Nucleosides and oligonucleotides containing 2'-ether groups.

The invention relates to ribo-nucleoside analogs whose 2'-OH group is etherified with hydroxyethyl or fluoroalkyl groups, a process for their preparation, oligonucleotides with these nucleosides and the use of the nucleosides for the production of oligonucleotides with the same or different nucleoside units in the molecule.

Nucleosides and oligonucleotides have found broad interest as antiviral agents or because of their ability to interact with nucleic acids ("antisense" oligonucleotides) and the biological activity associated with them, see for example Uhlmann, E., Peyman, A., Chemical Reviews 90: 543-584 (1990). To provide nucleosides with new properties or to improve the interaction of antisense oligonucleotides with natural nucleic acids and their stability towards nucleases, the sugar residues of nucleosides (or of the nucleotide units in oligonucleotides), or the intemucleotide phosphate bond in oligonucleotides has been modified in various ways, see for example Marquez, VE, Lim, MI, Medicinal Research Reviews 6: 1-40 (1986), Hélène, C., Toulmé, JJ, Biochimica et Biophysica Acta 1049: 99-125 (1990), English, U., Gauss, DH, Angewandte Chemie 103: 629-646 (1991), Matteucci, MD, Bischofberger, N., Annual Reports in Medicinal Chemistry 26:87 -296 (1991). Cook, PD, Anti-Cancer Drug Design 6: 585-607 (1991) and WO 91/06556 describe nucleosides which are modified on the 2'-OH group of the sugar. The modifications described lead to increased nuclease resistance; the longer the alkyl radical, the higher the nuclease resistance. A small increase in binding affinity is observed with small alkyl residues such as methyl, ethyl or propyl, but with longer chains the binding affinity drops drastically. Nucleosides with hydroxyethyl or fluoroalkyl groups as side chains on the 2'-OH group have so far never been incorporated into oligonucleotides. Surprisingly, the modifications according to the invention increase the binding affinity for the complementary RNA compared to unsubstituted alkyl chains of the same length. Based on the published data, this result was not to be expected. In analogy to the 2'-OH-modified oligoribonucleotides, the compounds according to the invention are also distinguished by a striking nuclease resistance. In addition, oligonucleotides which contain the nucleosides according to the invention have an increased cellular uptake and consequently have an improved bioavailability and activity in vivo.

The invention relates to compounds of the formula I.<chemistry id="chem0001" num="0001"><img file="EP0679657A2_D0001.tif" /></chemistry> wherein R₁ and R₂ independently represent hydrogen or a protecting group or R₁ has these meanings and R₂ represents a residue forming a phosphorus-containing nucleotide bridge group; B represents a purine or pyrimidine residue or an analog thereof; and R₃ for OH, F or (CF₂)_nCF₃ is where n is a number from 0 to 7.

If R₃ is OH, this hydroxy group can be protected with a group as defined for R₁ and R₂.

In a preferred embodiment, n stands for the number 0.

In a preferred embodiment, R₁ and R₂ represent hydrogen.

Protecting groups and methods for derivatizing the hydroxyl groups with such protecting groups are generally known in sugar and nucleotide chemistry and are described, for example, by Greene, BT, Protective Groups in Organic Synthesis, Wiley Interscience, New York (1991), by Sonveaux, E., Bioorganic Chemistry 14: 274-325 (1986) or by Beaucage, SL, Iyer, R., Tetrahedron 48: 2223-2311 (1992). Examples of such protective groups are: benzyl, methylbenzyl, dimethylbenzyl, methoxybenzyl, dimethoxybenzyl, bromobenzyl, 2,4-dichlorobenzyl; Diphenylmethyl, Di (methylphenyl) methyl, Di (dimethylphenyl) methyl, Di (methoxyphenyl) methyl, Di (dimethoxyphenyl) methyl, Triphenylmethyl, Tris-4,4 ', 4' '- tert.butylphenylmethyl, Di-p-anisylphenylmethyl, Tri (methylphenyl) methyl, tri (dimethylphenyl) methyl, methoxyphenyl (diphenyl) methyl, di (methoxyphenyl) phenylmethyl, tri (methoxyphenyl) methyl, tri (dimethoxyphenyl) methyl; Triphenylsilyl, alkyldiphenylsilyl, dialkylphenylsilyl and trialkylsilyl with 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-propyl-dimethylsilyl, t-butyl -dimethylsilyl, t-butyl-diphenylsilyl, n-octyl-dimethylsilyl, (1,1,2,2-tetramethylethyl) -dimethylsilyl; - (C₁-C₈-alkyl) ₂Si-O-Si (C₁-C₈-alkyl) ₂-, wherein alkyl means, for example, methyl, ethyl n- and i-propyl, n-, i- or t-butyl; C₂-C₁₂-, especially C₂-C₈ acyl, such as acetyl, propanoyl, butanoyl, pentanoyl, hexanoyl, benzoyl, methylbenzoyl, methoxybenzoyl, chlorobenzoyl and bromobenzoyl; R_S1-SO₂-, where R_S1 C₁-C₁₂-alkyl, especially C₁-C₆-alkyl, C₅- or C₆-cycloalkyl, phenyl, benzyl, C₁-C₁₂- and especially C₁-C₄-alkylphenyl, or C₁-C₁₂- and especially C₁-C₄-alkylbenzyl, or Halophenyl or halobenzyl means, for example, methyl, ethyl, propyl, butyl, phenyl, benzyl, p-bromo, p-methoxy and p-methylphenylsulfonyl; unsubstituted or substituted with F, Cl, Br, C₁-C₄-alkoxy, tri- (C₁-C₄-alkyl) silyl or C₁-C₄-alkylsulfonyl-substituted C₁-C₁₂-, preferably C₁-C₈-alkoxycarbonyl, for example methoxy, ethoxy -, n- or i-propoxy- or n-, i- or t-butoxycarbonyl, 2-trimethylsilylethoxycarbonyl, 2-methylsulfonylethoxycarbonyl, allyloxycarbonyl or unsubstituted or as substituted for alkoxycarbonyl phenyloxycarbonyl or benzyloxycarbonyl, for example methyl or methoxy or chlorophenyloxycarbonyl or benzyloxycarbonyl, and 9-fluorenylmethyloxycarbonyl. If R₁ and / or R₂ are alkyl, it can be substituted with F, Cl, Br, C₁-C₄alkoxy, phenyloxy, chlorophenyloxy, methoxyphenyloxy, benzyloxy, methoxybenzyloxy or chlorophenyloxy. R₁ and R₂ in formula I can be the same or different protecting groups.

