WO1998000434A1 - Modified oligonucleotides - Google Patents

Abstract

It is one object of the present invention to provide an oligonucleotide of formula (1): 5'-(U)n-3' in which U is an identical or different radical of a natural or a synthetic nucleoside, wherein the oligonucleotide comprises at least one modified nucleotide dimer comprising two nucleoside analogs connected via an amide-bond that has a certain configuration; the synthesis of these compounds and their use in pharmaceutical preparations.

Description

Modified oligonucieotides

The present invention relates to modified oligonucieotides comprising at least one nucleotide dimer with a modified backbone, to the modified nucleotide dimers in a certain configuration, processes for the preparation of these oligonucieotides or the nucleotide dimers, the use of these oligonucieotides or the nucleotide dimers and pharmaceutical preparations containing the modified oligonucieotides.

Nucleosides and oligonucieotides have acquired wide interest as antiviral active ingredients or because of their capability to interact with nucleic acids ("antisense" oligonucieotides) and the biological activity associated therewith, see, for example, Uhlmann & Peyman, Chemical Reviews (1990), 90, 543-584. To provide nucleosides having novel properties or to improve the interaction of antisense oligonucieotides with natural nucleic acids and their stability to nucleases, the sugar radicals of nucleosides (or the nucleotide units in oligonucieotides) or the internucleotide phosphate bond in oligonucieotides have been modified in very different ways.

Although several modifications have been performed already, as for example in WO-A- 9520597, the importance of a certain configuration at a certain position of the oligonucieotides, and its influence on the hybridization characteristics with DNA/RNA, has not been recognized. Accordingly, the current invention provides oligonucieotides in a certain configuration that are capable of a surprisingly strong hybridization to target RNA or DNA.

Detailed description of the invention

It is one object of the present invention to provide an oligonucleotide of formula 1

5'-(U)„-3' (1) in which U is an identical or different radical of a natural or a synthetic nucleoside, n is an integer from 2 to 200, preferably 2 to 100, more preferred 2 to 50 and most preferred 2 to 20 monomer units; and wherein the oligonucleotide of formula 1 comprises at least one structural unit of formula 2

wherein

R¹ is H, C₁-C₄alkyl or d-dalkoxy; preferred is H or d-C₄alkyl; more preferred is H or methyl; most preferred is H; R² is H, C C₄alkyl, phenyl, Cι-C₄alkyl-phenyl, C₃-C₉heteroaryl, Cι-C₄alkyl-C₃-C₉heteroaryl or an intercalator; wherein the aryl or heteroaryl is unsubstituted or substituted by OH,

R⁴, d-dalkoxy, -O-(CH₂-CH₂-O)_mR⁴, NR⁴ ₂ or NHR⁴; preferred is H, d-dalkyl, phenyl, CrC₄alkyl-phenyl or C₃-C₉heteroaryl; more preferred is H, methyl, ethyl or phenyl; most preferred is H, methyl or phenyl; R³ is C C₄alkyl, unsubstituted or substituted by OH, NR ₂ or NHR⁴; preferred is d-dalkyl; more preferred is methyl or ethyl; most preferred is methyl; R⁴ is H or d-C₄alkyl; preferred is methyl or ethyl; more preferred is methyl; X and Y are independent of one another, H , OH , OR⁴, O-d-C₄alkylNHR⁴, O-C

C₄alkylNR⁴ ₂, -O-(CH₂-CH₂-O)_mR⁴ or -O-CH₂-C(OR⁵)H-CH₂-OR⁶, -O-CH₂-C(OR⁵)H-CH₃; preferred is H, OH, OR⁴, O-d-dalkylNHR⁴, O-d-dalkylNR ₂, -O-(CH₂-CH₂-O)_mR⁴; more preferred is H, OH or OR⁴; O-CH₂CH₂NHR⁴, O-CH₂CH₂NR⁴ ₂, O-CH₂CH₂OR⁴; even more preferred is H , O-C H_3l O-CH₂CH₂OCH₃, O-CH₂CH₂NHCH₃, O -

CH₂CH₂N(CH₃)₂; and most preferred is H, O-CH₃ and O-CH₂CH₂OCH₃; R⁵ is H or Cι-Cι₀alkyl; preferred is H, CH₃ or d-C₄alkyl; more preferred is H, methyl or ethyl; R⁶ is H, CH₃ or an OH-protecting group; m is an integer from 1 to 4; preferred is 1 ; and A and B are, independent of one another, a purine or pyrimidine radical or an analogue thereof; with the proviso that if A and B are thymidine, R¹, R² and X are hydrogen and Y is methoxy, R³ is not methyl.

Beside the presence of one or more structural units of formula (2), the oligonucleotide may be further modified, e.g., by replacement of phosphodiester bonds with -thioate bonds.

Some examples of alkyl, alkoxy, hydroxyalkyl and aminoalkyl, as used throughout the specification, are methyl, ethyl and the isomers of propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl, and also the corresponding alkoxy, hydroxyalkyl and aminoalkyl radicals. The alkyl, alkoxy, hydroxyalkyl and aminoalkyl radicals preferably contain 1 to 4 C atoms like methyl, ethyl, n- and i-propyl, n-, i- and t-butyl, methoxy, ethoxy, aminomethyl, aminoethyl, hydroxymethyl and hydroxyethyl.

Examples of aminoalkyl are also aminomethyl, aminoethyl, 1 -aminoprop-2-yl or -3-yl, 1 -aminobut-2-yl or -3-yl or -4-yl, N-methyl- or N,N-dimethyl- or N-ethyl- or N,N-diethyl- or N-2-hydroxyethyl- or N,N-di-2-hydroxyethylaminomethyl or -aminoethyl or -aminopropyl or -aminobutyl. Examples of hydroxyalkyl are hydroxymethyl, 1 -hydroxyeth-2-yl, 1 -hydroxy- prop-2- or -3-yl, 1 -hydroxybut-2-yl, -3-yl or -4-yl.

Examples of C₆-Cιoaryl are naphthyl and phenyl, wherein phenyl is preferred. The heteroaryl preferably contains 1 to 3 heteroatoms selected from the group consisting of O, S and N, like thienyl, furyl, pyranyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl and pyridazinyl.

A preferred intercalator in connection with the present invention is anthraquinone substituted by a linker, the linker being preferably a chain of 2 to 7 atoms selected from the group consisting of C, N and O, like C₂-C₇alkyi. If A and/or B is a purine radical or an analogue thereof, it can be a radical of the formula 3, 4, 5 or 6.

in which

R⁸ and R⁹ independently of one another are H, OH, SH, NH₂, NHNH₂, NHOH, NHOalkyI having 1 to 12 C atoms, -N=CH-N(d-d₂alkyl)₂, F, Cl, Br, alkyl or hydroxyalkyl or aminoalkyl or alkoxy or alkylthio having 1 to 12 C atoms, preferably 1 to 4 C atoms; phenyl; benzyl; primary amino having 1 to 20 C atoms, preferably 1 to 12 C atoms and more preferably 1 to 4 C atoms or secondary amino having 2 to 30 C atoms, preferably 2 to 12 C atoms and more preferably 2 to 6 C atoms; and

R¹⁶ is as defined below.

The primary amino preferably contains 1 to 12 and particularly preferably 1 to 6 C atoms, and the secondary amino preferably 2 to 12 and particularly preferably 2 to 6 C atoms.

Some examples of alkyl, alkoxy, alkylthio, hydroxyalkyl and aminoalkyl, which preferably contain 1 to 6 C atoms, are methyl, ethyl and the isomers of propyl, butyl, pentyl and hexyl; and also corresponding alkoxy, alkylthio, hydroxyalkyl and aminoalkyl radicals. The alkyl, alkoxy, alkylthio, hydroxyalkyl and aminoalkyl radicals preferably contain 1 to 4 C atoms. Preferred alkyl, alkoxy, alkylthio, hydroxyalkyl and aminoalkyl radicals are methyl, ethyl, n- and i-propyl, n-, i- and t-butyl, methoxy, ethoxy, methylthio and ethylthio, aminomethyl, aminoethyl, hydroxymethyl and hydroxyethyl.

The primary amino and secondary amino can, for example, be radicals of the formula R¹³R¹ N, in which R¹³ and R¹⁴ are independently H, d-C₂oalkyl, -aminoalkyl or -hydroxyalkyl, preferably d-C₁₂alkyl, -aminoalkyl or -hydroxyalkyl and particularly preferably d-C₆alkyl, - aminoalkyl or -hydroxyalkyl; carboxyalkyl or carbalkoxyalkyl, where the carbalkoxy group contains 2 to 8 C atoms and the alkyl group contains 1 to 6, preferably 1 to 4, C atoms; C₂- doalkenyl, preferably C₂-C₁₂alkenyl and particularly preferably C₂-C₆alkenyl; phenyl, mono- or di(d-C₄alkyl- or -alkoxy)phenyl, benzyl, mono- or di(d-C₄alkyl- or -alkoxy)benzyl; or 1 ,2-, 1 ,3- or 1 ,4-imidazolyl-Cι-C₆alkyl; or R¹³ and R ⁴ together are tetra- or pentamethylene, 3- oxa-1 ,5-pentylene, -CH₂-NR ⁵-CH₂CH₂- or -CH₂CH₂-NR¹⁵-CH₂CH₂-, in which R¹⁵ is H or d- dalkyl. The amino group in the aminoalkyl is unsubstituted or substituted by one or two d- dalkyl or -C₁-C₄hydroxyalkyl groups. The hydroxyl group in hydroxyalkyl is unsubstituted or etherified with d-C₄alkyl.

Examples of alkyl have been given previously. Examples of aminoalkyl are aminomethyl, aminoethyl, 1 -aminoprop-2-yl or -3-yl, 1 -aminobut-2-yl or -3-yl or -4-yl, N-methyl- or N,N-di- methyl- or N-ethyl- or N,N-diethyl- or N-2-hydroxyethyl- or N,N-di-2-hydroxyethylamino- methyl or -aminoethyl or -aminopropyl or -aminobutyl. Examples of hydroxyalkyl are hydroxymethyl, 1-hydroxyeth-2-yl, 1 -hydroxyprop-2- or -3-yl, 1 -hydroxybut-2-yl, -3-yl or -4-yl. Examples of carboxyalkyl are carboxymethyl, carboxyethyl, carboxypropyl and carboxy- butyl, and examples of carbalkoxyalkyl are these carboxyalkyl groups esterified with methyl or ethyl. Examples of alkenyl are allyl, but-1 -en-3-yl or -4-yl, pent-3- or 4-en-1 -yl or -2-yl, hex-3- or -4- or -5-en-1 -yl or -2-yl. Examples of alkyl- and alkoxyphenyl or benzyl are methylphenyl, dimethylphenyl, ethylphenyl, diethylphenyl, methylbenzyl, dimethylbenzyl, ethylbenzyl, diethylbenzyl, methoxyphenyl, dimethoxyphenyl, ethoxyphenyl, diethoxyphenyl, methoxybenzyl, dimethoxybenzyl , ethoxybenzyl, diethoxybenzyl. Examples of imidazolylalkyi, in which the alkyl group preferably contains 2 to 4 C atoms, are 1 ,2-, 1 ,3- or 1 ,4-imidazolylethyl or -n-propyl or -n-butyl. R¹⁵ is preferably H, methyl or ethyl.

Preferred examples of primary amino and secondary amino are methyl-, ethyl-, dimethyl-, diethyl-, allyl-, mono- or di(1 -hydroxyeth-2-yl)-, phenyl- and benzylamino, acetylamino, isobutyrylamino, benzoyiamino, phenoxyacetylamino, 4-tert.-butylphenoxyacetylamino,

In a preferred embodiment R⁸ and R⁹ independently of one another are H, F, Cl, Br, OH, SH, NH₂, NHOH, NHNH₂, methyl, methylamino, dimethylamino, benzoyiamino, isobutyrylamino, methoxy, ethoxy, methylthio, phenoxyacetylamino, 4-tert.-butylphenoxyacetylamino,

Besides purine, some examples of analogues of the purine series are adenine, N-methyl- adenine, N-benzoyladenine, 2-methylthioadenine, 2-amino-6-chloropurine, 2-amino-6- methylthiopurine, 2-aminopurine, hypoxanthine, 2-aminoadenine, 6-hydroxypurine, guanine and N-isobutyrylguanine. More preferred are adenine, N-methyladenine, N-benzoyladenine, 2-methylthioadenine, 2-aminoadenine, 2-hydroxypurine, 2-amino-6-chloropurine, 2-amino-6-methylthiopurine, guanine, N-isobutyrylguanine, 2-aminopurine and hypoxanthine. Adenine, 2-aminoadenine, 2-aminopurine, guanine and hypoxanthine are particularly preferred.

