Patent 2087818 Summary

<br/> -53-<br/> THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE <br/>PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:<br/> 1. An oligonucleotide analog of the formula I <br/><IMG> <br/>and its physiologically tolerated salts, wherein<br/> R1 is hydrogen, C1-C18-alkyl, C2-C18-alkenyl, C2-C18-<br/>alkynyl, C2-C18-alkylcarbonyl, C3-C19-alkenylcarbonyl, <br/>C3-C19-alkynylcarbonyl, C6-C20-aryl, (C6-C14) -aryl- (C1-<br/>C8)-alkyl, or a radical of the formula II<br/> <IMG> <br/>R2 is hydrogen, C1-C18-alkoxy, halogen, azido <br/>or NH2;<br/> B is a conventional base in nucleotide chemistry; <br/>a is oxy or methylene;<br/>n is an integer from 1 to 100; <br/>W is oxo, thioxo or selenoxo; <br/>V is oxy, thio, or imino;<br/> Y is oxy, thio, imino or methylene;<br/> Y' is oxy, thio, imino, (CH2)m or V(CH2)m, where <br/>m is an integer from 1 to 18;<br/> X is hydroxyl or mercapto;<br/><br/>-54-<br/> U is hydroxyl, mercapto, SeH, C1-C18-alkoxy, C1-C18-<br/>alkyl, C6-C20-aryl, (C6-C14) -aryl- (C1-C8) -alkyl, NHR3, <br/>NR3R4 or a radical of the formula III <br/>(OCH2CH2)p O(CH2)q CH2R11 (III), where<br/> R3 is C1-C18-alkyl, C6-C20-aryl, (C6-C14) -aryl- (C1-C8) -<br/>alkyl, 2-(CH2)c-[NH(CH2)c]d-NR12R12, where c is an <br/>integer from 2 to 6 and d is an integer from 0 to 6, <br/>and each R12 independently of the other is hydrogen <br/>or C1-C6-alkyl or C1-C4-alkoxy-C1-C6-alkyl;<br/> R4 is C1-C18-alkyl, C6-C20-aryl, or (C6-C10)-aryl-(C1-C8)-<br/>alkyl, or, in the case of NR3R4, is, together with R3 <br/>and the nitrogen atom carrying them, a 5-6-membered <br/>heterocyclic ring, which can additionally contain a <br/>further hereto atom selected from the group <br/>comprising O, S, N,<br/>p is an integer from 1 to 100, <br/>q is an integer form 0 to 22,<br/> R11 is hydrogen or a functional group;<br/> Z = Z' are hydroxyl, mercapto, SeH, C1-C22-alkoxy, -O-<br/>(CH2)b-NR12R13, where b is an integer from 1 to 6, <br/>and R13 is C1-C6-alkyl or R12 and R13 together <br/>with the nitrogen atom carrying them form a 3-<br/>6-membered ring, C1-C18-alkyl, C6-C20-aryl, (C6-<br/>C14)-aryl-(C1-C8)-alkyl, (C6-C14)-aryl-(C1-C8)-<br/>alkoxy, where aryl includes heteroaryl, and <br/>aryl is optionally substituted by 1, 2 or 3 <br/>identical or different radicals selected from <br/>the group comprising carboxyl, amino, nitro, C1-<br/>C4-alkylamino, C1-C6-alkoxy, hydroxyl, halogen <br/>and cyano, C1-C18-alkylmercapto, NHR3, NR3R4, a <br/>radical of the formula III or a group which <br/>favors intracellular uptake or serves as the <br/>label for a DNA probe, or, during hybridization <br/>of the oligonucleotide analog to the target<br/><br/> - 55 -<br/>nucleic acid, attacks the latter with binding, <br/>crosslinking or cleavage;<br/>the curved bracket indicates that R2 and the neighboring <br/>phosphoryl residue can be located in the 2'- and 3' <br/>position or else the opposite way round in the 3'- and <br/>2'-position,<br/>where each nucleotide can be present in its D- or L-<br/>configuration and the base B can be located in the a- or <br/>.beta.-position, with the proviso that, if Z = hydroxyl, <br/>mercapto, methyl or ethoxy, at least one of the groups X, <br/>Y, Y', V and W is not hydroxyl, oxy or oxo, or R1 is not <br/>hydrogen.<br/> 2. An oligonucleotide analog as claimed in claim 1, wherein <br/>the base B is located in the .beta.