide-dendrimer conjugate according to claim 32, wherein p is 1.

40. An oligonucleotide-dendrimer conjugate according to claim 32, wherein X' is a

radical which is unsubstituted or is substituted by d-C₁₀alkoxy, F, Cl, Br, -CN, C₁- C₁₀alkyl, aryl, hydroxy-C₁-C₁₀alkyl, amino-C₁-C₁₀alkyl, OH, NR₁₂ or -NO₂ and which is selected from the group consisting of C₁-C₂₀alkylene, C₃-C₈cycloalkylene,

C₆-C₁₂arylene and C₇-C₁₂aralkylene.

41. An oligonucleotide-dendrimer conjugate according to claim 40, wherein X' is a

radical which is unsubstituted or is substituted by C₁-C₆alkoxy, F, Cl, Br, -CN, C₁- C₆alkyl, aryl, hydroxy-C₁-C₆alkyl, amino-C₁-C₆alkyl, OH, NR₁₂ or -NO₂ and which is selected from the group consisting of C₁-C₂₀alkylene. C₃-C₈cycloalkylene, C₆- C₁₂arylene and C₇-C₁₂aralkylene.

42. An oligonucleotide-dendrimer conjugate according to claim 40, wherein X' is a

radical which is unsubstituted or is substituted by hydroxy-C₁-C₁₀alkyl, amino-C₁- C₁₀alkyl or OH and is selected from the group consisting of C₁-C₂₀alkylene, C₃- C₈cycloalkylene, C₆-d₂arylene and C₁-C₁₂aralkylene.

43. An oligonucleotide-dendrimer conjugate according to claim 42, wherein X' is a

radical which is unsubstituted or substituted by hydroxy-C₁-C₆alkyl, amino-C₁-C₆alkyl or OH and which is selected from the group consisting of C₁-C₂₀alkylene, C₃-C₈cycloalkylene, C₆-C₁₂arylene and C₇-C₁₂aralkylene.

44. An oligonucleotide-dendrimer conjugate according to claim 43, wherein X' is a

radical which is unsubstituted or is substituted by hydroxy-C₁-C₂alkyl or OH and which is selected from the group consisting of C₁-C₁₀alkylene and C₇-C₁₀aralkylene.

45. An oligonucleotide-dendrimer conjugate according to claim 44, wherein X' is

selected from the group consisting of -(CH₂)₂-, -(CH₂)₆-, -(CH₂)₁₀-, -CH₂CH(OH)CH₂-,

-CH(CH₂OH)CH₂-, and

46. An oligonucleotide-dendrimer conjugate according to claim 32, wherein A is -O-, -NR₁₂-CO-NR₁₂-, -NR₁₂-, -NR₁₂-C(O)-O-, -C(O)O-, -C(O)NR₁₂-, -P(O)(OH)O-,

-OP(O)(OH)O-, -P(O)(OH)-NR₁₂- or -OP(O)(OH)-NR₁₂-.

47. An oligonucleotide-dendrimer conjugate according to claim 46, wherein A is -O-, -NR₁₂-CO-NR₁₂-, -NR₁₂-, -NR₁₂-C(O)-O-, -C(O)O- or -C(O)NR₁₂-.

48. An oligonucleotide-dendrimer conjugate according to claim 47, wherein A is -O-, -C(O)O- or -C(O)NH-.

49. An oligonucleotide-dendrimer conjugate according to claim 32, wherein m is 1.

50. An oligonucleotide-dendrimer conjugate according to claim 32, wherein A' is

-P(O)(OH)O- or -P(S)(OH)-.