If A or B in formula 2 is an analogous pyrimidine radical, it is preferably uracil, thymine or cytosine radicals of formulae 9 or 10

in which R¹⁶ and R¹⁷ independently of one another are is H, F, Cl, Br, CONH₂, alkyl, propinyl or hydroxyalkyl or aminoalkyl or alkoxy or alkylthio having 1 to 12 C atom; phenyl; benzyl; primary amino having 1 to 20 C atoms or secondary amino having 2 to 30 C atoms; the hydrogen atoms of the NH₂ group in formula 10 are unsubstituted or substituted by C C₆alkyl, benzoyl or benzyl; and the dihydro derivatives of the radicals of formulae 9 and 10:

R¹⁶ is preferably H, F, Cl, Br, d-dalkyl, C,-C₆alkenyl, d-C₆alkinyl, d-C₆hydroxyalkyl, d- C₆aminoalkyl, NHd-C₄alkyl, N(d-C₄alkyl)₂, propinyl; and more preferably H, F, Cl, Br, methyl, ethyl, or propinyl; and most preferably H, propinyl or methyl;

R¹⁷ is preferably H, F, Cl, Br, C C₆alkyl or d-C₆alkoxy, d-C₆hydroxyalkyl, d-C₆aminoalkyl, NH₂, NHd-dalkyl, N(d-C₄alkyl)₂, and propinyl; and more preferably H , F, Cl, Br, methyl, ethyl, and propinyl; and most preferably H, propinyl or methyl.

Some examples of pyrimidine analogues are uracil, thymine, cytosine, 5-fluorouracil, 5- chlorouracil, 5-bromouracil, 5-methylcytosine, 5-propinyluracil, 5-propinylcytosine and their base protected derivatives.

Especially preferred structural units are of formula B8, B28 and B49.

Another object of the present invention is a nucleoside dimer of the formula 12, that can be used, for example, as a building block for the construction of oligonucieotides as shown in formula 1.

wherein

R¹, R², R³, X,Y, m, A and B are as defined above;

R^1Θ and R¹⁹ independent of one another are H, an OH-protecting group or a phosphorus- containing, nucleotide-bridge-group-forming radical.

In a preferred embodiment R¹⁸ is H or an OH-protecting group and R¹⁹ is a phosphorus- containing, nucleotide-bridge-group-forming radical.

Suitable Protective groups and processes for derivatisation of the hydroxyl groups with such protective groups are generally known in sugar and nucleotide chemistry and described, for example, by B. T. Greene, Protective Groups in Organic Synthesis, Wiley Interscience, New York (1991 ). Examples of such protective groups are: linear or branched d-C₈alkyl, particularly d-dalkyl, for example methyl, ethyl, n- and i-propyl, n-, i- and t-butyl; C₇- Cι₈aralkyl, for example benzyl, methylbenzyl, dimethylbenzyl, methoxy benzyl, dimethoxy- benzyl, bromobenzyl, diphenylmethyl, di(methylphenyl)methyl, di(dimethylphenyl)methyl, di(methoxyphenyl)methyl, di(dimethoxyphenyl)methyl, trityl, tri(methylphenyl)methyl, tri(di- methylphenyl)methyl, methoxyphenyl(diphenyl)methyl, di(methoxyphenyl)phenylmethyl, tri(dimethoxyphenyl)methyl, tri(methoxyphenyl)methyl; triphenylsilyl, alkyldiphenylsilyl, dialkylphenylsilyl and trialkylsilyl having 1 to 20, preferably 1 to 12 and particularly preferably 1 to 8, C atoms in the alkyl groups, for example trimethylsilyl, triethylsilyl, tri-n- propylsilyl, i-propyldimethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, n-octyldimethylsilyl, (1 ,1 ,2,2-tetramethylethyl)dimethylsilyl; -(d-Cβalky sSi-O-SKd-Cβalkyl);.-, in which alkyl, for example, is methyl, ethyl, n- or i-propyi, n-, i- or t-butyl; C₂-C₁₂acyl, particularly C₂-C_θacyl, for example acetyl, propanoyl, butanoyl, pentanoyl, hexanoyl, benzoyl, methoxybenzoyl, methylbenzoyl, chlorobenzoyl and bromobenzoyl; R¹²-SO₂-, in which R¹² is d-Cι₂alkyl, particularly C C₆alkyl, C₅- or C₆cycloalkyl, phenyl, benzyl, Cι-Cι₂alkylphenyl and particularly Cι-C₄alkylphenyl, or d-C₁₂alkylbenzyl and particularly d-C₄alkylbenzyl, or halophenyl or halobenzyl, for example methyl-, ethyl-, propyl-, butyl-, phenyl-, benzyl-, p-bromo-, p- methoxy- or p-methylphenylsulfonyl; unsubstituted or F-, CI-, Br-, d-dalkoxy-, tri(d- C alkyl)silyl- or Cι-C₄alkylsulfonyl-substituted d-C₁₂alkoxycarbonyl, preferably d-C₈alkoxy- carbonyl, for example methoxy-, ethoxy-, n- or i-propoxy- or n-, i- or t-butoxycarbonyl, 2- trimethylsilylethoxycarbonyl, 2-methylsulfonylethoxycarbonyl, or phenoxycarbonyl or benzyl- oxycarbonyl which is unsubstituted or substituted as for alkoxycarbonyl, for example methyl- or methoxy- or chlorophenoxycarbonyl or -benzyloxycarbonyl, and also 9-fluorenylmethyl- oxycarbonyl.

If the protecting group is alkyl, it can be substituted by F, Cl, Br, Cι-C₄alkoxy, phenoxy, chlorophenoxy, methoxyphenoxy, benzyloxy, methoxybenzyloxy or chlorophenoxy.

In a preferred embodiment, the protective groups are, independently of one another, linear or branched d-C₄alkyl, C₇-dβaralkyl, trialkylsilyl having 1 to 12 C atoms in the alkyl groups; -(d-C₄alkyl)₂Si-O-Si(C C₄alkyl)₂ like (CH₃)₂Si-O-Si(CH₃)₂- and -(i-C₃H₇)₂Si-O-Si(iC₃H₇)₂-; C₂- C₈acyl, R¹²-SO₂-, in which R¹² is d-C₆alkyl; phenyl or benzyl unsubstituted or substituted with F, Cl or Br; Cι-C alkylphenyl; d-C alkylbenzyl; Cι-C₈alkoxycarbonyl; phenoxycarbonyl; benzyloxycarbonyl or 9-fluorenylmethoxycarbonyl. In a particularly preferred embodiment, the protective groups are methyl, ethyl, n- or i- propyl, n-, i- or t-butyl; benzyl, methylbenzyl, dimethylbenzyl, methoxybenzyl, dimethoxy- benzyl, bromobenzyl; diphenylmethyl, di(methylphenyl)methyl, di(dimethylphenyl)methyl, di(methoxyphenyl)methyl, di(methoxyphenyl)(phenyl)methyl, trityl, tri(methylphenyl)methyl, tri(dimethylphenyl)methyl, tri(methoxyphenyl)methyl, tri(dimethoxyphenyl)methyl; trimethyl- silyl, triethylsilyl, tri-n-propylsilyl, i-propyldimethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, n-octyldimethylsilyl, (1 ,1 ,2,2-tetramethylethyl)dimethylsilyl, -(i-C₃H₇)₂Si-O-Si(i-C₃H₇)₂-,-(CH₃)₂- Si-O-Si(CH₃)₂-; d-C₈acyl groups like acetyl, propanoyl, butanoyl, pentanoyl, hexanoyl, benzoyl, methylbenzoyl, methoxybenzoyi, chlorobenzoyl and bromobenzoyl; methyl-, ethyl-, propyl-, butyl-, phenyl-, benzyl-, p-bromo-, p-methoxy- and p-methylphenyisulfonyl; methoxy-, ethoxy-, n- or i-propoxy- or n-, i- or t-butoxycarbonyl, or phenoxycarbonyl, benzyloxycarbonyl, methyl- or methoxy- or chlorophenoxycarbonyl or -benzyloxycarbonyl or 9- fluorenylmethoxycarbonyl.

In an especially preferred embodiment R¹⁸ is

CN

,o.

CH, ,CH„

R¹⁹ is ^CH3 ^CH3

A phosphorus-containing, nucleotide-bridge-group-forming radical may correspond to formula P1 or P2

wherein

Y_a is hydrogen, Cι-Cι₂alkyl, C₆-C₁₂aryl, C₇-C₂₀aralkyl, C₇-C₂oalkaryl, -OR_b, -SR_b, secondary amino, O^*M⁺ or S^*M⁺;

X_a is oxygen or sulfur; R_a is hydrogen, M⁺, d-C₁₂alkyl, C₂-C₁₂alkenyl or C₆-Cι₂aryl, or the group R_aO- is N-hetero- aryl-N-yl having 5 ring members and from 1 to 3 nitrogen atoms; R_b is hydrogen, Cι-C ₂alkyl or C₆-d₂aryl; and

M⁺ is Na⁺, K⁺, Li⁺, NH₄ ⁺ or primary, secondary, tertiary or quaternary ammonium; alkyi, aryl, aralkyl and alkaryl in Y_a, R_a and R_b being unsubstituted or substituted by alkoxy, alkylthio, halogen, -CN, -NO₂, phenyl, nitrophenyl or halophenyl.

Y_a contains as secondary amino preferably from 2 to 12 and especially from 2 to 6 carbon atoms.

The secondary amino may be, for example, a radical of the formula R_cRdN, wherein R_c and R_d, are independently of one another is Cι-C₂₀-, preferably d-C₁₂- and especially C C₆- alkyl; d-C₂₀-, preferably Cι-C₁₂- and especially Ci-d-aminoalkyl; or Cι-C₂o-, preferably d- C₁₂- and especially d-C₆-hydroxyalkyl; carboxyalkyl or carbalkoxyalkyi, the carbalkoxy group containing from 2 to 8 carbon atoms and the alkyl group from 1 to 6, preferably from 1 to 4, carbon atoms; C₂-C₂₀-, preferably C₂-C₁₂- and especially C₂-C₆-alkenyl; phenyl, mono- or di-(Cι-C₄alkyl or d-C₄alkoxy)phenyl, benzyl, mono- or di-(C C₄alkyl or d-C alk- oxy)benzyl; or 1 ,2-, 1 ,3- or -imidazolyl-d-Cealkyl, or R_c and R_d together are tetra- or penta-methylene, 3-oxa-1 ,5-pentylene, -CH₂-NR_e-CH₂CH₂- o r - C H ₂CH₂-NR_e-CH₂CH₂-, wherein R_β is hydrogen or Cι-C₄alkyl. The amino group in aminoalkyl may be substituted by one or two d-C alkyl or d-C₄hydroxyalkyl groups. The hydroxy group in hydroxyalkyl may be etherified by d-dalkyl.

Primary, secondary, tertiary and quaternary ammonium for Y_a in connection with the definition of M⁺ is to be understood as being an ion of the formula R_fR_gR_hRjN⁺, wherein R₍ is

Cι-C₂₀-, preferably d-Cι₂- and especially Cι-C₆-alkyl, d-C₂₀-, preferably d-C₁₂- and especially d-Cβ-aminoalkyl, Cι-C₂o-, preferably d-C₁₂- and especially C C₆-hydroxyalkyl; carboxyalkyl or carbalkoxyalkyi, the carbalkoxy group containing from 2 to 8 carbon atoms and the alkyl group from 1 to 6, preferably from 1 to 4, carbon atoms; C₂-C₂₀-, preferably C₂- d₂- and especially C₂-C₆-alkenyl; phenyl, mono- or di-(d-C₄alkyl or d-C₄alkoxy)phenyl, benzyl, mono- or di-(d-C₄alkyl or Cι-C₄alkoxy)benzyl; or 1 ,2-, 1 ,3- or 1 ,4-imidazolyl-d- C₆alkyl, and R_g, R_h and Ri are each independently of the others hydrogen or have the definition of R,, or R₍ and R_g together are tetra- or penta-methylene, 3-oxa-1 ,5-pentylene, - CH₂-NR_e-CH₂CH₂- or -CH₂CH₂-NR_e-CH₂CH₂-, wherein R_e is hydrogen or C C₄alkyl, and R_h and R, each independently of the other have the definition of R,. The ammo group in aminoalkyl may be substituted by one or two C,-C alkyl or d-C₄hydroxyalkyl groups The hydroxy group in the hydroxyalkyl may be etherified by d-C₄alkyl.