-position, the nucleotides <br/>are present in the D-configuration, R 2 is located in the <br/>2'-position and a is oxy.<br/>3. An oligonucleotide analog as claimed in claim 1 or 2, <br/>wherein<br/> R1 is hydrogen, C1-C6-alkyl or a radical of the formula <br/>II;<br/> R 2 is hydrogen;<br/>n is an integer from 10 to 40; <br/>m is an integer from 1 to 6;<br/> U is hydroxyl, mercapto, C1-C6-alkoxy, C1-C6-alkyl, NR3R4 <br/>or NHR3, where<br/> R3 is C1-C8-alkyl or methoxyethyl, and B, W, V, Y, Y', X <br/>and Z are defined as in claim 1.<br/>4. An oligonucleotide analog as claimed in claim 3, wherein <br/>R1 is methyl.<br/> 5. An oligonucleotide analog as claimed in claim 3, wherein <br/>R2 is hydrogen.<br/><br/> - 56 -<br/>6. An oligonucleotide analog as claimed in claim 3, wherein <br/>n is an integer from 12 to 30.<br/>7. An oligonucleotide analog as claimed in claim 3, wherein <br/>m is the integer 1.<br/>8. An oligonucleotide analog as claimed in claim 3, wherein <br/>U is hydroxyl or C1-C6-alkyl.<br/> 9. An oligonucleotide analog as claimed in claim 3, wherein <br/>R3 is C1-C4-alkyl.<br/>10. An oligonucleotide analog as claimed in any one of claims <br/>1 to 9, wherein V, Y, and Y' have the meaning of oxy.<br/> 11. An oligonucleotide analog as claimed in any one of claims <br/>1 to 10, wherein W has the meaning of oxo.<br/>12. An oligonucleotide analog as claimed in any one of claims <br/>1 to 11, wherein U has the meaning of hydroxyl.<br/>13. An oligonucleotide analog as claimed in any one of claims <br/>1 to 11, wherein R1 is hydrogen.<br/>14. A process for preparing an oligonucleotide analog of the <br/>formula I as claimed in claim 1, wherein<br/>a) a nucleotide unit with a 3'(2')-terminal <br/>phosphorus(V) grouping and a free 5'-hydroxyl or <br/>mercapto group is reacted with a further nucleotide <br/>unit with in the 3' position a phosphorus(III) or <br/>phosphorus(V) grouping or an activated derivative <br/>thereof, or<br/>b) the oligonucleotide analog is constructed with <br/>fragments in a similar manner,<br/><br/>- 57 -<br/>and protective groups, which have been temporarily <br/>introduced in the oligonucleotides obtained according to <br/>(a) or (b) in order to protect other functions, are <br/>removed and the oligonucleotide analog of the formula I <br/>thus obtained is, where appropriate, converted into its <br/>physiologically tolerated salt.<br/>15. The use of an oligonucleotide analog as claimed in <br/>any one of claims 1 to 13 as an inhibitor of gene <br/>expression.<br/>16. The use of an oligonucleotide analog as claimed in <br/>any one of claims 1 to 13 as a probe for detecting <br/>nucleic acids or as an aid in molecular biology.<br/>17. A pharmaceutical preparation containing one or more <br/>oligonucleotide analogs of the formula I as claimed <br/>in any one of claims 1 to 13 together with <br/>physiologically tolerated adjuvants and/or <br/>excipients and/or together with other known active <br/>substances.<br/>