51. An oligonucleotide-dendrimer conjugate according to claim 32, wherein the bridging group B is a residue of the formula IIIa, IIIb or IIIc

52. An oligonucleotide-dendrimer conjugate according to claim 32, wherein the bridging group B is a residue of the formula IIId or IIIe

53. A compound of the formula IV dendrimer-X_p-[A-X']_n-R₁ (IV) in which dendrimer is the monovalent residue of a dendrimer of the first to tenth generation, X and X' are, independently of each other, a radical which is

unsubstituted or is substituted by C₁-C₁₀alkoxy, preferably C₁-C₆alkoxy, F, Cl, Br, - CN, C₁-C₁₀alkyl, preferably C₁-C₆alkyl, aryl, hydroxy-C₁-C₁₀alkyl, preferably hydroxy- C₁-C₆alkyl, amino-C₁-C₁₀alkyl, preferably amino-C₁-C₆alkyl, OH, NR₁₂ or -NO₂ and which is selected from the group consisting of C₁-C₂₀alkylene, C₂-C₁₂alkenylene, C₂-C₁₂alkynylene, C₃-C₈cycloalkylene, C₆-C₁₂arylene and Cτ-C₁₂aralkylene, A is -O-, - S-, -S-S-, -NR₁₂-CO-NR₁₂-, -NR₁₂-CS-NR₁₂-, -NR₁₂-, -NR₁₂-C(O)-O-, -C(O)O-, -C(O)S-, -C(O)NR₁₂-, -C(S)S-, -C(S)O-, -C(S)NR₁₂-, -SO₂NR₁₂-, -SO₂-, -P(O)(OH)O-, - OP(O)(OH)O-, -P(S)(SH)O-, -OP(S)(SH)O-, -P(S)(OH)O-, -OP(S)(OH)O-, -P(O)(OH)- NR₁₂-, -OP(O)(OH)-NR₁₂-, -P(S)(SH)-NR₁₂-, -OP(S)(SH)-NR₁₂-, -P(S)(OH)-NR₁₂- or - OP(S)(OH)-NR₁₂-, R₁₂ is H or C₁-C₁₀alkyl, preferably H or C₁-C₆alkyl; p is 0 or 1 ; n' is a number from 0 to 49, where, when more than one (A-X') unit is present, the meanings of Z and X' are identical or different in the individual units; and R₁ is a monovalent functional group.

54. A compound according to claim 53, wherein the monovalent functional group is

selected from the group consisting of -OR₁₀, -SR₁₀, -NCO, -NCS, -NHR₁₁, -C(O)OR₁₁, -C(O)SH, -C(O)Cl, -C(S)SR₁₁, -C(S)OR₁₁, -SO₃R₁₁, -SO₂CI, -OP(O)(OR)(OH), - OP(S)(OR)(OH), -OP(O)(SR)(SH), -OP(O)(OH), -OP(O)(SH),

-OP(OCH₃)N[CH(CH₃)₂]₂, -OP(OCH₂CH₂CN)N[CH(CH₃)₂]₂ and

P(OCH₂CH₂CN)N[CH(CH₃)₂]₂, where R is a phosphate protecting group, R₁₀ is H, -C(O)NH₂, -C(S)NH₂, -C₁-C₆alkyl, -C_xH_2x-NH₂, -C_xH_2x-SH or -(C_xH_2xO)_y-H and R₁₁ is H, -C₁-C₆alkyl, -C_xH_2x-NH₂, -C_xH_2x-SH or -(C_xH_2xO)_y-H, and x is a number from 2 to 6, and y is a number from 1 to 20.

55. A compound according to claim 54, wherein the functional group is selected from the group consisting of -OR₁₀, -SR₁₀, -NCO, -NCS, -NHR₁₁, -C(O)OR₁₁ and -P(O)(OH)₂.

56. A compound according to claim 55, wherein the functional group is selected from the group consisting of -NCS, -C(O)OR₁₁ and -P(O)(OH)₂.