In a particularly preferred embodiment, R₁ and R₂ represent benzyl, methylbenzyl, dimethylbenzyl, methoxybenzyl, dimethoxybenzyl, halogenated benzyl, in particular bromobenzyl; Diphenylmethyl, Di (methylphenyl) methyl, Di (dimethylphenyl) methyl, Di (methoxyphenyl) methyl, Di (methoxyphenyl) (phenyl) methyl, Triphenylmethyl, Tris-4,4 ', 4' '- tert.butylphenylmethyl, Di-p- anisylphenylmethyl, tri (methylphenyl) methyl, tri (dimethylphenyl) methyl, tri (methoxyphenyl) methyl, tri (dimethoxyphenyl) methyl; Trimethylsilyl, triethylsilyl, tri-n-propylsilyl, i-propyl-dimethylsilyl, t-butyl-dimethylsilyl, t-butyl-diphenylsilyl, n-octyl-dimethylsilyl, (1,1,2,2-tetramethylethyl) -dimethylsilyl, - ( CH₃) ₂Si-O-Si (CH₃) ₂-, - (i-C₃H₇) ₂Si-O-Si (i-C₃H₇) ₂-; Acetyl, propanoyl, butanoyl, pentanoyl, hexanoyl, benzoyl, methylbenzoyl, methoxybenzoyl, chlorobenzoyl and bromobenzoyl; Methyl, ethyl, propyl, butyl, phenyl, benzyl, p-bromo, p-methoxy and p-methylphenylsulfonyl; Methoxy-, ethoxy-, n- or i-propoxy- or n-, i- or t-butoxycarbonyl, or phenyloxycarbonyl, benzyloxycarbonyl, methyl- or methoxy- or chlorophenyloxycarbonyl or -benzyloxycarbonyl or 9-fluorenylmethyloxycarbonyl.

R₂ can as a phosphorus containing a nucleotide bridge group forming radical of the formula P1 or P2<chemistry id="chem0002" num="0002"><img file="EP0679657A2_D0002.tif" /></chemistry> where Y_a Hydrogen, C₁-C₁₂-alkyl, C₆-C₁₂-aryl, C₇-C₂₀-aralkyl, C₇-C₂₀-alkaryl, -OR_b, -SR_b, -NH₂, primary amino, secondary amino, O^⊖M^⊕ or S^⊖M^⊕ represents; X_a Means oxygen or sulfur; R_a Hydrogen, M^⊕, C₁-C₁₂-alkyl, C₂-C₁₂-alkenyl, C₆-C₁₂-aryl, or the group R_aO- represents N-heteroaryl-N-yl with 5 ring members and 1 to 3 N atoms; R_b Represents hydrogen, C₁-C₁₂ alkyl or C₆-C₁₂ aryl; and M^⊕ for Na^⊕, K^⊕, Li^⊕, NH₄^⊕ stands or represents primary, secondary, tertiary or quaternary ammonium; where alkyl, aryl, aralkyl and alkaryl in Y_a, R_a and R_b is unsubstituted or substituted with alkoxy, alkylthio, halogen, -CN, -NO₂, phenyl, nitrophenyl, or halophenyl.

Y_a preferably contains 1 to 12 and particularly preferably 1 to 6 carbon atoms as primary amino, and preferably 2 to 12 and particularly preferably 2 to 6 carbon atoms as secondary amino.

The primary amino and secondary amino can, for example, be residues of the formula R_cR_dAct N, where R_c stands for H or independently the meaning of R_d has, and R_d C₁-C₂₀-, preferably C₁-C₁₂- and particularly preferably C₁-C₆-alkyl, C₁-C₂₀-, preferably C₁-C₁₂- and particularly preferably C₁-C₆-aminoalkyl, C₁-C₂₀-, preferably C₁-C₁₂- and especially preferably C₁-C₆ hydroxyalkyl; Carboxyalkyl or carbalkoxyalkyl, where the carbalkoxy group contains 2 to 8 carbon atoms and the alkyl group 1 to 6, preferably 1 to 4 carbon atoms; C₂-C₂₀-, preferably C₂-C₁₂- and particularly preferably C₂-C₆-alkenyl; Phenyl, mono- or di- (C₁-C₄ alkyl or alkoxy) phenyl, benzyl, mono- or di- (C₁-C₄ alkyl or alkoxy) benzyl; or 1,2-, 1,3- or 1,4-imidazolyl-C₁-C₆-alkyl, or R_c and R_d together tetra- or pentamethylene, 3-oxa-1,5-pentylene, -CH₂-NR_e-CH₂CH₂- or -CH₂CH₂-NR₁₉-CH₂CH₂-, where R_e represents H or C₁-C₄-alkyl. The amino group in the aminoalkyl can be substituted with one or two C₁-C₄ alkyl or hydroxyalkyl groups. The hydroxyl group in the hydroxyalkyl can be etherified with C₁-C₄-alkyl.

Among primary, secondary, tertiary and quaternary ammonium is for Y_a in connection with the definition of M^⊕ an ion of formula R._fR_GR_HR_iN^⊕ to understand where R_f C₁-C₂₀-, preferably C₁-C₁₂- and particularly preferably C₁-C₆-alkyl, -aminoalkyl, -hydroxyalkyl; Carboxyalkyl or carbalkoxyalkyl, where the carbalkoxy group contains 2 to 8 carbon atoms and the alkyl group 1 to 6, preferably 1 to 4 carbon atoms; C₂-C₂₀-, preferably C₂-C₁₂- and particularly preferably C₂-C₆-alkenyl; Phenyl, mono- or di- (C₁-C₄ alkyl or alkoxy) phenyl, benzyl, mono- or di- (C₁-C₄ alkyl or alkoxy) benzyl; or 1,2-, 1,3- or 1,4-imidazolyl-C₁-C₆-alkyl, and R_G, R_H and R_i are independently hydrogen or the meaning of R_f have, or R_f and R_G together tetra- or pentamethylene, 3-oxa-1,5-pentylene, -CH₂-NR_e-CH₂CH₂- or -CH₂CH₂-NO_eRepresent -CH₂CH₂-, wherein R_e represents H or C₁-C₄-alkyl, and R_H and R_i independently of each other the meaning of R_f to have. The amino group in the aminoalkyl can be substituted with one or two C₁-C₄ alkyl or hydroxyalkyl groups. The hydroxyl group in the hydroxyalkyl can be etherified with C₁-C₄-alkyl.

Examples of carboxyalkyl are carboxymethyl, carboxyethyl, carboxypropyl and carboxybutyl, 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, ethoxybenzyl. Examples of imidazolylalkyl, in which the alkyl group preferably contains 2 to 4 carbon atoms, are 1,2-, 1,3- or 1,4-imidazolylethyl or -n-propyl or -n-butyl. R₁₉ preferably represents H, methyl or ethyl.

Preferred examples of primary amino and secondary amino are methyl, ethyl, dimethyl, diethyl, di-i-propyl, mono- or di- (1-hydroxy-eth-2-yl) -, phenyl- and benzylamino, acetylamino and Benzoylamino and piperidinyl, piperazinyl and morpholinyl.

Preferred examples of primary and secondary ammonium are methyl, ethyl, dimethyl, diethyl, di-i-propyl, mono- or di- (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 (C₁-C₄-alkyl) CH = CH-CH₂-; Examples of Y_a as aralkyl are phenyl-C_nH₂_n- With n equal to a number from 1 to 6, especially benzyl; Examples of Y_a as alkaryl are mono-, di- and tri (C₁-C₄-alkyl) phenyl. Preferred substituents are chlorine, bromine, methoxy, -NO₂, -CN, 2,4-dichlorophenyl and 4-nitrophenyl. Examples of R_b are 2,2,2-trichloroethyl, 4-chlorophenyl, 2-chlorophenyl and 2,4-dichlorophenyl; and examples of R_bPyrrol-N-yl, triazol-N-yl and benzotriazol-N-yl are O- as N-heteroaryl.