<br/>203r1818<br/> HOECHST AKTIENGESELLSCHAFT HOE 92/F 012 Dr.BO/pe<br/>Description<br/> Oligonucleotide analogs, their preparation and use<br/> The present invention relates to novel oligonucleotide<br/>analogs with useful physical, biological and<br/>pharmacological properties and a process for their<br/>preparation. Their application relates to their use as<br/>inhibitors of gene expression (antisense oligo-<br/>nucleotides, ribozymes, sense oligonucleotides and<br/>triplex-forming oligonucleotides), as probes for<br/>detecting nucleic acids and as aids in molecular biology.<br/>Oligonucleotides are being used to an increasing extent<br/>as inhibitors of gene expression (G. Zon, Pharmaceutical<br/>Research 5, 539 (1988); J. S. Cohen, Topics in Molecular<br/> and Structural Biology 12 (1989) Macmillan Press; C.<br/>Helene and J.J. Toulme, Biochimica et Biophysica Acta<br/>1049, 99 (1990); E. Uhlmann and A. Peyman, Chemical<br/>Reviews 90, 543 (1990)). Antisense oligonucleotides are<br/>nucleic acid fragments whose base sequence is<br/> complementary to an mRNA which is to be inhibited. This<br/>target mRNA can be of cellular, viral or other pathogenic<br/>origin. Suitable cellular target sequences are, for<br/>example, those of receptors, enzymes, immuno- modulators,<br/>ion channels or oncogenes. The inhibition of viral<br/>replication using antisense oligonucleotides has been<br/>described, for example, for RSV (Rous sarcoma virus),<br/>HSV-1 and -2 (herpes simplex virus type I and II), HIV<br/>(human immunodeficiency virus) and influenza viruses. In<br/>this context oligonucleotides are employed which are<br/> complementary to the viral nucleic acid. By contrast, the<br/>sequences of sense oligonucleotides are designed in such<br/>a way that these oligonucleotides bind ("capture")<br/>nucleic acid-binding proteins or nucleic acid-processing<br/>enzymes, for example, and thereby inhibit their biologi-<br/>cal activity (Helene, 1990). Viral targets which can be<br/><br/>2 0 8<br/>- 2 -<br/>mentioned here as examples are reverse transcriptase, DNA<br/>polymerase and transactivator proteins. Triplex-forming<br/>oligonucleotides generally have DNA as their target, and<br/>after binding to this DNA form a triple helical struc-<br/>ture. While generally the processing (splicing etc.) of<br/>mRNA and its translation into protein are inhibited using<br/>antisense oligonucleotides, triplex-forming oligonucleo-<br/>tides inhibit the transcription or replication of DNA<br/>(Helene et al., 1990, Uhlmann and Peyman, 1990). However,<br/>it is also possible to bind single-stranded nucleic acids<br/>in a first hybridization with an antisense oligonucleo-<br/>tide, with the formation of a double strand which then<br/>forms a triplex structure with a triplex-forming oligo-<br/>nucleotide in a second hybridization. In this case the<br/>antisense and triplex binding regions can be contained<br/>either in two separate oligonucleotides or in one oligo-<br/>nucleotide. The so-called ribozymes, which destroy the<br/>target RNA as a result of their ribonuclease activity<br/>(J.J. Rossi and N. Sarver, TIBTECH 8, 179 (1990)),<br/>represent a further application of synthetic oligonucleo-<br/>tides.<br/> Suitably labeled nucleic acid fragments are employed in<br/>DNA diagnostic investigation as so-called DNA probes for<br/>specific hybridization to a nucleic acid which is to be<br/> detected. Here, the specific formation of the new double<br/>strand is followed using labeling which preferably is not<br/>radioactive. In this way, genetic and malignant diseases,<br/>and diseases caused by viruses or other pathogens, can be<br/>detected.