Examples of carboxyalkyl are carboxymethyl, carboxyethyl, carboxypropyl and carboxy- butyl, and examples of carbalkoxyalkyi are those carboxyalkyl groups esterified by methyl or ethyl. Examples of alkenyl are allyl, but-1-en-3-yl or -4-yl, pent-3- or -4-en-1 -yl or -2-yl, hex- 3- or -4- or -5-en-1 -yl or -2-yl. Examples of alkyl- and alkoxy-phenyl and alkyl- and alkoxy- benzyl are methylphenyl, dimethylphenyl, ethylphenyl, diethylphenyl, methylbenzyl, dimethylbenzyl, ethylbenzyl, diethylbenzyl, methoxyphenyl, dimethoxyphenyl, ethoxyphenyl, diethoxyphenyl, methoxybenzyl, dimethoxybenzyl, ethoxybenzyl and diethoxybenzyl. Examples of imidazolylalkyi in which the alkyl group preferably contains from 2 to 4 carbon atoms are 1 ,2-, 1 ,3- or 1 ,4-ιmidazolyl-ethyl or -n-propyl or -n-butyl. R_e is preferably hydrogen, methyl or ethyl.

Preferred examples of primary ammo and secondary amino are methyl-, ethyl-, dimethyl-, diethyl-, diisopropyl, mono- or dι-(1 -hydroxy-eth-2-yl)-, phenyl- and benzyl-ammo, acetyl- amino and benzoyiamino and pipendinyl, piperazinyl and morpholinyl

Preferred examples of primary and secondary ammonium are methyl-, ethyl-, dimethyl-, diethyl-, diisopropyl-, mono- or dι-(1 -hydroxy-eth-2-yl)-, phenyl- and benzyl-ammonium

Examples of Y_a, R_a and R_b as alkyl are methyl, ethyl and the isomers of propyl, butyl, pentyl, hexyl, heptyl and octyl; examples of Y_a, R_a and R_b as aryl are phenyl and naphthyl; examples of R_a as alkenyl are allyl and (d-C₄alkyl)CH=CH-CH₂-, examples of Y_a as aralkyl are phenyl-C_nH_2π- wherein n is a number from 1 to 6, especially benzyl; examples of Y_a as alkaryl are mono-, di- and tri-(d-C a!kyl)phenyl. Preferred substituents are chlorine, bromine, methoxy, -NO₂, -CN, 2,4-dichlorophenyl and 4-nιtrophenyl. Examples of R_b are 2,2,2-trιchloroethyl, 4-chlorophenyl, 2-chlorophenyl and 2,4-dιchlorophenyl; and examples of R_bO- as N-heteroaryl are pyrrol-N-yl, tπazol-N-yl and benzotπazol-N-yl.

In an even more preferred form, R_a is β-cyanoethyl and Y_a is dι(ιsopropylamιno) ln an especially preferred form the dinucleoside analog is of formula C8, C28 or C49

The invention further relates to a process for the preparation of compounds of the formula 12, which is characterized in that a compound of the formula 14

wherein

R¹, X and A are as defined above; and

R²⁷ is H or an OH-protecting group as defined above; and

R²⁹ is H or an ester activating group like C₆F₅, p-NO₂-phenyl, hydroxybenzotriazol-1 -yl and

is reacted with a compound of the formula 15,

wherein

R², R³, Y and B are as defined above; and

R²⁸ is H, an OH-protecting group as defined above, or a phosphorus-containing, nucleotide- bridge-group-forming radical; if required (R²⁹ = H) in the presence of a condensing agent like, e.g., dicyclohexylcarbodi- imide, TBTU (benzotriazol-1 -yl-tetramethyluronium tetrafluoroborate) or HBTU (hexa- fluorophosphate).

Compounds of formulae 14 and 1 5 can be prepared, for example, according to De Mesmaeker et al., Angew. Chem. Int. Ed. Engl. (1994), 33, 226-229 or Pudlo & Townsend, Tetrahedron Lett. (1990), 31 , 3101.

The temperature in the synthesis reaction can be from -80 to 150°C, preferably 0 to 100°C.

In general, solvents are used which are protic and/or aprotic, and particularly preferably dipolar. Examples of solvents which can be employed on their own or as a mixture of at least two solvents are ethers (dibutyl ether, tetrahydrofuran, dioxane, diethylene glycol dimethyl ether, ethylene glycol dimethyl or diethyl ether, diethylene glycol diethyl ether, triethylene glycol dimethyl ether), halogenated hydrocarbons (methylene chloride, chloroform, 1 ,2- dichloroethane, 1 ,1 ,1 -trichloroethane, 1 ,1 ,2,2-tetrachloroethane), carboxylic acid esters and lactones (ethyl acetate, methyl propionate, ethyl benzoate, 2-methoxyethy! acetate, methoxymethyl acetate, γ-butyrolactone, δ-valerolactone, pivalolactone), carboxamides and lactams (N,N-dimethylformamide, N,N-diethylformamide, N,N-dimethylacetamide, tetra- methyiurea, hexamethylphosphoramide, γ-butyrolactam, e-caprolactam, N-methylpyrroli- done, N-acetylpyrrolidone, N-methylcaprolactam), sulfoxides (dimethyl sulfoxide), sulfones (dimethyl sulfone, diethyl sulfone, trimethyiene sulfone, tetramethylene sulfone), tertiary amines (triethylamine, N-methylpiperidine, N-methylmorpholine), aromatic hydrocarbons, for example benzene or substituted benzenes (chlorobenzene, o-dichlorobenzene, 1 ,2,4- trichlorobenzene, nitrobenzene, toluene, xylene) and nitriles (acetonitrile, propionitrile, benzonitrile, phenylacetonitrile), and also aliphatic or cycloaliphatic hydrocarbons (pentane, petroleum ether, hexane, cyclohexane and methylcyclohexane).

An object of the present invention is the use of a dimer of formula 12 for the preparation of oligonucieotides which comprise one or more identical or different dimer units of formula 12.

The oligonucieotides according to the invention can be prepared in a manner known per se by various processes, preferably on a solid support. For details see for example Gait, Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford (1984).

The oligonucieotides of the formula 1 and the dimeres of formula 12 can be used in a method of treatment. They have, e.g., antiviral and antiproliferative properties. The oligonucieotides and dimeres according to the invention have a surprisingly high stability to degradation by nucieases. A very good pairing with complementary nucleic acid strands, particularly of the RNA type, is also observed. The oligonucieotides according to the invention are therefore particularly suitable for antisense technology, i.e. for inhibition of the expression of undesired protein products due to the binding to suitable complementary nucleotide sequences in nucleic acids (see EP-A-266099, WO-A-8707300 and WO-A- 8908146). They can be employed for the treatment of infections and diseases, for example by blocking the expression of bioactive proteins at the nucleic acid stage (for example oncogenes) . The oligonucieotides according to the invention are also suitable as diagnostics and can be used as gene probes for the detection of viral infections or of genetically related diseases by selective interaction at the single- or double-stranded nucleic acid stage. In particular - due to the increased stability to nucieases - diagnostic use is not only possible in vitro but also in vivo (for example tissue samples, blood plasma and blood serum). Use possibilities of this type are described, for example, in WO-A- 9106556.

The invention relates to the use of the oligonucieotides according to the invention as diagnostics for the detection of viral infections or of genetically related diseases.

The invention also relates to the oligonucieotides of the formula 1 and dinucleosides of formula 12, according to the invention, for use in a therapeutic process for the treatment of diseases in mammals including humans by means of inactivation of nucleotide sequences in the body. The dose when administered to mammals of about 70kg body weight can be, for example, 0.01 to 1000mg per day. Administration is preferably effected parenterally, for example intravenously or intraperitoneally, in the form of pharmaceutical preparations.

The invention further relates to a pharmaceutical preparation comprising an effective amount of an oligonucleotide of the formula 1 or dimeres of formula (12) on its own or together with other active ingredients, a pharmaceutical carrier in a customary amount and, if appropriate, excipients.

The pharmacologically active oligonucieotides or dimeres according to the invention can be used in the form of parenterally administrable preparations or of infusion solutions. Solutions of this type are preferably isotonic aqueous solutions or suspensions, it being possible to prepare these before use, for example in the case of lyophilized preparations which contain the active substance on its own or together with a carrier, for example mannitol. The pharmaceutical preparations can be sterilized and/or contain excipients, for example preservatives, stabilisers, wetting and/or emulsifying agents, solubilisers, salts for regulating the osmotic pressure and/or buffers. The pharmaceutical preparations, which if desired can contain further pharmacologically active substances such as, for example, antibiotics, are prepared in a manner known per se, for example by means of conventional dissolving or iyophilizing processes, and contain about 0.1 % to 90%, in particular from about 0.5% to about 30%, for example 1 % to 5% of active substance(s).

The examples below illustrate the invention.

The following abbreviations are used in the examples:

Ac acetyl

Bn: benzyl

DMT: dimethoxy trityl

HV: high vacuum

Me: methyl pMeOBOM (p-methoxyphenyl)-methoxymethyl

(MeO)Bn (p-methoxyphenyl)-methyl nBu NF: tetrabutyl ammonium fluoride

O-Ac: acetate Ph: phenyl pMeOBOM: p-methoxybenzyloxybenzyl

RT: room temperature

T: thymin-1 -yl tBuPh₂Si: tert. butyldiphenylsilyl

Ts: p-toiuenesulfonyl

TTTr: tris tert. butyl trityl

A) Preparation of Modified Nucleosides and Dinucleotide Analogs Example A1 : Preparation of compound (Aβ)

(I) For preparation of the aldehyde I see: J. Lebreton, A. De Mesmaeker, A. Waldner Synlett,

1994, 54.

A solution of dry CeCI₃ (31 .8 g, 1 28.7 mmol) in THF (300 ml) at -78°C is treated with CHgMgBr (46.8 ml, 3M solution in Et₂0, 140.4 mmol) and stirred for 2.5 h at -78°C. A solution of aldehyde I (5.6 g, 11.7 mmol) is added and stirring is continued for 2 h at -78°C. The reaction mixture is poured into a saturated, aqueous solution of KHSO₄ and extracted with CH₂CI₂ (3x). The combined organic layers are washed with Brine, dried (Na₂SO₄), concentrated and purified by flash chromatography (silica, 25-50% EtOAc in hexane to give compound A1 (3.3 g, 56%).

¹H NMR (250 MHz, CDCI₃): δ = 6.2 (m, 1 H, H-C(1 ')), mixture of diastereomers.

To a solution of compound A1 (3.0 g, 6.06 mmol) in pyridine (20 ml) is added MeSO₂CI and the reaction is stirred at 0°C for 1.5 h. The reaction mixture is diluted with CH₂CI₂ (100 ml), washed with aqueous citric acid and brine, dried (Na₂SO₄), concentrated and purified by flash chromatography (50% EtOAc in hexane) to give compound A2 (2.16 g, 63%).

¹H NMR (250 MHz, CDCI₃): δ = 2.9 (2s, 3H, CH₃SO₂), mixture of diastereomers. Ms(FD): 573 (M)

To a solution of compound A2 (2.0 g, 3.48 mmol) in DMF (10 ml) is added NaN₃ (1.704 mg, 26.2 mmol) and the reaction mixture is stirred for 6 h at 65°C. The reaction mixture is poured into a saturated, aqueous solution of NH₄CI and extracted with EtOAc (3x). The combined organic layers are washed with Brine, dried (Na₂SO₄), concentrated and purified by flash chromatography (silica, 35-40% EtOAc in hexane) to give compound A3 (1 .32 g, 72%).