In a particularly preferred embodiment, R means_a β-cyanoethyl and represents Y_a Di (i-propylamino).

If B represents a purine residue or an analogue thereof, it can be a residue of the formula II, IIa, IIb, IIc, IId, IIe or IIf,<chemistry id="chem0003" num="0003"><img file="EP0679657A2_D0003.tif" /></chemistry><chemistry id="chem0004" num="0004"><img file="EP0679657A2_D0004.tif" /></chemistry><chemistry id="chem0005" num="0005"><img file="EP0679657A2_D0005.tif" /></chemistry><chemistry id="chem0006" num="0006"><img file="EP0679657A2_D0006.tif" /></chemistry> where R_b1 represents H, Cl, Br, OH or -O-C₁-C₁₂-alkyl, and R_b2, R_b3 and R_b5 independently of one another H, OH, SH, NH₂, NHNH₂, NHOH, NHO-C₁-C₁₂-alkyl, -N = CH-N (C₁-C₁₂-alkyl) ₂, -N = CH-azacycloalkyl, F, Cl, Br, C₁-C₁₂-alkyl, hydroxy-C₁-C₁₂-alkyl, amino-C₁-C₁₂-alkyl, C₁-C₁₂-alkoxy, benzyloxy, C₁-C₁₂-alkylthio, the hydroxyl and amino groups being unsubstituted or substituted by a protective group, Phenyl, benzyl, primary amino with 1 to 20 carbon atoms or secondary amino with 2 to 30 carbon atoms, R_b4 Hydrogen, CN or -C≡CR_b7, R_b6 and R_b7 Represent hydrogen or C₁-C₄ alkyl.

Suitable protecting groups have been mentioned previously. Preferred protecting groups are C₁-C₈ acyl groups, such as acetyl, propionyl, butyroyl and benzoyl. R_b6 preferably represents H or methyl.

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 carbon atoms, are methyl, ethyl and the isomers of propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl , and corresponding alkoxy, alkylthio, hydroxyalkyl and aminoalkyl radicals. The alkyl, alkoxy, alkylthio, hydroxyalkyl and aminoalkyl particularly preferably contains 1 to 4 carbon 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 residues of the formula R_a1R_a2Act N, where R_a1 stands for H or independently the meaning of R_a2 has, and R_a2 C₁-C₂₀-, preferably C₁-C₁₂- and particularly preferably C₁-C₆-alkyl, -aminoalkyl, -hydroxyalkyl; Carboxyalkyl or carbalkoxyalkyl, where the carbalkoxy group contains 2 to 8 carbon atoms and the alkyl group 1 to 6, preferably 1 to 4 carbon atoms; C₂-C₂₀-, preferably C₂-C₁₂- and particularly preferably C₂-C₆-alkenyl; Phenyl, mono- or di- (C₁-C₄ alkyl or alkoxy) phenyl, benzyl, mono- or di- (C₁-C₄ alkyl or alkoxy) benzyl; or 1,2-, 1,3- or 1,4-imidazolyl-C₁-C₆-alkyl, or R_a1 and R_a2 together tetra- or pentamethylene, 3-oxa-1,5-pentylene, -CH₂-NR_a3-CH₂CH₂- or -CH₂CH₂-NO_a3Represent -CH₂CH₂-, wherein R_a3 represents H or C₁-C₄-alkyl. The amino group in the aminoalkyl can be substituted with one or two C₁-C₄ alkyl or hydroxyalkyl groups. The hydroxyl group in the hydroxyalkyl can be etherified with C₁-C₄-alkyl.

Examples of alkyl have been given previously. Examples of aminoalkyl are aminomethyl, aminoethyl, 1-aminoprop-2-yl or -3-yl, 1-amino-but-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-hydroxy-eth-2-yl, 1-hydroxy-prop-2- or -3-yl, 1-hydroxy-but-2-yl, -3-yl or -4-yl. Examples of carboxyalkyl are carboxymethyl, carboxyethyl, carboxypropyl and carboxybutyl, 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, ethoxybenzyl. Examples of imidazolylalkyl, in which the alkyl group preferably contains 2 to 4 carbon atoms, are 1,2-, 1,3- or 1,4-imidazolylethyl or -n-propyl or -n-butyl. R_a3 preferably represents H, methyl or ethyl.

Preferred examples of primary amino and secondary amino are methyl, ethyl, dimethyl, diethyl, allyl, mono- or di- (1-hydroxy-eth-2-yl) -, phenyl- and benzylamino, acetylamino, isobutyrylamino and benzoylamino .

In a preferred embodiment, R_b1 Represents hydrogen. In another preferred embodiment, R_b5 Represents hydrogen. In a further preferred embodiment, R is_b2 and R_b3 independently of one another H, F, Cl, Br, OH, SH, NH₂, NHOH, NHNH₂, methylamino, dimethylamino, benzoylamino, isobutyrylamino, methoxy, ethoxy and methylthio.

In addition to purine xanthine, hypoxanthine, adenine, N-methyladenine, N-benzoyladenine, 2-methylthioadenine, 2-aminoadenine, 6-hydroxypurine, 2-amino-6-chloropurine, 2-amino-6-methylthiopurine, some examples of analogs of the purine series are , Guanine, N-isobutyrylguanine. Adenine, 2-aminoadenine and guanine and their base-protected derivatives are particularly preferred.

If B in formula I represents a pyrimidine residue, it is preferably an uracil, thymine or cytosine residue of the formula III, IIIa, IIIb or IIIc<chemistry id="chem0007" num="0007"><img file="EP0679657A2_D0007.tif" /></chemistry><chemistry id="chem0008" num="0008"><img file="EP0679657A2_D0008.tif" /></chemistry> where R_b6 H or C₁-C₄ alkyl and R_b8 H, OH, SH, NH₂, NHNH₂, NHOH, NHO-C₁-C₁₂-alkyl, -N = CH-N (C₁-C₁₂-alkyl) ₂, -N = CH-azacycloalkyl, F, Cl, Br, C₁- C₁₂-alkyl, hydroxy-C₁-C₁₂-alkyl, amino-C₁-C₁₂-alkyl, C₁-C₁₂-alkoxy, benzyloxy, C₁-C₁₂-alkylthio, where the hydroxyl and amino groups are unsubstituted or substituted by a protecting group, phenyl, Are benzyl, primary amino having 1 to 20 C atoms, secondary amino having 2 to 30 C atoms, C₁-C₁₂ alkenyl or C₁-C₁₂ alkynyl, and the NH₂ group in formula IIIb is unsubstituted or substituted by C₁-C₆-alkyl, benzoyl or a protective group, and the dihydro derivatives of the radicals of the formulas III, IIIa, IIIb and IIIc. R preferably represents_b8 in formula III H, C₁-C₆-alkyl or hydroxyalkyl, C₂-C₆-alkenyl or -alkynyl, F, Cl, Br, NH₂, benzoylamino, mono- or di-C₁-C₆-alkylamino. Preferably R represents_b8 in formula IIIb and IIIc are H, C₁-C₆-alkyl or alkoxy or -hydroxyalkyl, C₂-C₆-alkenyl or -alkynyl, F, Cl, Br, NH₂, benzoylamino, mono- or di-C₁-C₆-alkylamino.