<br/> In their naturally occurring form, oligonucleotides are<br/>little, or not at all, suited for the majority of the<br/>said applications. They have to be chemically modified so<br/>that they are suitable for the specific requirements. In<br/>order that oligonucleotides can be employed in biological<br/>systems, for example for inhibition of viral replication,<br/>they must fulfil the following preconditions:<br/><br/>2087818<br/>- 3 -<br/>1. They must possess a sufficiently high degree of<br/>stability under in vivo conditions, that is in serum<br/>as well as intracellularly.<br/>2. They must be able to pass through the cell and<br/>nuclear membranes.<br/>3. They must bind to their target nucleic acid in a<br/>base-specific manner under physiological conditions<br/>in order to exert the inhibitory effect.<br/> These preconditions are not essential for DNA probes;<br/>however, these oligonucleotides must be derivatized in a<br/>manner which permits detection, for example by<br/>fluorescence, chemiluminescence, colorimetry or specific<br/>staining, (Beck and Koster, Anal. Chem. 62, 2258 (1990)).<br/>Chemical alteration of the oligonucleotides usually takes<br/> place by altering the phosphate backbone, the ribose unit<br/>or the nucleotide bases in an appropriate manner (Cohen,<br/>1989; Uhlmann and Peyman, 1990). A further method, which<br/>is frequently employed, is the preparation of oligo-<br/>nucleotide 5'-conjugates by reacting the 5'-hydroxyl<br/>group with appropriate phosphorylation reagents. Oligo-<br/>nucleotides which are only modified at the 5'-end have<br/>the disadvantage that they are degraded in serum. if, on<br/>the other hand, all the internucleotide phosphate<br/>radicals are altered, the properties of the oligo-<br/>nucleotides are often drastically changed. For example,<br/>the solubility of the methylphosphonate oligonucleotides<br/>in aqueous medium is diminished, as is their ability to<br/>hybridize. Phosphorothioate oligonucleotides have non-<br/>specific effects, so that, for example, homooligomers are<br/>also active against viruses.<br/> The object is, therefore, to prepare oligonucleotide<br/>analogs with specific activity, increased serum stability<br/>and good solubility.<br/>,r?;<br/><br/>2007818<br/>4 -<br/> The invention relates to oligonucleotide analogs of the<br/>formula I<br/> R V<br/>= o<br/>Y R2<br/>U-P-V<br/> II<br/>L W<br/> n<br/> Y R2<br/>1<br/> Z-P-X<br/>11<br/>W<br/> and their physiologically tolerated salts, where<br/> Rl is hydrogen, C,-C1e-alkyl, preferably C1-C6-alkyl,<br/>CZ-C18-alkenyl, C2-C18-alkynyl, C2-C,8-alkylcarbonyl,<br/>C,-Clg-alkenylcarbonyl, C,-Cl,-alkynylcarbonyl,<br/>CS-C20-aryl, ( C6-C14 )-aryl- ( C1-Ce )-alkyl, or a<br/>radical of the formula II<br/> Z P Zr (II);<br/>W<br/>R2 is hydrogen, hydroxyl, C,-C18-alkoxy, halogen,<br/>azido or NH2;<br/> B is a conventional base in nucleotide chemistry,<br/>for example natural bases such as adenine,<br/>cytosine, guanine and thymine or unnatural bases<br/>such as purine, 2,6-diaminopurine, 7-<br/>deazaadenine,7-deazaguanine,N4 N4 -ethanocytosine,<br/>N6N6-ethano-2,6-diaminopurine,pseudoisocytosine;<br/>a is oxy or methylene;<br/> n is an integer from 1 to 100, preferably 10 to 40;<br/>W is oxo, thioxo or selenoxo;<br/> V is oxy, thio or imino;<br/> Y is oxy, thio, imino or methylene;<br/> Y' is oxy, thio, imino, ( CH2 ) m or V( CH2 ) m, where<br/>m is an integer from 1 to 18, preferably from 1 to<br/>6;<br/> X is hydroxyl or mercapto;<br/><br/>2087818<br/>- 5 -<br/> U is hydroxyl, mercapto, SeH, C1-CY8-alkoxy,<br/>preferably C1-C6-alkoxy, C1-C1e-alkyl, preferably<br/>C1-C6-alkyl, C6-C20-aryl, ( C6-Cla ) -aryl- ( Cl-CB ) -<br/>alkyl, NHR3, NR3R or a radical of the formula III<br/> ( OCHZCH2 ) PO ( CH2 ) yCH2R11 ( I I I), where<br/> R3 is C1-C1e-alkyl, preferably C1-CB-alkyl, C6-CZO-<br/>aryl, ( C6-C,a ) -aryl- ( C1-CB ) -alkyl, - ( CHZ ) .-<br/>[ NH ( CHz ).] d-NR iz Riz, where c is an integer from 2 to<br/>6 and d is an integer from 0 to 6, and each R 12<br/>. 10 independently of the other is hydrogen or C1-C6-<br/>alkyl or C1-C,-alkoxy-C,-C6-alkyl, preferably<br/>methoxyethyl;<br/> R is C,-C18-alkyl, preferably C1-Ce-alkyl and<br/>particularly preferably Cl-C4-alkyl, C6-C20-aryl or<br/>( C6-Clo )-aryl- ( C1-C8 )-alkyl, or, in the case of<br/> NR'R , is, together with R3 and the nitrogen atom<br/>carrying them, a 5-6-membered heterocyclic ring,<br/>which can additionally contain a further hetero<br/>atom selected from the group comprising 0, S, N,<br/>p is an integer from 1 to 100, preferably 3 to 20<br/>and particularly preferably 3 to 8,<br/>q is an integer from 0 to 22, preferably 0 to 15,<br/>R" is hydrogen or a functional group such as<br/>hydroxyl, amino, NHR13, COOH, CONHz, COORaz or<br/> halogen, where R12 is C1-C4-alkyl, preferably<br/>methyl;<br/> Z Z' are hydroxyl, mercapto, SeH, C1-CaZ-alkoxy,<br/>preferably C6-C18-alkoxy, -O- ( CH2 ) b-NR12R13, where b<br/>is an integer from 1 to 6, and R13 is C1-C6-alkyl<br/>or R12 and R13, together with the nitrogen atom<br/>carrying them, form a 3-6-membered ring, C1-C,e-<br/>alkyl, preferably C1-Ce-alkyl, C6-C2O-aryl, (C6-<br/>Cõ ) -aryl- ( C1-CB ) -alkyl, preferably ( C6-C,o ) -aryl-<br/>( C1-C4 ) -alkyl, ( C6-C14 ) -aryl- ( C1-CB ) -alkoxy,<br/> pref erably ( C6-C,o )-aryl- ( C1-C4 )-alkoxy, where aryl<br/>includes heteroaryl, and aryl is optionally<br/>substituted by 1, 2 or 3 identical or different<br/>radicals selected from the group comprising<br/>k . . . , , , , . - _.. .<br/><br/>2087818<br/>- 6 -<br/>carboxyl, amino, nitro, C1-C4-alkylamino, C1-<br/>C 6 -<br/>alkoxy, hydroxyl, halogen and cyano, C1-C18-alkyl-<br/>mercapto, NHR3, NR3R , a radical of the formula<br/> III or a group which favors intracellular uptake<br/>or serves as the label for a DNA probe, or,<br/>during hybridization of the oligonucleotide<br/>analog to the target nucleic acid, attacks the<br/>latter with binding, crosslinking or cleavage,<br/>and<br/>the curved bracket indicates that R 2 and the neighboring<br/>phosphoryl residue can be located in the 2'- and 3'-<br/>position or else the opposite way round in the 3'- and<br/>2'-position,<br/>where each nucleotide can be present in its D- or L-<br/>configuration and the base B can be located in the a- or<br/>,B-position, with the proviso that, if Z = hydroxyl,<br/>mercapto, methyl or ethoxy, at least one of the groups X,<br/>Y, Y', V and W is not hydroxyl, oxy or oxo, or R1 is not<br/> hydrogen.<br/> Preferred are oligonucleotide analogs of the formula I<br/>and their physiologically tolerated salts, where the base<br/>B is located in the ,B-position, the nucleotides are<br/>present in the D-configuration, R2 is located in the 2'-<br/> position and a is oxy.