57. A process for preparing an oligonucleotide-dendrimer conjugate according to claim 1 , which comprises reacting a compound of the formula IV according to claim 53 with a compound of the formula Va

R₁-[A-X']_n-A'_m-oligonucleotide (Va) in which X' is a radical which is unsubstituted or is substituted by C₁-C₁₀alkoxy, F, Cl, Br, -CN, C₁-C₁₀alkyl, aryl, hydroxy-C₁-C₁₀alkyl, amino-C₁-C₁₀alkyl, OH, NR₁₂ or -NO₂ and which is selected from the group consisting of C₁-C₁₀alkylene, C₂-C₁₂alkenylene, C₂-C₁₂alkynylene, C₃-C₈cycloalkylene, C₆-C₁₂arylene and C₇-C₁₂aralkylene, A and A' are,independently of each other, -O-, -S-, -S-S-, -NR₁₂-CO-NR₁₂-, -NR₁₂-CS-NR₁₂-. -NR₁₂-, -NR₁₂-C(O)-O-, -C(O)O-, -C(O)S-, -C(O)NR₁₂-, -C(S)S-, -C(S)O-, -C(S)NR₁₂-, - SO₂NR₁₂-, -SO₂-, -P(O)(OH)O-, -OP(O)(OH)O-, -P(S)(SH)O-, -OP(S)(SH)O-.

-P(S)(OH)O-, -OP(S)(OH)O-, -P(O)(OH)-NR₁₂-, -OP(O)(OH)-NR₁₂-, -P(S)(SH)-NR₁₂-, - OP(S)(SH)-NR₁₂-, -P(S)(OH)-NR₁₂- or -OP(S)(OH)-NR₁₂-, R₁₂ is H or C₁-C₁₀alkyl; m is 0 or 1 ; oligonucleotide is a natural, modified or synthetic sequence which is composed of natural, modified or synthetic deoxynucleosides or peptide nucleic acid building blocks which are linked via internucleotide bridges and which encompasses a region which is complementary to a target nucleic acid; n" is a number from 0 to 49, where, when more than one (A-X') unit is present, the meanings of A and X' being identical or different in the individual units; and R_1', is a monovalent functional group.

58. An oligonucleotide-dendrimer conjugate according to claim 1 for use in a therapeutic process for treating diseases in homoiothermic animals including man.

59. A pharmaceutical preparation which comprises an effective quantity of an oligonucleotide-dendrimer conjugate according to claim 1 either alone or together with other active compounds, a pharmaceutical excipient and auxiliary substances, if desired.

60. The use of an oligonucleotide-dendrimer conjugate according to claim 1 as a

diagnostic agent for detecting viral infections or genetically determined diseases.

61. A dendrimer-oligonucleotide conjugate according to any one of claims 1 to 52,

wherein the oligonucleotide has the sequence 5'-TCCCGCCTGTGACATGCATT-3'

(SEQ. ID. NO. 5) and the nucleosides are linked via -P(O)S--.

62. A dendrimer-oligonucleotide conjugate according to claim 61 , which is selected from the group of compounds of the formula (VI) and (VII) (SEQ. ID. NO. 5, nucleosides linked via -P(O)S--, ("PS"))

63. An oligonucleotide-dendrimer conjugate according to claim 62, wherein r is 1.

64. An oligonucleotide-dendrimer conjugate according to claim 62 or 63, wherein s is 2.

65. A dendrimer-oligonucleotide conjugate according to any one of claims 1 to 52, wherein the oligonucleotide has the sequence 5'-GTTCTCGCTGGTGAGTTTCA-3'

(SEQ. ID. NO. 6) and the nucleosides are linked via -P(O)S--.

66. A dendrimer-oligonucleotide conjugate according to claim 65, which is selected from the group of compounds of the formula (VIII) and (IX) (SEQ. ID. NO. 6, nucleosides linked via -P(O)S--, ("PS"))

67. An oligonucleotide-dendrimer conjugate according to claim 66, wherein r is 1.

68. An oligonucleotide-dendrimer conjugate according to claim 66 or 67, wherein s is 2.