R_b6 preferably represents H or methyl. R_b8 in formula III preferably denotes H, F, Cl, Br, NH₂, NHCH₃, N (CH₃) ₂, C₁-C₄-alkyl, C₂-C₄-alkenyl or C₂-C₄-alkyn-1-yl. R_b8 in formula IIIb and IIIc preferably represents H, C₁-C₄-alkyl, especially methyl, C₂-C₄-alkenyl, especially vinyl or C₂-C₄-alkyn-1-yl, especially 1-propin-1-yl, or NH₂, NHCH₃ or (CH₃) ₂N.

Some examples of pyrimidine analogs are uracil, thymine, cytosine, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, dihydrouracil, 5-methylcytosine, 5-propinthymine and 5-propynecytosine.

The invention further relates to a process for the preparation of compounds of the formula I,<chemistry id="chem0009" num="0009"><img file="EP0679657A2_D0009.tif" /></chemistry> wherein R₁ and R₂ independently represent hydrogen or a protecting group; and B represents a purine or pyrimidine residue or an analog thereof; and<ul id="ul0001" list-style="none"><li>(a) R₃ means OH, which is characterized in that a compound of formula IVa<chemistry id="chem0010" num="0010"><img file="EP0679657A2_D0010.tif" /></chemistry> wherein R₁₄ and R₁₅ represent the same or different protective groups and B represents a purine or pyrimidine radical or an analog thereof, functional groups in the base radical B being protected by protective groups, reacted in an inert solvent with a compound of the formula A.         X-CH₂-COOR₄ (A), wherein R₄ is C₁-C₄ alkyl and X is Cl, Br, I, tosyl-O or mesyl-O; and the ester function is subsequently reduced with NaBH₄ or LiAlH₄, the OH group which forms can be temporarily protected with a group as defined for R₁;</li><li>(b) R₃ F means which is characterized in that a compound of the formula I in which R₃ is OH is reacted with a compound of the formula B.<chemistry id="chem0011" num="0011"><img file="EP0679657A2_D0011.tif" /></chemistry></li><li>(c) R₃ - (CF₂)_nIs -CF₃, where n is a number from 0 to 7, which is characterized in that a compound of the formula IVa is reacted with a compound of the formula C, D or E.         CH≡C- (CF₂)_n-CF₃ (C)         CH₂ = CH- (CF₂)_n-CF₃ (D)<chemistry id="chem0012" num="0012"><img file="EP0679657A2_D0012.tif" /></chemistry> and then the optionally present double bond or chlorinated double bond catalytically to CH₂CH₂ (CF)_nCF₃ reduced;</li><li>(d) R₃ OH, F or - (CF₂)_n-CF₃ means which is characterized in that a compound of formula IVb<chemistry id="chem0013" num="0013"><img file="EP0679657A2_D0013.tif" /></chemistry> wherein R₁₄ and R₁₅ have the meaning mentioned above and A is a leaving group, preferably alkoxy, acyloxy, mesyl-O, tosyl-O and particularly preferably OCH₃, OCOCH₃ and benzoyloxy, by one of the processes described in (a) to (c) substituted on the 2'-OH group and then introduces the base radical B by substitution in a manner known per se [E. Lukevics, A. Zablocka, Nucleoside Synthesis, Ellis Horwood, New York (1991)]; and optionally splits off the protective groups R₁₄ and R₁₅.</li></ul>

The compounds of the formulas IVa and IVb, A, B, C, D and E are known, some are commercially available or can be prepared by known or analogous processes.

Inert solvents are, for example, hydrocarbons, halogenated hydrocarbons, alkylated carboxamides and lactams, ethers, nitriles such as acetonitrile, dialkyl sulfones or sulfoxides or cyclic sulfones and sulfoxides.

The reaction temperatures in process steps (a) to (d) are between -50 to 200 ° C, preferably between 0 to 90 ° C.

In addition to B, the reactions are advantageously carried out in the presence of bases, for example alkali metal hydrides, alcoholates, hydroxides, carbonates, trialkylamines or diazabicycloundecene.

The compounds of the formula I are isolated and purified by methods known per se, such as, for example, precipitation or crystallization and filtration and chromatographic methods.

Oligonucleotides can be built up from the compounds of formula I, which, because of their interaction with nucleic acids, have valuable biological activities and can be used as active pharmaceutical ingredients or as diagnostics.

The invention furthermore relates to the use of the compounds of the formula I for the preparation of oligonucleotides which contain the same or different monomer units of compounds of the formula I, but at least one monomer unit of compounds of the formula I in combination with monomer units of other natural or synthetic nucleosides, wherein the oligonucleotides contain 2 to 200 monomer units. The oligonucleotides preferably contain 2 to 100, particularly preferably 2 to 50, and particularly preferably 4 to 30 monomer units. Oligonucleotides which contain the same or different monomer units of compounds of the formula I are preferred. Oligonucleotides which additionally contain monomer units of synthetic or natural nucleosides which are derived from D-ribose or 2-deoxyribose are also preferred.

Another object of the invention are oligonucleotides of formula V.         5'-U- (OYOV-)_xOYOW-3 '(V), where x is a number from 0 to 200 and Y is a nucleotide bridge group, U, V and W each represent the same or different radicals of natural or synthetic nucleosides and at least one of the radicals U, V and / or W is a radical of the formula VI<chemistry id="chem0014" num="0014"><img file="EP0679657A2_D0014.tif" /></chemistry> means, and B and R₃ have the meanings given for the compounds of formula I including the preferences and examples.

A preferred bridging group Y is the group -P (O) O which occurs in natural oligonucleotides^⊖-. Examples of other bridge groups are P (O) S^⊖-, -P (S) S^⊖-, -P (O) R₁₆-, P (O) NR₁₇R₁₈, or -CH₂-, wherein R₁₆ represents H or C₁-C₆-alkyl and R₁₇ and R₁₈ independently of one another have the meaning of R₁₆. In formula V, x is preferably a number from 0 to 100, particularly preferably a number from 1 to 50 and particularly preferably a number from 3 to 29. The residues of the formula VI can be attached terminally or in the nucleotide sequence, it being possible for all or more, for example 2 to 5, residues of the formula VI to follow one another, or the residues of the formula VI can be bound between residues of natural or synthetic nucleosides, or mixed forms of these distributions can be present in the nucleotide sequence.

A very particularly preferred embodiment is oligonucleotides of the formula V, in which x represents a number from 2 to 50, preferably 2 to 30, Y for the group -P (O) O^⊖- stands, U, V and W each mean the same or different residues of a natural nucleoside and at least one of the residues U, V or W corresponds to formula VI. Suitable natural nucleosides are adenosine, cytidine, guanosine, uridine, 2-aminoadenine, 5-methylcytosine, 2'-deoxyadenosine, 2'-deoxycytidine, 2'-deoxyguanosine and thymidine. Adenine, cytosine, guanine, thymine and uracil are particularly worth mentioning as natural nucleoside bases. The radicals of the formula VI can be bonded terminally or in the nucleotide sequence, it being possible for all or more, for example 2 to 5, identical or different radicals of the formula VI to follow one another, or identical or different radicals of the formula VI are bound between radicals of natural nucleosides , or mixed forms of these distributions are present in the nucleotide sequence. In another preferred embodiment of oligonucleotides of the formula V, all radicals U, V and W correspond to the same or different radicals of the formula VI. X preferably represents a number from 3 to 29 and preferably contains a total of 1 to 12 radicals of the formula VI.