<br/> Particularly preferred are oligonucleotide analogs of the<br/>formula I, where<br/> R1 is hydrogen, C,-C6-alkyl, in particular methyl, or<br/>a radical of the formula II;<br/> R2 is hydrogen or hydroxyl, in particular hydrogen;<br/>n is an integer from 10 to 40, in particular 12 to<br/>30;<br/>m is an integer from 1 to 6, in particular 1;<br/>u is hydroxyl, mercapto, C,-C6-alkoxy, C1-C6-alkyl,<br/> NR3R or NHR3, in particular hydroxyl or Cl-C6-<br/>alkyl, where<br/>,. , , ,. .<br/><br/>_ 7 -<br/> R3 is C,-CB-alkyl, preferably CI-C,-a1ky1, or<br/>methoxyethyl, and B, W, V, Y, Y', X and Z have<br/>the abovementioned meaning.<br/> Especially preferred are oligonucleotide analogs of the<br/>formula I, where V, Y' and Y have the meaning of oxy.<br/>Additionally particularly preferred are oligonucleotide<br/>analogs of the formula I, where V, Y, Y' and W have the<br/>meaning of oxy or oxo.<br/> Very particularly preferred are oligonucleotide analogs<br/>of the formula I, where V, Y, Y', W and U have the<br/>meaning of oxy, oxo or hydroxyl.<br/> Furthermore, oligonucleotide analogs of the formula I<br/>are preferred, where R' is hydrogen.<br/> Especially preferred are oligonucleotide analogs of the<br/>formula I, where U, V, W, X, Y' and Y have the meaning<br/>of oxy, oxo or hydroxyl and R' is hydrogen.<br/> The residues which occur repeatedly, such as R2, B, a,<br/>W, V, Y, U, R3, R', p, q and Z, can, independently of each<br/>other, have identical or different meanings, i.e. each V,<br/> for example, is, independently of the others, oxy, thio<br/>or imino.<br/> Halogen is preferably fluorine, chlorine or bromine.<br/>Heteroaryl is understood to mean the radical of a mono-<br/>cyclic or bicyclic (C3-C9)-heteroaromatic, which contains<br/> one or two N atoms and/or an S or an 0 atom in the ring<br/>system.<br/> Examples of groups which favor intercellular uptake are<br/>various lipophilic radicals such as -0-(CH2),-CH3, where x<br/>is an integer from 6-18, -0-(CHZ)õ-CH=CH-(CH2)m-CH31 where n<br/>and m are independently of each other an integer from 6 to<br/>12, -0- ( CH2CH2O ) 4- ( CHZ ) 9-CH3, -0- ( CH2CH2O ) 8- ( CH2 )13-CH3 and<br/>-O- ( CH2CHZ0 ),- ( CH2 )15-CH37 and also steroid residues, such as<br/>cholesteryl, and conjugates which make use of natural<br/>carrier systems, such as bile acid, folic acid, 2-(N-<br/>alkyl, N-alkoxy)-aminoanthraquinone and conjugates of<br/><br/>2087818<br/>-s-<br/>mannose and peptides of the corresponding receptors,<br/>which lead to receptor-mediated endocytosis of the<br/>oligonucleotides, such as EGF (epidermal growth factor),<br/>bradykinin and PDGF (platelet derived growth factor).<br/> Labeling groups are understood to mean fluorescent<br/>groups, for example of dansyl (= N-dimethyl-l-amino-<br/>naphthyl-5-sulfonyl) derivatives, fluorescein derivatives<br/>or coumarin derivatives, or chemiluminescent groups, for<br/>example of acridine derivatives, as well as the<br/>digoxigenin system, which is detectable by ELISA, the<br/>biotin group, which is detectable by the biotin/avidin<br/>system, or linker arms with functional groups which allow<br/>a subsequent derivatization with detectable reporter<br/>groups, for example an aminoalkyl linker, which is<br/>reacted with an acridinium active ester to form the<br/>chemiluminescent probe. Typical labeling groups are:<br/>0 \ 0 OH<br/>~<br/>H<br/> COOH<br/>/<br/>\<br/>N H (CH2)X-O<br/>.