¹H NMR (250 MHz, CDCI₃): δ = 6.45 (m, 1 H, H-C(1 ')), mixture of diastereomers. Ms(CI): 537 (M+NH4)

A4 A4a

To a solution of compound A3 (1 .3 g, 2.51 mmol) in MeOH (40 ml) is added SnCI₂2 H₂O (2.54 g, 1 1.3 mmol). The reaction mixture is stirred for 28 h at 25°C. The reaction mixture is neutralized with saturated, aqueous solution of Na₂CO₃ and concentrated. The mixture is diluted with saturated, aqueous solution of Na₂CO₃ and extracted with CH₂CI₂ (3x). The combined organic layers are washed with Brine, dried (Na₂SO₄), concentrated and purified by flash chromatography (silica, 5-10 % MeOH in CH₂CI₂) to give the two diastereomeric compounds A4a (R-C(5') configuration,458.8 mg, 37%) and A4 (S-C(5'), configuration 133.8 mg, 1 1 %).

A4a: ¹H NMR (500 MHz, CDCI₃): δ = 3.79 dd, 1 H, H-C(4')); MS(EI): 494 (M+H) A4: ¹H NMR (500 MHz, CDCI₃): δ = 3.70 dd, 1 H, H-C(4')); MS(EI): 494 (M+H)

A solution of carboxylic acid II (cf. A. De Mesmaeker, A. Waldner, J. Lebreton, P. Hoffmann, V. Fritsch, R. M. Wolf, S. M. Freier, Angew. Chem. Int. Ed. 1994, 33, 226.) (142 mg, 0.272 mmol, dried over P₂O₅ on HV, 16.0 h) in CH₃CN (2 ml) is treated with Et₃N (30 mg, 0.299 mmol), O-(1 -benztriazol-1 -yl)-N,N,N,N-tetramethyluroniumtetrafluoroborat (95 mg, 0.299 mmol) and hydroxybenztriazol (18 mg, 0.135 mmol). The reaction mixture is stirred for 2 h. A solution of amine A4a (133 mg, 0.271 mmol, dried over P₂O₅ on HV, 16.0 h) in CH₃CN (2 ml) and Et₃N (30 mg, 0.299 mmol) are added to the reaction mixture and stirring is continued for 3 h. The reaction mixture is poured into aqueous, saturated NaH₂PO - solution and concentrated. The aqueous phase is extracted with CH₂CI₂ (3x), the combined organic layers are washed with aqueous, saturated NaH₂PO -solution, brine, dried (Na₂SO₄), concentrated and purified by flash chromatography (5% MeOH in CH₂CI₂) to give compound A5 (268 mg, 99 %).

¹H NMR (500 MHz, CDCI₃): δ = 6.23, 5.58 (2dd, 2H, 2x H-C(1')); MS(EI): 996 (M-H)

A solution of compound A5 (265 mg, 0.266 mmol) in THF (3 ml) is treated with TBAF (0.58 ml of 1 .0 M solution in THF, 0.58 mmol) and stirred at 25°C for 4.5 h. The reaction is concentrated and purified by flash chromatography (10 - 20% MeOH in CH₂CI₂) to give compound A6 (123 mg, 89%). ¹H NMR (400 MHz, D₆-DMSO): δ = 6.07, 5.94 (2dd, 2H, 2x H-C(1 ')); MS(EI): 520 (M-H)

A solution of compound A6 (120 mg, 0.230 mmol) in pyridine (3 ml) is treated with 4,4'- dimethoxytriphenylmethylchloride (233 mg, 0.690 mmol) and stirred for 24 h at 25°C. The reaction mixture is poured into aqueous, saturated NaHCO₃-solutioπ, extracted with CH₂CI₂ (3x), the organic layers are washed with brine, dried (Na₂SO ), concentrated, coevaporated with toluene (3x) and purified by flash chromatography (10-20% MeOH in EtOAc, 1 % Et N) to give compound A7 (151 mg, 80 %).

H NMR (250 MHz, CDCI3): δ = 6.08, 5.85 (2dd, 2H, 2x H-C(1 ')); MS(EI): 822 (M-H)

Alcohol A7 (108 mg, 0.130 mmol), dissolved in CH₂CI₂ (2ml), is added to a solution of di- isopropylammon i um tetrazolide ( 1 5 mg, 0.088 mmol) and cyanoethoxy-bis- diisopropylamino-phosphine (58 mg, 0.195 mmol) in CH₂CI₂ (2 ml) at 25°C. The reaction mixture is stirred for 3 h, poured into aqueous, saturated NaHCO₃-solution, extracted with CH₂CI₂ (3x), the organic layers are washed with brine, dried (Na₂SO₄), concentrated, and purified by flash chromatography (1 -10 % MeOH in EtOAc, 1 % Et₃N) to give compound A8 (120 mg, 90 %).

³¹P NMR (101 MHz, CDCI₃): δ = 149.3, 149.0 ( 2 diastereomers); MS(EI): 1023 (M-H)

Example A2: Preparation of compound A28)

A solution of compound III (cf. D. C. Baker, D. Horton, CG. Tindal Methods Carbohydr. Chem. 1972, 7, 3) (47.5 g, 0.182 mol) in THF (70 ml) is added to a suspension of NaH (8.76 g, 55%, 0.201 mol, washed with hexane) in THF (1 10 ml) at 0°C. The reaction is stirred for 1.0 h at 0°C and 0.5 h at 25°C. Benzylbromide (46.7 g, 0.273 mol) and Bu₄NI (3.36 g, 9.1 mmol) are added to the reaction mixture and stirring is continued for 1.0 h at 25°C. The reaction mixture is poured into a saturated, aqueous solution of NH CI and extracted with EtOAc (3x) . The combined organic layers are washed with Brine, dried (Na₂SO ), concentrated and purified by flash chromatography (silica, 20% EtOAc in hexane) to give compound A9 (55.0 g, 86%)

¹H NMR (500 MHz, CDCI₃): δ = 1.60, 1.40, 1.38, 1.37 (4s, 12H, CH₃); MS (FD): 350 (M)

Compound A9 (55.0 g, 0.157 mol) is dissolved in AcOH/H₂O (9/1 , 1 105 ml) and stirred for 2.0 h at 40°C. The reaction mixture is concentrated coevaporated with toluene (3x) and purified by flash chromatography (silica, 65% EtOAc in hexane) to give diol A10 (29.0 g, 60%).

¹H NMR (500 MHz, CDCI₃): δ = 1.60, 1.37 (2s, 6H, CH₃); MS(FD): 310 (M)

A solution of compound A10 (29.0 g, 93.5 mmol) in pyridine (250 ml) is treated with toluene- 4-sulfonyl-chloride (25.0 g, 130.9 mmol) and DMAP (1.1 g, 9.4 mmol) at 0°C. The reaction is stirred for 4.0 h at 25°C, quenched with MeOH (1 1 ml), stirred for additional 0.3 h, concentrated, coevaporated with toluene (2x) and purified by flash chromatography to give compound A11 (36.8 g, 85%)

¹H NMR (500 MHz, CDCI₃): δ = 1.57, 1.35 (2s, 6H, CH₃); MS(FD): 464 (M)

A solution of compound A11 (1 1.7 g, 25.3 mmol) in DME (83 ml, degassed with Argon) is treated with Nal (11.4 g, 76.0 mmol), Bu₃SnH (1 1.1 g, 38.0 mmol) and AIBN (410 g, 0.25 mmol) and stirred for 1 .0 h at 80°C. The reaction mixture is adsorbed onto silica gel, concentrated and purified by flash chromatography (silica, 30% EtOAc in hexane) to give compound A12 (7.5 g, 73%).

¹H NMR (400 MHz, CDCI₃): δ = 1.60, 1.37 (2s, 6H, CH₃); 1.23 (d, J = 6 Hz, 3H, H-C(6')) MS (CI): 312 (M+NH₄ ⁺)

A solution of compound A12 (12,5 g, 42.6 mmol) in pyridine (125 ml) at 0°C is treated with toluene-4-sulfonyl-chloride (20.3 g, 106 mmol) and DMAP (520 g, 4.3 mmol). The reaction is slowly heated to 70°C and stirred for 3.0 h. The reaction mixture is poured into aqueous, saturated NH₄CI solution, extracted with CH₂CI₂ (3x), dried (Na₂SO ) concentrated and purified by flash chromatography (silica, 25 - 35% EtOAc in hexane) to give compound A13 (15,9 g, 84%)

¹H NMR (500 MHz, CDCI₃): δ = 1.34 (d, J = 6 Hz, 3H, H-C(6')); 1.32 (s, 3H, CH₃) MS(CI): 448 (M ), 357 (M-PhCH₂)

A solution of compound A13 (15.9 g, 36.0 mmol) in DMF (120 ml) is treated with NaN₃ (4.6 g, 71 .2 mmol) and stirred at 80°C for 3.0 h. The reaction mixture is poured into Brine and extracted with EtOAc (3x) The combined organic layers are dried (Na₂SO ), concentrated and purified by flash chromatography (silica, 20% EtOAc in hexane) to give compound A14 (10.6 g, 93%)

¹H NMR (500 MHz, CDCI₃): δ = 1 60 (s, 3H, CH₃); 1 44 (d, J = 7 Hz, 3H, H-C(6')), 1.38 (s, 3H, CH₃), MS(EI) 320 (M+H⁺)

5)

A solution of compound A14 (5.0 g, 15 7 mmol) in CH₂CI₂ (25 ml) at 0°C is treated with H₂O (2.9 ml) and CF₃COOH (5.8 ml). The reaction mixture is stirred for 9.0 h at 25°C, cooled to 0°C and carefully treated with solid NaHCO₃ The reaction mixture is stirred for 0.3 h diluted with CH₂CI₂ and washed with CH₂CI₂ The aqueous phase is extracted with CH₂CI₂ (2x), the combined organic layers are dried (Na₂SO₄) and concentrated to give compound A15 (4.4 g, 100%) A small fraction is purified by flash chromatography (silica, 3% MeOH in CH₂CI₂) for analysis

R, = 0 35, 0 27 (silica, 4% MeOH in CH₂CI₂)

A solution of crude compound A15 (4.4 g, 15.8 mmol) in pyridine (50 ml) is treated with Ac₂O (8.1 g, 79.0 mmol) and DMAP (0.2 g, 1.6 mmol). The reaction mixture is stirred for 0.5 h at 25°C, poured into saturated, aqueous solution of NH CI and extracted with CH₂CI₂ (3x). The combined organic layers are dried (Na₂SO₄), concentrated and purified by flash chromatography (silica, 15 - 20% EtOAc in hexane) to give compound A16 (4.7 g, 92%, mixture of anomers (3.5:1 by ¹H NMR))

¹H NMR of less polar, major anomer (500 MHz, CDCI₃): δ = 2.14, 2.1 1 (2s, 6H, OAc); 1 .41 (d, J = 7 Hz, 3H, H-C(6')); MS(FD): 363 (M)

A solution of compound A16 (4.1 g, 12.0 mmol) and thymine (2.1 g, 16.8 mmol) in CH₃CN (40 ml) is treated with N,O-bis(trimethylsilyl)acetamid (5.8 g, 28.4 mmol) and stirred for 0.5 h at 50°C. Trimethylsilyltrifluoromethane-sulfonate (5.7 g, 25.8 mmol) is added to the reaction mixture and stirring is continued for 3.0 h at 50°C. The reaction mixture is cooled to 25°C, poured into saturated, aqueous NaHCO₃ solution and extracted with CH₂CI₂ (3x). The combined organic layers are dried (Na₂SO₄) , concentrated and purified by flash chromatography (silica, 50% EtOAc in hexane) to give compound A17 (4.42 g, 80%).

'H NMR (250 MHz, CDCI₃): δ = 2.15 (s, 3H, OAc); 1 .95 (s, 3H, CH₃); 1.42 (d, 3H, H-C(6')) 8)

A solution of compound A17 (10.6 mg, 24.6 mmol) in DMF (70 ml) at 0°C is treated with DBU (7.5 g, 49.2 mmol) and a solution of p-methoxybenzyloxymethylchloride (8.3 g, 44.3 mmol) in DMF (30 ml). The reaction mixture is stirred for 2.0 h (0°C - 25°C), concentrated and purified by flash chromatography (30 - 50% EtOAc in hexane) to give compound A18 (12.3 g, 87%).