The oligonucleotides according to the invention can be prepared in a manner known per se by various methods in optionally automated DNA synthesizers which can be purchased together with procedural instructions. In the case of the bridge group -P (O) O^⊖For example, the phosphorus triester process, the phosphite triester process or the H-phosphonate process can be used, which are familiar to the person skilled in the art. In the phosphite triester process, for example, one can proceed in such a way that the nucleosides of the formula I, in which R₁ and R₂ each denote H, are reacted with a protective group reagent, for example 4,4'-dimethoxytriphenylmethyl chloride, to give a nucleoside of the formula F.<chemistry id="chem0015" num="0015"><img file="EP0679657A2_D0015.tif" /></chemistry> and binds the compound of formula F to a solid support material, for example to Controlled Pore Glass (CPG), which contains long-chain alkylamino groups, using a "linker", for example succinic anhydride. In a separate process, the hydroxyl group of the compound of formula F is derivatized, for example to a phosphoramidite using R'OP [N (i-propyl) ₂)] ₂ to a compound of formula G.<chemistry id="chem0016" num="0016"><img file="EP0679657A2_D0016.tif" /></chemistry> where R 'represents, for example, β-cyanoethyl.

After splitting off the protective group, such as the DMT group of the material bound to the support, coupling -N (i-C₃H₇) ₂ is coupled with the compound of the formula F, blocking any free hydroxyl groups (capping) and then oxidizing that formed phosphite to phosphate. After deprotection of the dimer, the reaction cycle is repeated with a compound of formula G until an oligomer with the desired number of monomer units has been synthesized, and the product is detached from the support material. In this way, oligonucleotides are obtained in which all residues U, V and W according to formula V consist of residues of formula VI. In this way, oligonucleotides with any monomer units can be produced in any sequence, depending on the use of synthetic, natural and nucleoside building blocks according to the invention in the individual reaction cycles.

The compounds of formula I according to the invention, in which R₁ and R₂ each denote H, have antiviral and antiproliferative properties and can accordingly be used as medicaments. The oligonucleotides according to the invention also have a high stability against degradation by nucleases. Their excellent pairing with complementary nucleic acid strands, especially of the RNA type, is particularly surprising. In addition, they show an unexpectedly high cellular uptake. The oligonucleotides according to the invention are therefore particularly suitable for antisense technology, that is to say for the inhibition of the expression of unwanted protein products by binding to suitable complementary nucleotide sequences of mRNA (EP 266,099, WO 87/07300 and WO 89/08146). They can be used to treat infections and diseases, for example by blocking the expression of bioactive proteins at the nucleic acid level (for example oncogenes). The oligonucleotides according to the invention are also suitable as diagnostics and can be used as gene probes for the detection of viral infections or genetically caused diseases by selective interaction at the level of single or double-stranded nucleic acids ("gene probes"). In particular - due to the increased stability towards nucleases - a diagnostic application is possible not only in vitro but also in vivo (for example tissue samples, blood plasma and blood serum). Such possible uses are described, for example, in WO 91/06556.

Another object of the invention relates to the use of the oligonucleotides according to the invention as diagnostics for the detection of viral infections or genetically caused diseases.

Another object of the invention also relates to the nucleosides of the formula I according to the invention and the oligonucleotides of the formula V for use in a therapeutic method for the treatment of diseases in warm-blooded animals, including humans, by inactivating nucleotide sequences in the body. The dose when administered to warm-blooded animals of approximately 70 kg body weight can be, for example, 0.01 to 1000 mg per day. Administration is preferably carried out in the form of pharmaceutical preparations parenterally, for example intravenously or intraperitoneally.

The invention further relates to a pharmaceutical preparation containing an effective amount of a nucleoside of the formulas I or an oligonucleotide of the formulas V alone or together with other active ingredients, a pharmaceutical carrier material preferably in a significant amount and optionally auxiliaries.

The pharmacologically active nucleosides and oligonucleotides according to the invention can be used in the form of parenterally administrable preparations or infusion solutions. Such solutions are preferably isotonic aqueous solutions or suspensions, these being able to be prepared before use, for example in the case of lyophilized preparations which contain the active substance alone or together with a carrier material, for example mannitol. The pharmaceutical preparations can be sterilized and / or contain auxiliaries, 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 contain further pharmacologically active substances, such as, for example, antibiotics, are produced in a manner known per se, for example by means of conventional solution or lyophilization processes, and contain about 0.1% to 90%, in particular about 0 , 5% to about 30%, for example 1% to 5% active substance (s).

The following examples illustrate the invention. The 1 H-NMR spectra are based on the numbering of the carbon atoms in the following cyclic carbon skeletons: Output connections:<chemistry id="chem0017" num="0017"><img file="EP0679657A2_D0017.tif" /></chemistry> Nucleosides (examples):<chemistry id="chem0018" num="0018"><img file="EP0679657A2_D0018.tif" /></chemistry>

Abbreviations used in the text and in the formulas:<dl id="dl0001"><dt>DMF</dt><dd>Dimethylformamide</dd><dt>ClBnCl₂</dt><dd>2,4-dichlorobenzyl chloride</dd><dt>Bn</dt><dd>Benzyl</dd><dt>Ac</dt><dd>Acetyl</dd><dt>φ</dt><dd>Phenyl</dd><dt>BSA</dt><dd>N, N-bistrimethylsilylacetamide</dd><dt>DBU</dt><dd>Diazabicyclo [5.4.0] undec-7-ene</dd><dt>BOM-Cl</dt><dd>Benzyloxymethyl chloride</dd><dt>DMTCl</dt><dd>4,4'-dimethoxytrityl chloride</dd><dt>THF</dt><dd>Tetrahydrofuran</dd></dl>

A) Preparation of nucleoside analogs

Example A1:

28.0 g of 1-methylribose are added dropwise at 60 ° C. to an initial charge of 13.5 g of NaH in 130 ml of DMF. After the end of the H₂ development, 110.0 g of ClBnCl₂ are added dropwise. The reaction mixture is stirred at 25 ° C for 16 hours. In order to destroy any NaH still present, methanol is carefully added dropwise and the reaction mixture is then poured onto ice / water. The lumpy precipitate is filtered off and washed well with acetonitrile. Compound (A1) is obtained.<chemistry id="chem0019" num="0019"><img file="EP0679657A2_D0019.tif" /></chemistry> 1 H-NMR (250 MHz, CDCl₃): The HC (l) proton appears as a singlet at 5.0 ppm. MS: 638 (M⁺)

Example A2:

65.9 g of the product prepared in Example A1 are dissolved in 600 ml of methylene chloride and cooled to 0 ° C. Then 121 ml of SnCl₄ in 800 ml of methylene chloride are added dropwise and the mixture is left to stand at 3 ° C. After 26 hours another 2 ml of SnCl₄ are added. After a total of 35 hours, the reaction solution is carefully poured onto 700 ml of a saturated NaHCO₃ solution. After dilution with 400 ml of methylene chloride, the Sn-containing precipitate is filtered off. The organic phase of the filtrate is dried with MgSO₄ and evaporated to the compound (A2).<chemistry id="chem0020" num="0020"><img file="EP0679657A2_D0020.tif" /></chemistry> 1 H-NMR (250 MHz, CDCl₃): The HC (1) proton appears at 4.90 ppm as a doublet with J = 5 Hz.

Example A3:

125.9 g of the product obtained in Example A2 are dissolved in 1 l of pyridine. 25.5 g of acetic anhydride and 1 g of 4-dimethylaminopyridine are added at 20 ° C. The mixture is then stirred for 17 hours. The reaction mixture is taken up in 1 l of water, acidified with concentrated hydrochloric acid and extracted with ethyl acetate. The extract is dried with MgSO₄ and evaporated. Finally, the residue is crystallized with hexane. The connection (A3) is obtained.<chemistry id="chem0021" num="0021"><img file="EP0679657A2_D0021.tif" /></chemistry> 1 H-NMR (250 MHz, CDCl₃): 5.15 [d, J = 4.5 Hz, <u>H</u>-C (1)]; 3.50 (s, OC<u>H</u>₃); 2.17 (s, OCOC<u>H</u>₃); MS: 522 (M⁺) [α]_{Well (D)}= 87.4 ± 1.0 °, CHCl₃ (0.998%)

Example A4:

24th g of thymine are slurried in 100 ml of 1,2-dichloroethane. After adding 116.4 g of BSA, the mixture is heated under reflux until a clear solution is obtained. The mixture is then cooled to 50 ° C. and 50 g of the product prepared in Example A3 and 27.5 g of trimethylsilyl trifluoromethanesulfonate are added. It is stirred for 20 hours at 70 ° C and then poured onto 300 ml of NaHCO₃ solution and filtered. After extraction with dichloroethane, it is dried with MgSO₄ and evaporated. Finally, the residue is crystallized with methanol. The connection (A4) is obtained.<chemistry id="chem0022" num="0022"><img file="EP0679657A2_D0022.tif" /></chemistry> 1 H-NMR (250 MHz, CDCl₃): 8.25 (s, N<u>H</u>); 6.10 [d, J = 4.5 Hz,<u>H</u>-C (1 ')]; 2.13 (s, OCOC<u>H</u>₃); 1.66 (s, C<u>H</u>₃) MS: 616 (M⁺)

Example A5:

85 g of the product prepared in Example A4 are suspended in 850 ml of acetonitrile. 24.2 g DBU and 24.9 g BOM-Cl are added dropwise at room temperature. After stirring for 20 hours, the reaction mixture is poured into water and extracted with ethyl acetate. The extract is dried with MgSO₄ and evaporated. The connection (A5) is obtained.<chemistry id="chem0023" num="0023"><img file="EP0679657A2_D0023.tif" /></chemistry> 1 H-NMR (250 MHz, CDCl₃): 6.05 [d, J = 4.5 Hz, <u>H</u>-C (1 ')]; 5.5 (AB, C<u>H</u>₂); 5.37 [dd, HC (2 ')]; 2.13 (s, OCOC<u>H</u>₃); 1.55 (s, C<u>H</u>₃) MS: 736 (M⁺)

Example A6

106 g of the product produced in Example A5 are suspended in 1 l of THF. 26 g of a 30% NaOCH₃ / CH₃OH solution are added dropwise. After stirring for 2.5 hours, the reaction solution is poured into water, saturated aqueous sodium chloride solution is added and the mixture is extracted with ethyl acetate. After drying with MgSO₄, the extract is evaporated. Compound (A6) is obtained.<chemistry id="chem0024" num="0024"><img file="EP0679657A2_D0024.tif" /></chemistry> 1 H-NMR (250 MHz, CDCl₃): 5.93 [d, J = 5 Hz, <u>H</u>-C (1 ')]; 5.5 (AB, C<u>H</u>₂); 3.03 (d, J = 6.5 Hz, O<u>H</u>); 1.72 (s, C<u>H</u>₃) MS: 694 (M⁺)

Example A7

79.4 g of the product obtained in Example A6 are dissolved in 800 ml of THF. After adding 3.3 g of NaH, the mixture is boiled briefly and then 21 g of methyl bromoacetate are added dropwise at 40 ° C. The reaction mixture is stirred at 60 ° C. for a total of 27 hours, 1 g of NaH and 2 ml of methyl bromoacetate being added after 16 hours and after 20 hours. Finally the reaction mixture is poured onto water and extracted with ethyl acetate. The extract is dried with MgSO₄ and evaporated. This gives compound (A7).<chemistry id="chem0025" num="0025"><img file="EP0679657A2_D0025.tif" /></chemistry> 1 H-NMR (250 MHz, CDCl₃): 7.70 [s, <u>H</u>-C (6)]; 5.92 [s,<u>H</u>-C (1 ')]; 5.48 (AB, C<u>H</u>₂); 3.75 (s, OC<u>H</u>₃); 1.58 (s, C<u>H</u>₃). MS: 766 (M⁺).

Example A8

37 g of the product obtained according to Example A7 are dissolved in 400 ml of THF. 1.5 g of LiBH₄ are added in portions at 20 ° C. and the mixture is stirred for 1 hour. The reaction mixture is then carefully poured onto 500 ml of water and neutralized with 32 ml of 2N aqueous hydrochloric acid. After extraction with ethyl acetate and evaporation, compound (A8) is obtained.<chemistry id="chem0026" num="0026"><img file="EP0679657A2_D0026.tif" /></chemistry> 1 H-NMR (250 MHz, CDCl₃): 7.65 [s, <u>H</u>-C (6)]; 5.96 [s,<u>H</u>-C (1 ')]; 5.50 (AB, C<u>H</u>₂); 2.57 (broad s, O<u>H</u>); 1.60 (s, C<u>H</u>₃). MS: 738 (M⁺).

Example A9

20.0 g of the product prepared in Example A8 are dissolved in 200 ml of THF and hydrogenated over 2 g of Pd / C (5%) at 25 ° C. and under normal pressure for 4.5 hours (H₂ uptake 102%). After filtration and evaporation of the filtrate, the residue is dissolved in 170 ml of methanol and adjusted to a pH of 11 with a 30% NaOCH₃ / CH₃OH solution. After 24 hours, the mixture is poured onto 250 ml of water, acidified with 2 N aqueous hydrochloric acid and extracted with ethyl acetate. The extract is dried with MgSO₄ and evaporated. Compound (A9) is obtained.<chemistry id="chem0027" num="0027"><img file="EP0679657A2_D0027.tif" /></chemistry> 1 H-NMR (250 MHz, CDCl₃): 9.24 (s, N<u>H</u>); 7.90 [s,<u>H</u>-C (6)]; 5.99 [s,<u>H</u>-C (1 ')]; 2.68 (t, OH); 1.60 (s, C<u>H</u>₃). MS: 618 (M⁺).