R_f = 0.27 (silica, 33% EtOAc in hexane)

¹H NMR (250 MHz, CDCI₃): δ = 3.79 (s, 3H, OCH₃); 2.15 (s, 3H, OAc); 1.95 (s, 3H, CH₃)

A solution of compound A18 (12.3 g, 21 .3 mmol) in MeOH (120 ml) at 0°C is treated with NaOMe (4.6 g, 85.2 mmol) and stirred for 1.0 h at 0°C. The reaction mixture is poured into aqueous, saturated NH₄CI-solution, extracted with CH₂CI₂ (3x), dried (Na₂SO ), adsorbed on Silica gel and purified by flash chromatography (50% EtOAc in hexane) to give compound A19 (10.8 g, 94%)

¹H NMR (500 MHz, CDCI₃): δ = 3.80 (s, 3H, OCH₃); 1.94 (d, J = 1 Hz, 3H, CH₃) MS(CI): 555 (M+NH₄ ⁺), 538 (M+H⁺)

To a solution of compound A19 (10.3 g, 19.1 mmol) in THF (100 ml) at 0°C is added NaH (2.3 g, 57.3 mmol) and the reaction mixture is stirred for 0.5 h at 0°C. Mel is added to the reaction mixture and stirring is continued for 1.0 h at 0°C. The reaction mixture is poured into aqueous, saturated NH₄CI-solution, extracted with CH₂CI₂ (3x), the combined organic layers are dried (Na₂SO ), concentrated and purified by flash chromatography (30% EtOAc in hexane) to give compound A20 (10.8 g, 100%)

¹H NMR (500 MHz, CDCI₃): δ = 3.79 (s, 3H, ArOCH₃); MS(CI): 569 (M+NH₄ ⁺), 552 (M+H⁺)

To a solution of compound A20 (2.0 g, 3.63 mmol) in MeOH (3 ml) is added SnCI H₂O at 0°C and the reaction is stirred for 16.0 h (0 - 25°C). The reaction mixture is poured into saturated, aqueous NaHCO₃-solution and extracted with CH₂CI₂ (3x) . The combined organic layers are dried (Na₂SO ), concentrated and purified by flash chromatography (5% MeOH in CH₂CI₂) to give compound A21 (1.4 g, 71%).

Η NMR (500 MHz, CDCI₃): δ = 3.80 (s, 3H, ArOCH₃); 3.54 (s, 3H, OCH₃); MS(CI): 526 (M+H⁺)

A solution of carboxylic acid IV (344 g, 0.62 mmol, dried over P₂O₅ on HV, 16.0 h) in CH₃CN (6 ml) is treated with Et₃N (70 mg, 0.685 mmol) , O-(1 -benztriazol-1 -yl)-N,N,N,N- tetramethyluroniumtetrafluoroborat (220 mg, 0.685 mmol) and hydroxybenztriazol (42 mg, 0.312 mmol). The reaction mixture is stirred for 1.5 h. A solution of amine A21 (327 mg, 0.632 mmol, dried over P₂O₅ on HV, 16.0 h) in CH₃CN (6 ml) and Et₃N (94 mg, 0.935 mmol) are added to the reaction mixture and stirring is continued for 0.5 h. The reaction mixture is poured into aqueous, saturated NaH₂PO -soiution and concentrated. The aqueous phase is extracted with CH₂CI₂ (3x), the combined organic layers are washed with aqueous, saturated NaH₂PO₄-solution, brine, dried (Na₂SO ), concentrated and purified by flash chromatography (1 -2.5%MeOH in CH₂CI₂) to give compound A22 (539 mg, 90%).

'H NMR (500 MHz, CDCI₃): δ = 3.41 (2s, 6H, 2x OCH₃); 3.74 (3H, Ar-OCH₃); MS(EI): 1058 (M-H⁺)

To a solution of compound A22 (770 mg, 0.727 mmol) in CH₂CI₂ (10 ml) and H₂O (1 ml) is added DDQ (430 mg, 1.89 mmol) in portions during 2 h and the reaction mixture is stirred for additional 0.5 h. The reaction mixture is filtered though celite, concentrated and purified by flash chromatography (5% MeOH in CH₂CI₂). The chromatographed compound (mixture of product and hemiaminal) is dissolved in CH₂CI₂ and rapidly stirred with saturated, aqueous Na₂CO₃ solution. The organic phase is separated from the aqueous phase, dried (Na₂SO₄) and concentrated to give compound A23 (636 mg, 96%).

¹H NMR (500 MHz, CDCI₃): δ =3.40 and 3.43 (2s, 6H, 2x OCH₃); MS(CI): 909 (M )

A solution of compound A23 (630 mg, 0.693 mmol) in THF (8 ml) is treated with TBAF (1.04 ml of 1 .0M solution in THF, 1.04 mmol) and stirred at 25°C for 1.5 h. The reaction is concentrated and purified by flash chromatography (5 - 7% MeOH in CH₂CI₂) to give compound A24 (393 mg, 85%).

H NMR (500 MHz, CDCI₃): δ = 3.56 and 3.40 (2s, 6H, 2x OCH₃); MS(CI): 689 (M+NH4), 672 (M+H)

A solution of compound A24 (383 mg, 0.57 mmol) degassed with argon, is treated with Pd/C (10%, 76 mg) and stirred under an H₂-atmosphere for 21 h. The reaction vessel is flushed with argon , filtered through celite, concentrated and purified by flash chromatography (15% MeOH in CH₂CI₂) to give compound A25 (290 mg, 88%).

¹H NMR (250 MHz, CD₃OD): δ =3.47 and 3.45 (2s, 6H, 2x OCH₃); MS(EI): 580 (M-H)

A solution of compound A26 (288 mg, 0.496 mmol) in pyridine (3.5 ml) is treated with 4,4'- dimethoxytriphenylmethylchloride (406 mg, 1.20 mmol) and Et₃N (152 mg, 1 .50 mmol) and stirred for 4 h at 25°C. The reaction mixture is poured into aqueous, saturated NaHCO₃- solution, extracted with CH₂CI₂ (3x) , dried (Na₂SO₄), concentrated, coevaporated with toluene (2x) and purified by flash chromatography (7% MeOH in CH₂CI₂, 1 % Et₃N) to give compound A27 (380 mg, 87%).

¹H NMR (500 MHz, CDCI₃): δ = 3.52 and 3.49 (2s, 6H, 2x OCH₃); MS(EI): 882(M-H)

Alcohol A27 (300 mg, 0.339 mmol) and di-isopropylammonium tetrazolide (437 mg, 2.55 mmol) are dried for 12 h (HV), dissolved in CH₂CI₂ (10 ml) and treated with cyanoethoxy-bis- diisopropylamino-phosphine (460 mg, 1.53 mmol). The reaction mixture is stirred for 6 h at 25°C. The reaction is concentrated, dissolved in CH₂CI₂ and precipitated in cold pentane. The mother liquor is concentrated and remaining product is precipitated. The precipitates are washed with pentane and purified by flash chromatography (3% MeOH, 1 % Et₃N in CH₂CI₂) to give phosphoramidite A28 (350 mg, 95%, 1 :1 mixture of diastereomers).

³¹ P NMR (101 MHz, CDCI₃): δ = 151.3, 150.2; MS(EI): 1082(M-H)

Example A3: Preparation of compound (A49)

A solution of compound III (20 g, 0.077 mol) in THF (70 ml) is added to a suspension of NaH (3.69 g, 55%, 0.085 mol, washed with hexane) in THF (130 ml) at 0°C. The reaction is stirred for 1.0 h at 0°C and 0.5 h at 25°C. 4-methoxybenzylchloride (18 g, 0.1152 mol) and Bu₄NI (1.42 g, 3.8 mmol) are added to the reaction mixture and stirring is continued for 48 h at 25°C. The reaction mixture is poured into a saturated, aqueous solution of NH₄CI and extracted with EtOAc (3x). The combined organic layers are washed with Brine, dried (Na₂SO₄), concentrated and purified by flash chromatography (silica, 25-35% EtOAc in hexane) to give compound A29 (15.24 g, 52%)

H NMR (500 MHz, CDCI₃): δ = 3.79 (3H, OCH₃); MS (El): 379 (M-H)

Compound A29 (15.0 g, 0.039 mol) is dissolved in AcOH/H₂O (9/1 , 1 105 ml) and stirred for 2.0 h at 40°C. The reaction mixture is concentrated coevaporated with toluene (2x). The material is dissolved in CH₂CI₂, washed with aqueous NaHCO_3l dried with Na₂SO , concentrated and purified by flash chromatography (silica, 65% EtOAc in hexane) to give diol A30 (11.9 g, 89%)

'H NMR (500 MHz, CDCI₃): δ = 3.82 (3H, OCH₃); MS(EI): 339 (M-H)

A solution of compound A30 (1 1 .76 g, 34.6 mmol) in pyridine (100 ml) is treated with toluene-4-sulfonyl-chloride (9.23 g, 48 mmol) and DMAP (0.42 g, 3.5 mmol) at 0°C. The reaction is stirred for 4.0 h at 25°C, quenched with MeOH (1 1 ml), stirred for additional 0.3 h, concentrated, coevaporated with toluene (2x) and purified by flash chromatography to give compound A31 (17.8 g, 89%)

¹H NMR (500 MHz, CDCI₃): δ = 3.82 (3H, OCH₃); MS(CI): 512 (M + NH₄)

A solution of compound A31 (15.15 g, 31 mmol) in DME (200 ml, degassed with Argon) is treated with Nal (13.8 g, 92.0 mmol), Bu₃SnH (13.53 g, 46.5 mmol) and AIBN (1.2 g, 6.2 mmol) and stirred for 1 .0 h at 80°C. The reaction mixture is adsorbed onto silica gel, concentrated and purified by flash chromatography (silica, 30-50% EtOAc in hexane) to give compound A32 (7.9 g, 79%) ¹H NMR (400 MHz, CDCI₃): δ = 3.81 (3H, OCH₃); MS (Cl): 342 (M+NH₄)

A solution of compound A32 (7.9 g, 24 mmol) in pyridine (80 ml) at 0°C is treated with toluene-4-sulfonyl-chloride (1 1 .62 g, 61 mmol) and DMAP (293 mg, 2.4 mmol). The reaction is heated to 70°C and stirred for 3.0 h. The reaction mixture is poured into aqueous, saturated NH₄CI solution, extracted with CH₂CI₂ (3x), dried (Na₂SO ) concentrated and purified by flash chromatography (silica, 25 - 35% EtOAc in hexane) to give compound A33 (9.14 g, 80%)

¹H NMR (500 MHz, CDCI₃): δ = 3.82 (3H, OCH₃)

A solution of compound A33 (8.94 g, 18.7 mmol) in DMF (90 ml) is treated with NaN₃ (3.65 g, 56 mmol) and stirred at 70°C for 16 h. The reaction mixture is poured into Brine and extracted with EtOAc (3x). The combined organic layers are dried (Na₂SO₄), concentrated and purified by flash chromatography (silica, 15-20% EtOAc in hexane) to give compound A34 (6.0 g, 92%).

¹H NMR (500 MHz, CDCI₃): δ = 3.85 (3H, OCH₃); MS(CI): 367 (M+NH₄)

A solution of compound A34 (6.0 g, 17.2 mmol) in 90% AcOH (90 mi) is stirred for 5 h at 80°C and 16 h at 25°C, cooled to 0°C and carefully treated with solid NaHCO₃. The reaction mixture is concentrated, coevaporated with toluene (2x) and purified by flash chromatography (silica, 35-50% EtOAc in hexane) to give A35 (5.2 g, 98%).

¹H NMR (500 MHz, CDCI₃): δ = 3.83 (3H, OCH₃)

A solution of crude compound A35 (3.3 g, 10.7 mmol) in pyridine (30 ml) is treated with Ac₂O (5.45 g, 53.0 mmol) and DMAP (0.13 g, 1.01 mmol). The reaction mixture is stirred for 0.5 h at 25^CC, poured into saturated, aqueous solution of NH₄CI and extracted with CH₂CI₂ (3x). The combined organic layers are dried (Na₂SO₄), concentrated and purified by flash chromatography (silica, 25 - 35% EtOAc in hexane) to give compound A36 (4.12 g, 98%, mixture of anomers (2.5:1 by ¹H NMR))

¹H NMR of less polar, major anomer (500 MHz, CDCI₃): δ = 3.81 (3H, OCH₃)

A solution of compound A36 (4.12 g, 10.5 mmol) and thymine (1.72 g, 13.7 mmol) in CH₃CN (40 ml) is treated with N,O-bis(trimethylsilyl)acetamid (4.7 g, 23.1 mmol) and stirred for 0.5 h at 50°C. Tπmethylsilyltrifluoromethane-sulfonate (4.67 g, 21 mmol) is added to the reaction mixture and stirring is continued for 3.5 h at 50°C. The reaction mixture is cooled to 25°C, poured into saturated, aqueous NaHCO₃ solution and extracted with CH₂CI₂ (3x). The combined organic layers are dried (Na₂SO₄) , concentrated and purified by flash chromatography (silica, 50% EtOAc in hexane) to give compound A37 (4.13 g, 86%).