Example A10

4.2 g of the product prepared in Example A9 are dissolved in 50 ml of pyridine and, after adding 2.4 g of acetic anhydride, the mixture is stirred at room temperature for 19 hours. The solution is poured onto 100 ml of 2N HCl and extracted with ethyl acetate. The extract is washed with 2N HCl and water, dried over MgSO₄ and evaporated. Compound (A10) is obtained.<chemistry id="chem0028" num="0028"><img file="EP0679657A2_D0028.tif" /></chemistry> 1 H-NMR (250 MHz, CDCl₃): 9.28 (s, N<u>H</u>); 7.67 [s,<u>H</u>-C (6)]; 5.95 [s,<u>H</u>-C (1 ')]; 2.00 (s, C<u>H</u>₃); 1.60 (s, C<u>H</u>₃). MS: 663 (M + H) ⁺.

Example A11

4.2 g of the product prepared in Example A10 are hydrogenated in 50 ml of methanol in the presence of 2.09 g of anhydrous sodium acetate over 0.8 g of Pd / C (5%) at 35 ° C. and under normal pressure. After 58 hours the hydrogenation mixture is filtered and evaporated. To remove salts, the residue is chromatographed on a small frit using silica gel (ethyl acetate / methanol 9: 1). Compound (A11) is obtained.<chemistry id="chem0029" num="0029"><img file="EP0679657A2_D0029.tif" /></chemistry> 1 H-NMR (250 MHz, DMSO): 10.1 (s, N<u>H</u>); 7.61 [s,<u>H</u>-C (6)]; 5.64 [d, J = 6 Hz,<u>H</u>-C (1 ')]; 1.70 (s, CH₃); 1.57. MS: 379 (M + Cl) ⁻.

Example A12

2.27 g of the product prepared in Example A11 are taken up twice in pyridine and evaporated. It is taken up again in 30 ml of pyridine and 2.57 g of DMTCl are added. After stirring at room temperature for 20 hours, the reaction mixture is diluted with 250 ml of ethyl acetate and poured onto 50 ml of water. The organic phase is dried with MgSO₄ and concentrated. The residue is chromatographed on silica gel (toluene / ethyl acetate / triethylamine 49: 49: 2). Compound (A12) is obtained.<chemistry id="chem0030" num="0030"><img file="EP0679657A2_D0030.tif" /></chemistry> 1 H-NMR (250 MHz, CDCl₃): 9.22 (s, NH); 7.70 [s,<u>H</u>-C (6)]; 5.94 [d, J = 1.5 Hz,<u>H</u>-C (1 ')]; 3.78 (s, OC<u>H</u>₃); 2.19 (s, C<u>H</u>₃); 1.36 (s, C<u>H</u>₃). MS: 706 (M + NH₄) ⁺.

Example A13

2.70 g of the product obtained in Example A12 are added to an initial charge of 0.93 g of diisopropylammonium tetrazolide, 1.51 g of 2-cyanoethyl-N, N, N ', N'-tetraisopropylphosphorodiamidite and 30 ml of methylene chloride. The reaction mixture is stirred for 17 hours at room temperature and then poured onto a saturated aqueous NaHCO₃ solution. The organic phase is dried with MgSO₄ and evaporated. The residue is chromatographed on silica gel (ethanol / ethyl acetate 1: 1 with 2% addition of triethylamine). The foam obtained is dissolved in 1 ml of methyl t-butyl ether and added dropwise at 0 ° C. in pentane. Compound (A13) (diastereoisomers, 1: 1) is obtained.<chemistry id="chem0031" num="0031"><img file="EP0679657A2_D0031.tif" /></chemistry> 1 H-NMR (250 MHz, CDCl₃): 7.74 [s, <u>H</u>-C (6)] and 7.68 [s, <u>H</u>-C (6)]; 6.08 [d, J = 4 Hz,<u>H</u>-C (1)]; 5.97 [d, J = 4 Hz,<u>H</u>-C (1 ')]; 31 P NMR (CDCl3): 150.174 and 150.038 MS: 847 (M + H) ⁺

Example 14:

20.6 g of the product obtained in Example A8 are dissolved in 200 ml of CH₂Cl₂. 4.35 g of diethylamino-sulfur trifluoride (DAST) are added dropwise at 5 ° C. and the reaction mixture is stirred at 5 ° C. for 3 hours. Then the solution is poured onto 300 ml of saturated NaHCO₃ solution and extracted with CH₂Cl₂. The extract is dried over MgSO₄ and evaporated. The residue is chromatographed (silica gel, toluene / ethyl acetate 1: 1). Compound (A14) is obtained.<chemistry id="chem0032" num="0032"><img file="EP0679657A2_D0032.tif" /></chemistry> 1 H-NMR (250 MHz, CDCl₃): 7.68 [s, HC (6)]; 5.92 [s,<u>H</u>-C (1 ')]; 5.50 [AB, CH₂] 1.58 (s, CH₃). ¹⁹F-NMR (CDCl₃): -223.74 (t, CH₂<u>F</u>). MS: 758 (M + NH₄) ⁺.

Example A15

1.30 g of the product obtained in Example A14 are dissolved in 26 ml of THF and hydrogenated over 0.65 g of Pd / C (5%) at 20 ° C. and normal pressure. After 0.5 hours, the catalyst is filtered off and the filtrate is evaporated. The residue is taken up in methanol (15 ml) and adjusted to pH 11 with NaOMe / MeOH solution. After stirring for 20 hours, the mixture is poured onto 20 ml of water and extracted with methyl acetate. After evaporation, compound (A15) is obtained.<chemistry id="chem0033" num="0033"><img file="EP0679657A2_D0033.tif" /></chemistry> 1 H-NMR (250 MHz, CDCl₃): 8.99 (s, N<u>H</u>); 7.61 [s,<u>H</u>-C (6)]; 5.88 [d, J = 1.5 Hz,<u>H</u>-C (1 ')]; 1.52 [s, C<u>H</u>₃]. MS: 621 (M + H) ⁺.

Example A16

Analogously to the working instructions (A11), (A12) and (A13), the compound (A15) is converted into the phosphoramidite (A16) (diastereoisomers, 1: 1).<chemistry id="chem0034" num="0034"><img file="EP0679657A2_D0034.tif" /></chemistry> 1 H-NMR (250 MHz, cDCl₃): 7.75 [s, HC (6)] and 7.68 [s, HC (6)]; 6.06 [d, J = 4 Hz, HC (1 ')] and 6.00 [d, J = 4 Hz, HC (1')]. 31 P NMR (CDCl3): 150.107.

Example A17

:

20.0 g of compound (A6) are dissolved in 200 ml of THF and kept at 60 ° C. with 0.83 g of NaH (100%) until the evolution of H₂ has ended. After cooling, 48.0 g of trifluoropropyne are pressed into this solution in an autoclave and heated to 50.degree. After 48 hours, the reaction mixture is concentrated in half, then poured onto water and extracted with ethyl acetate. The extract is dried (MgSO₄) and evaporated. The residue is chromatographed on silica gel (n-hexane / ethyl acetate 4: 1). Compound (A17) is obtained.<chemistry id="chem0035" num="0035"><img file="EP0679657A2_D0035.tif" /></chemistry> 1 H-NMR (250 MHz, CDCl₃): 7.72 [s, HC (6)]; 6.80 (d, J = 8Hz, C<u>H</u>= C); 5.82 [s, HC (1 ')]; 5.48 (AB, CH₂); 4.85 (m, c<u>H</u>= C); 1.58 (s, CH₃). MS: 789 (M + H) ⁺.

Example A18

10.0 g of compound (A17) are dissolved in 200 ml of THF and hydrogenated over 2 g of Pd / C at room temperature and normal pressure. After 1 hour the catalyst is filtered off and the filtrate is evaporated. The residue is aftertreated with NaOMe analogously to Example A15 and chromatographed (silica gel; n-hexane / ethyl acetate 2: 1). Compound (A18) is obtained.<chemistry id="chem0036" num="0036"><img file="EP0679657A2_D0036.tif" /></chemistry> 1 H NMR (CDCl₃): 9.00 (s, NH); 7.66 [s, HC (6)]; 5.93 [d, J = 1.5 Hz, HC (1 ')]; 1.61 (s, CH₃). ¹⁹F-NMR (CDCl₃): -65.23. MS: 705 (M + Cl) ⁻.

Example A19

8.9 g of compound (A6) are dissolved in 90 ml of THF and briefly boiled with 0.34 g of NaH (100%). After the H₂ evolution has subsided, 5.1 g of 1,1,2-trichloro-3,3,3-trifluoropropene are added dropwise at 20 ° C. and the mixture is then stirred at 55 ° C. for 5 hours. The reaction mixture is poured onto water and extracted with ethyl acetate. After evaporation, the mixture is chromatographed (silica gel, toluene / ethyl acetate 4: 1). Compound (A19) is obtained as a cis / trans (about 1: 1) mixture.<chemistry id="chem0037" num="0037"><img file="EP0679657A2_D0037.tif" /></chemistry> 1 H-NMR (CDCl₃): 7.63 and 7.60 [each s, each HC (6)]; 5.91 and 5.87 [each s, each HC (1 ')]; 1.61 and 1.57 (each s, each CH₃). MS: 891 (M + Cl) ⁻.

Example A20

If the compound (A18) or (A19) is deprotected analogously to the examples above and converted into the phosphoramidite, the compound (A20) (diastereoisomers 1: 1) is obtained.<chemistry id="chem0038" num="0038"><img file="EP0679657A2_D0038.tif" /></chemistry> 1 H-NMR (CDCl₃): 8.6 (broad s, NH); 7.74 and 7.69 [each s, each HC (6)]; 5.97 and 5.94 [each d, J = 4 Hz, each HC (1 ')]. 31 P NMR (CDCl3): 150.287 and 150.035 ppm.

Example B

Production of oligonucleotides

Oligonucleotides are used using the dimethoxytritylated and 3'-activated [3 '- (β-cyanoethoxy-di (i-propylamino) phosphoramidite) l nucleosides or such naturally activated nucleosides are bound to a solid support (controlled pore glass, CPG) and the synthesis is carried out on a DNA synthesizer (Applied Biosystems, model 380 B, standard phosphoramidite chemistry and iodine oxidation) according to the manufacturer's standard protocols [see also "Oligonucleotide synthesis a practical approach "MJ Gait; IRL Press 1984 (Oxford-Washington DC)]. After the coupling of the last nucleoside building block, the 5'-protected oligonucleotide is detached from the support overnight with simultaneous cleavage of all other protective groups by treatment with concentrated aqueous ammonia and then using "reverse phase" using 50 mM ammonium acetate buffer (pH 7) / acetonitrile. HPLC cleaned. The 5'-dimethoxytrityl protective group is then cleaved off by treatment with 80% aqueous acetic acid for 20 minutes, the oligonucleotide is precipitated with ethanol and isolated by centrifugation. The purity of the oligonucleotide is checked by gel electrophoresis (polyacrylamide), its identity using matrix-assisted laser desorption time-of-flight mass spectroscopy (MALDI-TOF MS).

Example C1:

Affinity; Interaction of the oligonucleotides (antisense) with complementary oligoribonucleotide sequences (sense)

The interaction of the oligonucleotides with the corresponding base-complementary oligomers of the natural ribonucleotides is determined by recording UV melting curves and the T determined from them_mValues ​​characterized. This standard method is described, for example, by Marky, LA, Breslauer, KJ, Biopolymers 26: 1601-1620 (1987). A solution of the oligonucleotides and the corresponding base-complementary natural oligoribonucleotides is prepared in 10 mM phosphate buffer, 100 mM NaCl, 0.1 mM EDTA, pH = 7.0 (c = 4 × 10 / M / oligonucleotide) and the change in absorbance at 260 nm depending on the temperature (15 to 95 ° C) recorded From the melting curves obtained, the T_mValue determined (Table 3).<tables id="tabl0001" num="0001"><img file="EP0679657A2_D0039.tif" /></tables><tables id="tabl0002" num="0002"><img file="EP0679657A2_D0040.tif" /></tables>

Example D2

Specificity; Interaction of the oligonucleotide with base-complementary oligoribonucleotides, in which a wrong nucleoside (Y) was incorporated

Solutions of the oligonucleotide are prepared with the corresponding base-complementary oligonucleotides of the sequences r (GGA CCG GAA YGG TAC GAG) in 10 mM phosphate buffer, 100 mM NaCl, 0.1 mM EDTA, pH 7, (c = 4-10⁻⁶ M / oligonucleotide ) and measures the change in absorbance at 260 nm depending on the temperature (15 ° C to 95 ° C). The T becomes from the curves_mValue determined. The results are shown in Table 4.<tables id="tabl0003" num="0003"><img file="EP0679657A2_D0041.tif" /></tables>

Example C3:

Nuclease stability; Enzymatic hydrolysis of various oligonucleotides of sequence d (TCC AGG TGT CCG ttt C)

14 µg each of the synthetic oligonucleotide and the corresponding natural oligomer are incubated in 200 µl 10% heat-inactivated serum from calf fetuses at 37 ° C (c = 70 µg / ml). After 0.5; 1; 2; 4; 6, 24 and 48 hours, 15 ul of the reaction solution are quenched by adding to 25 ul 9 M urea and trisborate buffer (pH 7) and stored at - 20 ° C until measurement . The quenched reaction solutions are separated by means of polyacrylamide gel electophoresis and the cleavage products are determined via the phosphorus content (phospho-imagers method). The ratio R the sum of the concentrations of the fully intact oligonucleotide (c_n^(t)) and the fragment created by splitting off the natural C building block from the 3 'end (c_n-1^(t)) at a given time t to the initial concentration of the completely intact oligonucleotide at time t = 0 (c_n⁽⁰⁾) R = (c_n^(t) + c_n-1^(t)) / c_n⁽⁰⁾ is plotted against time. The half-lives τ determined in the process_1/2 - these are the times for which R = 0.5 -<chemistry id="chem0039" num="0039"><img file="EP0679657A2_D0042.tif" /></chemistry><chemistry id="chem0040" num="0040"><img file="EP0679657A2_D0043.tif" /></chemistry><chemistry id="chem0041" num="0041"><img file="EP0679657A2_D0044.tif" /></chemistry>