'H NMR (250 MHz, CDCI₃): δ = 3.82 (3H, OCH₃); MS(EI): 458 (M-H)

p(M

A solution of compound A37 (4.13 g, 10 mmol) in DMF (30 ml) at 0°C is treated with DBU (2.74 g, 18.0 mmol) and a solution of p-methoxybenzyloxymethylchloride (3.02 g, 16.2 mmol) in DMF (10 ml). The reaction mixture is stirred for 2.0 h (0°C - 25°C), concentrated and purified by flash chromatography (50% EtOAc in hexane) to give compound A38 (5.12 g, 93%)

¹H NMR (250 MHz, CDCI₃): δ = 3.80 and 3.81 (2s, 6H, 2x OCH₃); MS(CI): 627 (M+NH4)

p(M

A solution of compound A38 (5.2 g, 8.4 mmol) in MeOH (50 ml) at 0°C is treated with NaOMe (1.82 g, 33.6 mmol) and stirred for 1.0 h at 25°C. The reaction mixture is poured into aqueous, saturated NH₄CI-solution, extracted with CH₂CI₂ (3x), dried (Na₂SO₄), adsorbed on silica gel and purified by flash chromatography (50% EtOAc in hexane) to give compound A39 (4.47 g, 94%)

'H NMR (500 MHz, CDCI₃): δ = 3.80 and 3.83 (2s, 6H, 2x OCH₃); MS(CI): 602 (M+CI)

To a solution of compound A39 (3.0 g, 5.3 mmol) in THF (30 ml) at 0°C is added NaH (381 mg, 15.9 mmol) and the reaction mixture is stirred for 0.5 h at 0°C. Mel (7.52g, 53 mmol) is added to the reaction mixture and stirring is continued for 2.5 h at 0°C. The reaction mixture is poured into aqueous, saturated NH CI-solution, extracted with CH₂CI₂ (3x), the combined organic layers are dried (Na₂SO ), concentrated and purified by flash chromatography (50% EtOAc in hexane) to give compound A40 (3.04, 99%).

¹H NMR (500 MHz, CDCI₃): δ = 3.80 and 3.81 (2s, 6H, 2x OCH₃); MS(CI): 616 (M+CI)

To a solution of compound A40 (3.4 g, 5.2 mmol) in CH₂CI₂/H₂O (33 ml, 10:1 ) is added DDQ (4.93 g, 21 .7 mmol) in portions during 1.5h. The reaction is stirred for an additional 1 h at 25°C, filtered though Celite, concentrated and purified by flash chromatography (silica, 60- 80% EtOAc in hexane) to give compound A41 (1.25g, 77%).

¹H NMR (500 MHz, CDCI₃): δ = 3.60 (3H, OCH₃); MS(EI): 310 (M-H)

A solution of compound A41 (1 .25 g, 4.0 mmol) and imidazol (554 mg, 8 mmol) in CH₂CI₂ (20 ml) at 0°C is treated with t-butyldiphenylchlorosilane (1.76 g, 6.4 mmol) and stirred for 4h at 25°C. The reaction is quenched with MeOH (2 ml), stirred for 0.25 h, poured into aqueous, saturated NH₄CI-solution, extracted with CH₂CI₂ (3x), the combined organic layers are washed with saturated, aqueous NaHCO₃ solution, dried (Na₂SO₄), concentrated and purified by flash chromatography (35% EtOAc in hexane) to give compound A42 (1.85, 86%)

¹H NMR (500 MHz, CDCI₃): δ = 3.32 (3H, OCH₃)

To a solution of compound A42 (1.85 g, 3.45 mmol) in CH₃CN (20 ml) is added triazol (5.35 g, 77.5 mmol), Et₃N (8.2 g, 81 mmol) and the reaction is cooled to 0°C. POCI₃ (1.32 g, 8.6 mmol) is added slowly and the reaction is stirred for 0.5 h at 25°C. The reaction mixture is poured into saturated, aqueous NaHCO₃ solution, extracted with CH₂CI₂ (3x), the combined organic layers are washed with brine, dried (Na₂SO₄), concentrated and purified by flash chromatography (50% EtOAc in hexane) to give compound A43 (1.91 , 94%).

¹H NMR (500 MHz, CDCI₃): δ = 3.52 (3H, OCH₃)

To a solution of compound A43 (1.91 g, 3.2 mmol) in dioxane (20 ml) is added NH₃ (10 ml, 25% in H₂O) and the reaction mixture is heated at 60°C for 0.5 h. The reaction mixture is concentrated, poured into saturated, aqueous NaHCO₃ solution, extracted with CH₂CI₂ (3x), the combined organic layers are washed with brine, dried (Na₂SO ), concentrated and purified by flash chromatography (6% MeOH in CH₂CI₂) to give compound A44 (1.53, 86%).

¹H NMR (500 MHz, CDCI₃): δ = 3.45 (3H, OCH₃); MS(CI): 583 (M+CI)

To a solution of compound A44 (1.53 g, 2.8 mmol) in MeOH (15 ml) is added pyridine (1.1 1 g, 14 mmol) and N-methyl pyrolidone dimethylacetal (2.03 g, 14 mmol) and the reaction mixture is stirred at 25°C for 3 h. The reaction mixture is concentrated, coevaporated with toluene (2x) and purified by flash chromatography (4% MeOH in CH₂CI₂) to give compound A45 (1.41 , 80%).

¹H NMR (500 MHz, CDCI₃): δ = 2.82 (s, 3H, N-CH₃); MS(EI): 630 (M+H)

To a solution of compound A45 (897.6 mg, 1 .42 mmol) in MeOH (1 0 ml) is added SnCI₂2H₂O (1.83 g, 8.31 mmol) in portions during 2h. The reaction is stirred for additional 3 h at 25°C. The reaction mixture is carefully quenched with saturated, aqueous NaHCO₃ solution, concentrated, redissolved in CH₂CI₂. The organic layer is washed with brine, dried (Na₂SO₄), and concentrated to give crude compound A46 (620 mg, 72%).

¹H NMR (500 MHz, CDCI₃): δ = 5.88 (1 H,d,H-C(1 '))

A solution of carboxylic acid IV (572 mg, 1.01 mmol, dried over P₂O₅ on HV, 16.0 h) in CH₃CN (8 ml) is treated with Et₃N (1 12 mg, 1.1 1 mmol), O-(1 -benztriazol-1 -yl)-N,N,N,N- tetramethyluroniumtetrafluoroborat (356 mg, 1 .1 1 mmol) and hydroxybenztriazol (68 mg, 0.505 mmol). The reaction mixture is stirred for 2 h. A solution of amine A46 (610 mg, 1.01 mmol, dried over P₂O₅ on HV, 16.0 h) in CH₃CN (5 ml) and Et₃N (153 mg, 1.51 mmol) are added to the reaction mixture and stirring is continued for 17 h. The reaction mixture is poured into aqueous, saturated NaH₂PO₄-solution and concentrated. The aqueous phase is extracted with CH₂CI₂ (3x), the combined organic layers are washed with aqueous, saturated NaH₂PO₄-solution, brine, dried (Na₂SO₄), concentrated and purified by flash chromatography to give compound A47 (652 mg, 80 %).

¹H NMR (500 MHz, CDCI₃): δ = 3.19, 3.12, 3.05 (3s, 9H, 2x OCH₃, NCH₃); MS(EI): 1036 (M-H⁺)

A solution of compound A47 (650 mg, 0.59 mmol) in THF (10 ml) is treated with TBAF (1.34 ml of 1 .0M solution in THF, 1 .34 mmol) and stirred at 25°C for 4.5 h. The reaction is concentrated and purified by flash chromatography (5 - 20% MeOH in CH₂CI₂) to give compound A48 (341 mg, 88%).

¹H NMR (500 MHz, CDCI₃): δ = 1.18 (d, 3H, CH₃); MS(CI): 662 (M+H⁺)

A solution of compound A48 (335 mg, 0.506 mmol) in pyridine (10 ml) is treated with 4,4'- dimethoxytriphenylmethylchloπde (265 mg, 0.76 mmol) and stirred for 22 h at 25°C. The reaction mixture is poured into aqueous, saturated NaHCO₃-solution, extracted with CH₂CI₂ (3x), the organic layers are washed with brine, dried (Na₂SO₄), concentrated, coevaporated with toluene (3x) and purified by flash chromatography (10-20% MeOH in EtOAc, 1 % EtsN) to give compound A49 (345 mg, 71%).

¹H NMR (500 MHz, CDCI₃): δ = 3.77 (2s, 6H, 2x ArOCH₃); 1.13 (d, 3H, CH₃) MS(EI): 962 (M-H⁺)

Alcohol A49 (338 mg, 0.355 mmol), dissolved in CH₂CI₂ (5ml), is added to a solution of di- isopropylammonium tetrazohde (67 mg, 0.0.391 mmol) and cyanoethoxy-bis-dnsopropyl- amino-phosphine (235 mg, 0.78 mmol) in CH₂CI₂ (10 ml) at 25°C. The reaction mixture is stirred at RT for 2 h and is then poured into aqueous, saturated NaHCO₃-solution, extracted with CH₂CI₂ (3x), the organic layers are washed with brine, dried (Na₂SO ), concentrated, and purified by flash chromatography (2-10 % MeOH in EtOAc, 1% Et₃N) to give compound A50 (366 mg, 88 %).

31

P NMR (101 MHz, CDCI₃): δ = 151.7, 150.8 (1 :1 mixture of diastereomers).

Example A4: Preparation of compound (A54)

A solution of carboxylic acid IX (636 mg, 1 .21 mmol, dried over P₂O₅ on HV, 1 6.0 h) in CH₃CN (6 ml) is treated with Et₃N (138 mg, 1.33 mmol), O-(1 -benztriazol-1 -yl)-N,N,N,N- tetramethyluroniumtetrafluoroborat (430 mg, 1.33 mmol) and hydroxybenztriazol (82 mg, 0.61 mmol). The reaction mixture is stirred for 1 h. A solution of amine A4 (600 mg, 1.21 mmol, dried over P₂O₅ on HV, 16.0 h) in CH₃CN (4 ml) and Et₃N (138 mg, 1.33 mmol) are added to the reaction mixture and stirring is continued for 3 h. The reaction mixture is poured into aqueous, saturated NaH₂PO -solution and concentrated. The aqueous phase is extracted with CH₂CI₂ (3x), the combined organic layers are washed with aqueous, saturated NaH₂PO₄-solution, brine, dried (Na₂SO ), concentrated and purified by flash chromatography (2-5% MeOH in CH₂CI₂) to give compound A51 (1 .14 g, 94 %). 'H NMR (500 MHz, CDCI₃): δ = 6.31 , 6.18 (2dd, 2H, 2x H-C(1 ')); MS(EI): 996 (M-H)

A solution of compound A51 (700 mg, 0.70 mmol) in THF (5 ml) is treated with TBAF (1.54 ml of 1 .0M solution in THF, 1 .54 mmol) and stirred at 25°C for 4 h. The reaction is concentrated and purified by flash chromatography (12 - 15% MeOH in CH₂CI₂) to give compound A52 (316 mg, 86%).

¹H NMR (400 MHz, CD₃OD): δ = 6.21 , 6.07 (2dd, 2H, 2x H-C(1 ')); MS(EI): 520 (M-H)

A solution of compound 52 (290 mg, 0.56 mmol) in pyridine (3 ml) is treated with 4,4'- dimethoxytriphenymethyllchloride (568 mg, 1.68 mmol) in portioned and stirred for 24 h at 25°C. The reaction mixture is poured into aqueous, saturated NaHCO₃-solution, extracted with CH₂CI₂ (3x), the organic layers are washed with brine, dried (Na₂SO₄), concentrated, coevaporated with toluene (3x) and purified by flash chromatography (5-10% MeOH in CH₂CI₂ , 1 % Et₃N) to give compound 53 (328 mg, 71 %).

¹H NMR (250 MHz, CD₃OD): δ = 6.25 (m, 1 H, H-C(1 ')); MS(EI): 822 (M-H)

Alcohol A53 (315 mg, 0.38 mmol), dissolved in CH₂CI₂ (2ml), is added to a solution of di- isopropylammonium tetrazolide (44 mg, 0.256 mmol) and cyanoethoxy-bis- diisopropylamino-phosphine (172 mg, 0.0.573 mmol) in CH₂CI₂ (2 ml) at 25°C. The reaction mixture is stirred for 5 h, poured into aqueous, saturated NaHCO₃-solution, extracted with CH₂CI₂ (3x), the organic layers are washed with brine, dried (Na₂SO₄), concentrated, and purified by flash chromatography (1-10 % MeOH in EtOAc, 1 % Et₃N) to give compound A54 (365 mg, 93 %).

31

P NMR (101 MHz, CDCI₃): δ = 149.8, 148.5 ( 2 diastereomers); MS(EI): 1023 (M-H) B: Synthesis of oligonucieotides

Each oligonucleotide is prepared on an ABI 390 DNA synthesizer using standard phos- phoramidite chemistry according to Gait, M.J., Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford (1984) but with prolonged coupling times (10 min). Dimeth- oxytrityl oligonucieotides are purified by reverse phase HPLC (column: Nucleosil RPC₁₈, 10 μ, 10x 250 mm; eluent A: 50 mM triethylammonium acetate (TEAA), pH 7.0; eluent B: 50mM TEAA, pH 7.0 in 70 % acetonitrile; elution with gradient from 15 % to 45 % B in 45 min). After purification by HPLC the oligodeoxynucleotides are controlled by capillary gel electrophoresis (concentration: 1 OD/ml, injection: 2 kV, 3 sec, separation: 9kV, capillary: effective length 30 cm, inner diameter 100 μm, polyacryiamide 10 % T, buffer: 100 mM H₃PO₄, 100 mM Tris, 2 mM EDTA, 7 M urea pH 8.8) . The molecular weight of each oligodeoxynucleotide is checked by mass spectroscopy [MALDI-TOF: Pieles, U., Zϋrcher, W., Schar, M., Moser, H., Nucl. Acids Res. 21 :3191 (1993)]. The oligodeoxynucleotide is desorbed using 2,4,6-trihydroxyacetophenone as a matrix (detection of negatively charged ions) with diammonium hydrogen citrate as additive (25mM final concentration).

Oligonucieotides synthesized:

SEQ 1 : 5 ' -GpCpGpTsTpTsTpTsTpTsTpTsTpGpCpG-3 '

SEQ 2: 5 ' TpTpTpTsTpCpTpCpTpCpTpCpTpCpT-3 ' p is an usual phosphordiester bond

C: Properties of oligonucieotides

The thermal denaturation (T_m) of DNA/RNA hybndes is performed at 260 nm using a Gilford Response II Spectrophotometer (Ciba-Corning Diagnostics Corp. , Oberlm, OH) . Absorbance versus temperature profiles are measured at 4 μM of each strand in 10 mM phosphate pH 7.0 (Na salts), 100 mM total [Na⁺] (supplemented as NaCl), 0.1 mM EDTA. T_m's are obtained from fits of absorbance versus temperature curves to a two-state model with linear slope baselines [Freier, S.M., Albergo, D.D., Turner, D.H., Biopolymers 22:1 107- 1 131 (1982)]. All values are averages of at least three experiments. The absolute experimental error of the T_m values is ± 0.5°C.

Binding to the complementary RNA strand (Δt_m / modification compared to wildtype)

R³ X Y conf. SEQ 1 SEQ 2

CH₃ H H (S) + 1.4 + 1.0

CH₃ H H (R) - 4.9 - 3.6

H H H - - 0.9 + 0.4

From these examples it is evident that a change in the configuration from (R) to (S) causes a dramatic increase in T_m. Surprisingly, Δt_m for the (S) configuration is even better than Δt_m in case of no substitution (R³=H). This clearly shows that it is important to have a R³ that is not hydrogen and that is bound in (S) configuration.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Novartis AG

(B) STREET: Schwarzwaldallee 215

(C) CITY: Basel

(E) COUNTRY: Switzerland

(F) POSTAL CODE (ZIP): 4058

(G) TELEPHONE: +41 61 696 11 11 (H) TELEFAX: + 41 61 696 79 76 (I) TELEX: 962 991

(ii) TITLE OF INVENTION: Modified oligonucieotides

(iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oligonucleotide" ( ix ) FEATURE :

(A) NAME/KEY: misc_feature

(B) LOCATIONS..5

(D) OTHER INFORMATIO :/note= "modified backbone"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATIONS..7

(D) OTHER INFORMATION: /note= "modified backbone"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATIONS..9

(D) OTHER INFORMATION:/note= "modified backbone"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 10..11

(D) OTHER INFORMATION:/note= "modified backbone"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 12..13

(D) OTHER INFORMATION:/note= "modified backbone"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GCGTTTTTTT TTTGCG 16

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oligonucleotide"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATIONS..5

(D) OTHER INFORMATION: /note= "modified backbone"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

TTTTTCTCTC TCTCT 15

Claims

What is claimed is:

1 . An oligonucleotide of formula 1

5'-(U)_n-3' (1), in which U is an identical or different radical of a natural or a synthetic nucleoside, n is an integer from 2 to 200; and wherein the oligonucleotide of formula 1 comprises at least one structural unit of formula 2

wherein

R¹ is H, d-dalkyl or Cι-C alkoxy;

R² is H, d-C alkyl, phenyl, C C₄alkyl-phenyl, C₃-C heteroaryl, C,-C₄alkyl-C₃-C₉hetero- aryl or an intercalator; wherein the aryl or heteroaryl is unsubstituted or substituted by OH, R⁴, d-C₄alkoxy, -O-(CH₂-CH₂-O)_mR⁴, NR⁴ ₂ or NHR⁴; R³ is C C₄alkyl, unsubstituted or substituted by OH, NR⁴ ₂ or NHR⁴; R⁴ is H or Cι-C₄alkyl; X and Y are independent of one another, H, OH, OR⁴, O-d-dalkylNHR⁴, O-C,-

C₄alkylNR⁴ ₂, -O-(CH₂-CH₂-O)_mR⁴ or -O-CH₂-C(OR⁵)H-CH₂-OR⁶; or -O-CH₂-C(OR⁵)H-

CH₃; R⁵ is H, CH₃ or d-doalkyl; R⁶ is H, CH₃ or an OH-protecting group; m is an integer from 1 to 4; A and B are, independent of one another, a purine or pyrimidine radical or an analogue thereof; with the proviso that if A and B are thymidine, R¹, R² and X are hydrogen and Y is methoxy, R³ is not methyl.

2. The oligonucleotide of claim 1 wherein the intercalator is anthraquinone connected via a linker.

3. The oligonucleotide of claim 2 wherein the linker is a chain of 2 to 7 atoms selected from the group consisting of C, N and O.

4 . The oligonucleotide according to claim 1 in which A and/or B as a purine radical or an analogue thereof is a radical of formula 3, 4, 5 or 6

in which

R⁸ and R⁹ independently of one another are H, OH, SH, NH₂, NHNH₂, NHOH, NHOalkyI having 1 to 12 C atoms, -N=CH-N(C₁-C₁₂alkyl)₂, F, Cl, Br, alkyl or hydroxyalkyl or aminoalkyl or alkoxy or alkylthio having 1 to 12 C atoms; R¹⁶ is H, F, Cl, Br, CONH₂, alkyl, propinyl or hydroxyalkyl or aminoalkyl or alkoxy or alkylthio having 1 to 12 C atom.

5. The oligonucleotide according to claim 4, in which the primary amino contains 1 to 12 C atoms and the secondary amino 2 to 12 C atoms.

6. The oligonucleotide according to claim 4, in which the primary amino and secondary amino are radicals of the formula R¹³R¹ N in which R¹³ and R¹⁴ are independently H, Ci- C₂₀alkyl, -aminoalkyl or -hydroxyalkyl; carboxyalkyl or carbalkoxyalkyi, where the carbalkoxy group contains 2 to 8 C atoms and the alkyl group contains 1 to 6, C atoms; C₂-C₂₀alkenyl; phenyl, mono- or di(d-C₄alkyl- or -alkoxy)phenyl, benzyl, mono- or di(Cι- dalkyl- or -alkoxy)benzyl; or 1 ,2-, 1 ,3- or 1 ,4-imidazolyl-Cι-C₆alkyl; or R¹³ and R ¹⁴ together are tetra- or pentamethylene, 3-oxa-1 ,5-pentylene, -CH₂-NR¹⁵-CH₂CH₂- or -CH₂CH₂-NR¹⁵-CH₂CH₂-, in which R¹⁵ is H or Cι-C₄alkyl; and the amino group in the aminoalkyl is unsubstituted or substituted by one or two d-C₄alkyl or -d-C hydroxyalkyl groups; and the hydroxyl group in hydroxyalkyl is unsubstituted or etherified with d- dalkyl.

7. The oligonucleotide according to claim 5, in which the primary amino and secondary amino are selected from the group consisting of methyl-, ethyl-, dimethyl-, diethyl-, allyl-, mono- or di(hydroxyeth-2-yl)-, phenyl-, benzyl-, acetyl-, isobutyryi-, benzoyiamino, phenoxyacetylamino, 4-tert.-butylphenoxyacetylamino, N=CH-N(CH₃)₂, N=CH-N(C H₉)₂, and

8. The oligonucleotide according to claim 4, in which R⁸ and R⁹, independent of one another, are H , F, Cl , Br, OH , SH , NH₂, NHOH, NHNH₂, methyl, methylamino, dimethylamino, benzoyiamino, methoxy, ethoxy, methylthio, phenoxyacetylamino, 4- tert.-butylphenoxyacetylamino, N=CH-N(CH₃)₂,

9. The oligonucleotide according to claim 1 , in which A or B are independent of one another a purine radical or a radical of a purine analogue from the series consisting of adenine, N-methyladenine, N-benzoyladeniπe, 2-methylthioadenine, 2-amino-6-chloro- purine, 2-amino-6-methylthiopurine, 2-aminopurine, hypoxanthine, 2-aminoadenine, 2- hydroxypurine, guanine, N-isobutyrylguanine and .

10. The oligonucleotide according to claim 1 , in which A or B are independent of one another a purine radical or a radical of a purine analogue from the series consisting of adenine, 2-aminoadenine, 2-aminopurine, guanine and hypoxanthine.

11. The oligonucleotide according to claim 1 , in which A or B are independent of one another an analogous pyrimidine radical like a uracil, thymine or cytosine radical of the formulae 9 or 10

in which R ⁶ and R¹⁷ independently of one another are H, F, Cl, Br, alkyl, alkenyl, alkinyl, propargyl or hydroxyalkyl or aminoalkyl or alkoxy or alkylthio having 1 to 12 C atoms, phenyl, benzyl, primary amino having 1 to 20 C atoms or secondary amino having 2 to 30 C atoms, and the hydrogen atoms of the NH₂ group in formula 10 are unsubstituted or substituted by d-C₆alkyl, benzoyl or benzyl; and the dihydro derivatives of the radicals of formulae 9 and 10.

12. The oligonucleotide according to claim 10, in which R ⁶ is H, F, Cl, Br, d-C₆alkyl, d- dalkenyl, d-C₆alkinyl, d-C₆hydroxyalkyl, d-daminoalkyl, NHC,-C₄alkyl, N(d- C₄alkyl)₂, propinyl.

13. The oligonucleotide according to claim 10, in which R¹⁶ is H, F, Cl, Br, methyl, ethyl, and propinyl.

14. The oligonucleotide according to claim 10, in which R¹⁷ is H, F, Cl, Br, d-C₆alkyl, Cι- C₆alkoxy, d-Cehydroxyalkyl, d-C₆aminoalkyl, NH₂, NHd-C₄alkyl, N(C₁-C alkyl)_2l or propinyl.

15. The oligonucleotide according to claim 10, in which R¹⁷ is H, F, Cl, Br, methyl, ethyl, or propinyl.

16. The oligonucleotide according to claim 10, wherein R ⁶and R¹⁷ are H, methyl or propinyl.

17. The oligonucleotide according to claim 1 , in which A and B are independent of one another as the radical of a pyrimidine analogue are derived from uracil, thymine, cytosine, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, 5-methyicytosine, 5-propinyluracil, and 5-propinylcytosine.

18. The oligonucleotide according to claim 1 , comprising at least one structural unit of formula 2 wherein

R¹ is H or d-C₄alkyl;

R² is H, d-dalkyl, phenyl, d-C alkyl-phenyl or C₃-C₉heteroaryl;

R³ is d-C₄alkyl;

R⁴ is methyl or ethyl;

X and Y are independent of one another, H, OH, OR⁴, O-d-C₄alkylNHR⁴, O-d-

C₄alkylNR ₂, -O-(CH₂-CH₂-O)_mR⁴;

R⁵ is H or d-C₄alkyl.

19. The oligonucleotide according to claim 1 , wherein R¹ is H or methyl;

R² is H, methyl, ethyl or phenyl;

R³ is methyl or ethyl;

R⁴ is methyl;

X and Y are independent of one another, H , OH or OR⁴; O-CH₂CH₂NHR⁴, O-

CH₂CH₂NR⁴ ₂, O-CH₂CH₂OR⁴; ι5

R is H or C,-C₄alkyl.

20. The oligonucleotide according to claim 1 , wherein R¹ is H;

R is H, methyl or phenyl;

R^J methyl;

X and Y are i ndependent of one another, H , O-CH₃, O-CH₂CH₂OCH₃, O -

CH₂CH₂NHCH₃, O-CH₂CH₂N(CH₃)₂; R⁵ H or methyl.

21. The oligonucleotide according to claim 1 , wherein the structural unit of formula 2 is of formula B8, B28 or B49

22. The oligonucleotide of claim 1 wherein n is 2 to 100.

23. The oligonucleotide of claim 1 wherein n is 2 to 50.

24. The oligonucleotide of claim 1 wherein n is 2 to 20.

25. A nucleoside dimer of formula 12

wherein

R¹ is H, d-C₄alkyl or d-dalkoxy; R² is H, d-C₄alkyl, d-C alkoxy, phenyl, d-C alkyl-phenyl, C₃-C₉heteroaryl, Cι-C alkyl- C₃-C₉heteroaryl or an intercalator; wherein the aryl or heteroaryl is unsubstituted or substituted by OH, R⁴, C C₄alkoxy, -O-(CH₂-CH₂-O)_mR⁴, NR ₂ or NHR⁴;

R³ is d-dalkyl, unsubstituted or substituted by OH, NR⁴ ₂ or NHR⁴;

R⁴ is H or d-dalkyl;

X and Y are independent of one another, H, OH, OR⁴, O-d-dalkylNHR⁴, O-d- C₄alkylNR ₂, -O-(CH₂-CH₂-O)_mR⁴ or -O-CH₂-C(OR⁵)H-CH₂-OR⁶; or -O-CH₂-C(OR⁵)H- CH₃;;

R⁵ is H or d-Cioalkyl;

R⁶ is H, CH₃ or an OH-protecting group; is an integer from 1 to 4;

A and B are, independent of one another, a purine or pyrimidine radical or an analogue thereof;

R^1S and R¹⁹ are H, an OH-protecting group or a radical forming a phosphorus-containing nucleotide bridging group; with the proviso that if A and B are thymidine, Ri, R² and X are hydrogen and Y is methoxy, R³ is not methyl.

26. The nucleoside dimer according to claim 25 wherein R¹⁸ is H or an OH-protecting group and R¹⁹ is a phosphorus-containing, nucleotide-bridge-group-forming radical.

27. The nucleoside dimer according to claim 25 wherein the OH-protecting group is linear or branched d-C₈alkyl; C₇-C_1βaralkyl; triphenylsilyl, alkyldiphenylsilyl, dialkylphenylsilyl or trialkylsilyl having 1 to 20 C atoms in the alkyl groups; -(d-Cβalkyl^Si-O-SKd-Cβalkyl);-. C₂-C₁₂acyl, R¹²-SO₂-, in which R¹² is d-C₁₂alkyl, C₅- or C₆cycloalkyl, phenyl, benzyl, C Cι₂alkylphenyl, d-C₁₂alkylbenzyl, or is d-d₂alkoxycarbonyl, phenoxycarbonyl, benzyloxycarbonyl, methylphenoxycarbonyl or methylbenzyloxycarbonyl which is unsubstituted or substituted by F, Cl, Br, d-C₄alkoxy, tri(Cι-C₄alkyl)silyl or Cι-C alkylsulfonyl, or 9- fluorenylmethoxycarbonyl.

28. The nucleoside dimer according to claim 27, wherein the OH-protecting group is linear or branched C C₄alkyl, C₇-d₈aralkyl, trialkylsilyl having 1 to 12 C atoms in the alkyl groups; -(CH₃)₂Si-O-Si(CH₃)₂-; -(i-C₃H₇)₂Si-O-Si(iC₃H₇)₂-; C₂-C₈acyl; R^1Z-S0₂-, in which R¹² is d-dalkyl; phenyl or benzyl unsubstituted or substituted with F, Cl or Br; d- dalkylphenyl, Cι-C alkylbenzyl; d-C₈alkoxycarbonyl; phenoxycarbonyl; benzyloxycarbonyl or 9-fluorenylmethoxycarbonyl.

29. The nucleoside dimer according to claim 27, wherein the OH-protecting group is methyl, ethyl, n- or i-propyl, n-, i- or t-butyl; benzyl, methylbenzyl, dimethylbenzyl, methoxy- benzyl, dimethoxybenzyl, bromobenzyl; diphenylmethyl, di(methylphenyl)methyl, di(di- methylphenyl)methyl, di(methoxyphenyl)methyl, di(methoxyphenyl)(phenyl)methyl, trityl, tri(methylphenyl)ethyl, tri(dimethylphenyl)methyl, tri(methoxyphenyl)methyl, tri(dime- thoxyphenyl)methyl; trimethylsilyl, triethylsilyl, tri-n-propylsilyl, i-propyldimethylsilyl, t- butyldimethylsilyl, t-butyldiphenylsilyl, n-octyldimethylsilyl, (1 ,1 ,2,2-tetramethylethyl)di- methylsiiyl, -(CH₃)₂Si-O-Si(CH₃)₂-, -(i-C₃H₇)₂Si-O-Si(iC₃H₇)₂-; acetyl, propanoyl, butanoyl, pentanoyl, hexanoyl, benzoyl, methyl benzoy I, methoxybenzoyl, chlorobenzoyl or bromo- benzoyl; methyl-, ethyl-, propyl-, butyl-, phenyl-, benzyl-, p-bromo-, p-methoxy- or p- methylphenylsulfonyl; methoxy-, ethoxy-, n- or i-propoxy- or n-, i- or t-butoxycarbonyl, or phenoxycarbonyl, benzyloxycarbonyl, methyl- or methoxy- or chlorophenoxycarbonyl or - benzyloxycarbonyl or 9-fluorenylmethoxycarbonyl.

30. A nucleoside dimer according to claim 25 wherein the phosphorus-containing, nucleotide-bridge-group-forming radical may correspond to formula P1 or P2

I I

^Y-P = _a (P1), Λ (^P2)

OR_a Y_a OR_a wherein Y_a is hydrog en , Cι-Cι₂alkyl, C₆-Cι₂aryl, C_T-C∞aralkyl, C₇-C₂₀alkaryl, -OR_b, -S R_b, secondary amino, O^"M⁺ or S^"M⁺; X_a is oxygen or sulfur; R_a is hydrogen, M⁺, C C₁₂alkyl, C₂-C₁₂alkenyl or C₆-Cι₂aryl, or the group R_aO- is N- heteroaryl-N-yl having 5 ring members and from 1 to 3 nitrogen atoms; R_b is hydrogen, C Cι₂alkyl or C₆-C₁₂aryl; and ⁺ is Na⁺, K⁺, Li⁺, NH₄ ⁺ or primary, secondary, tertiary or quaternary ammonium; alkyl, aryl, aralkyi and alkaryl in Y_a, R_a and R_b being unsubstituted or substituted by alkoxy, alkylthio, halogen, -CN, -NO₂, phenyl, nitrophenyl or halophenyl.

31. A nucleotide dimer according to claim 30 wherein R_a is β-cyanoethyl and Y_a is di(iso- propyl)amino.

32. A nucleoside dimer according to claim 25, wherein R¹ is H or d-dalkyl;

R² is H, d-dalkyl, phenyl, C C₄alkyl-phenyl or C₃-C₉heteroaryl;

R³ is d-C₄alkyl;

R⁴ is methyl or ethyl;

X and Y are independent of one another, H, OH, OR⁴, -O-(CH₂-CH₂-O)_mR⁴;

R⁵ is H or d-dalkyl.

33. A nucleoside dimer according to claim 25, wherein R¹ is H or methyl;

R² is H, methyl, ethyl or phenyl;

R³ is methyl or ethyl;

X and Y are independent of one another, H , OH or OR⁴; O-CH₂CH₂NHR⁴, O-

CH₂CH₂N(CH₃)₂, O-CH₂CH₂OCH₃; R^s is H or d-dalkyl.

34. A nucleoside dimer according to claim 25, wherein R¹ is H;

R² is H, methyl or phenyl; R³ methyl; R⁵ is H or methyl;

X and Y are independent of one another, H , O-C H₃, O-CH₂CH₂OCH₃, O- CH₂CH₂NHCH₃, O-CH₂CH₂N(CH₃)₂;

35. A nucleoside dimer according to claim 25, selected from the group consisting of compounds of formula C8, C28 and C49

36. A process for the preparation of a nucleoside dimer according to claim 25 which comprises: a compound of the formula 14

wherein

R is H or Cι-C₄alkyl;

X is H, OH, OR⁴, O-d-dalkylNHR⁴, O-d-C₄alkylNR⁴ ₂, -O-(CH₂-CH₂-O)_mR⁴ or -O-

CH₂-C(OR⁵)H-CH₂-OR⁶; R⁴ is H or Cι-C₄alkyl; R⁵ is H or d-C₁₀alkyl; R⁶ is H, CH₃ or an OH-protecting group; m is an integer from 1 to 4; A is a purine or pyrimidine radical or an analogue thereof.

R is H or an OH-protecting group;

,29

R is H or an ester activating group;

is reacted with a compound of the formula 15 60 -

wherein

R² is H, d-C₄alkyl, d-dalkoxy, phenyl, C C₄alkyl-phenyl, C₃-C₉heteroaryl, d-

C₄alkyl-C₃-C₉heteroaryl or an intercalator; wherein the aryl or heteroaryl is unsubstituted or substituted by OH, R⁴, d-dalkoxy, -O-(CH₂-CH₂-O)_mR⁴, NR⁴ ₂ or NHR⁴; R³ is Cι-C₄alkyl, unsubstituted or substituted by OH, NR⁴ ₂ or NHR⁴; Y is H, OH, OR⁴, O-d-dalkylNHR⁴, O-C,-C₄alkylNR⁴ ₂, -O-(CH₂-CH₂-O)_mR⁴ or -O-

CH₂-C(OR⁵)H-CH₂-OR⁶; R⁴ is H or d-dalkyi; R⁵ is H or d-doalkyl; R⁶ is H or an OH-protecting group; m is an integer from 1 to 4;

B is a purine or pyrimidine radical or an analogue thereof. R²⁸ is H or an OH-protecting group.

37. The use of a nucleoside dimer according to claim 25 for the preparation of oligonucieotides according to claim 1.

38. The use of an oligonucleotide according to claim 1 as a diagnostic for the detection of viral infections or genetically related diseases.

39. The oligonucleotide according to claim 1 for use in a therapeutic process for the treatment of diseases in mammals including humans by means of interaction with nucleotide sequences in the body.

40. A pharmaceutical preparation comprising an effective amount of an oligonucleotide according to claim 1 on its own or together with other active ingredients , a pharmaceutical carrier and, if appropriate, excipients.

41. The nucleoside dimer according to claim 25 for use in a therapeutic process for the treatment of diseases in mammals including humans.