<br/>0<br/>Fluorescein derivative<br/>(x = 2-18, preferably 4-6)<br/><br/> 2087818<br/>9 -<br/> CH3<br/>0 0<br/>0 N-(CH2}x-N-<br/>H H<br/>Acridinium ester<br/> 0 0-CH2 ).-0-<br/>x - 2-18, preferably 4<br/>COOR<br/> R- 8 or C1-C4-alkyl 5 =fluoreacein= for x 4 and R CB3)<br/> Fluorescein derivative<br/>Y.' R= H or amino-protective group<br/>0<br/>R NN H<br/>s y;<br/> 0<br/> Biotin conjugate "biotin" for R Fmoc)<br/>., ~<br/><br/>2037818<br/>-<br/>0<br/>0<br/>\<br/> HO<br/> OH<br/>0 0 =<br/> H<br/>Digoxigenin conjugate<br/> Oligonucleotide analogs which bind to nucleic acids or<br/>intercalate and/or cleave or crosslink contain, for<br/>5 example, acridine, psoralen, phenanthridine,<br/>naphthoquinone, daunomycin or chloroethylaminoaryl<br/>conjugates. Typical intercalating and crosslinking<br/>residues are:<br/>-0-(CHZ)x \ IN<br/>10 Acridine derivative x 2 12, preferably 4<br/>OCH3<br/>;,.<br/> -S-(CH2)x-NH N<br/>,<br/>CI<br/> x = 2-12, preferably 4<br/><br/>2087818<br/>-~~-<br/> C H 3 = CH2X-(CH2)2X-<br/>:<br/>0 0 0 CH<br/> C H 3 X. -NH or -o- Trimethylpsoralen conjugate "psoralen" for X 0)<br/> NH N i I 0<br/> N<br/>Phenanthroline conjugate<br/>41,<br/> 0-<br/>H N<br/> 0<br/>0<br/>0 0<br/>.;;.<br/> Psoralen conjugate<br/>0<br/> N H-~~ 0<br/>CI<br/> 0<br/>Naphthoquinone conjugate<br/> ' : 1 _ _ _<br/><br/>2087818<br/>- 12 -<br/>0 OH 0<br/> CH<br/>OH<br/> OCH30 OH 0<br/>0<br/>KCH3<br/> HO<br/> NH<br/> Daunomycin derivative<br/>~ ~<br/>C I -CH2CH<br/> N (CH2),-0-<br/>H3C<br/> X<br/>x 1-18, X alkyl, halogen, NO21 CN, -C-R<br/>'I<br/>~ ~<br/>C I -CH2CH<br/> N (CHZ)x-0<br/>C I -CHZCHZ<br/> x<br/>x 1-18, X alkyl, halogen, NO2, CN, -C-R-<br/>p<br/><br/>2 0 3~~8 13<br/>- 13 -<br/> The morpholinyl and the imidazolidinyl radicals may be<br/>mentioned as examples of NR3R4 groups in which R3 and R ,<br/>together with the nitrogen atom carrying them, form a 5-<br/>to 6-membered heterocyclic ring, which additionally<br/>contains a further hetero atom.<br/> The invention is not limited to a- and 8-D- or L-<br/>ribofuranosides, a- and ,B-D- or L-deoxyribofuranosides<br/>and corresponding carbocyclic 5-membered ring analogs,<br/>but is also valid for oligonucleotide analogs which are<br/>composed of other sugar components, for example ring-<br/>expanded and ring-contracted sugars, acyclic sugar<br/>derivatives or suitable sugar derivatives of another<br/>type. Furthermore, the invention is not limited to the<br/>derivatives of the phosphate radical which are cited by<br/>way of example in formula I, but also relates to the<br/>~=: known dephospho derivatives.<br/> As for the synthesis of biological oligonucleotides, the<br/>preparation of oligonucleotide analogs of the formula I<br/>takes place in solution or preferably on a solid phase,<br/>optionally with the aid of an automatic synthesis<br/>apparatus.<br/> However, solid phase synthesis of oligonucleotides with<br/>a phosphate or phosphate ester radical at the 3'-end is<br/>not possible by the standard phosphoramidite chemistry of<br/> Caruthers (M.D. Matteucci and M.H. Caruthers, J. Am.<br/>Chem. Soc. 103, 3185 (1981)), since the first nucleotide<br/>unit is bound to the solid support via the 3'-hydroxyl<br/>group and for this reason oligonucleotides with a 3'-<br/>hydroxyl group always result from these syntheses.<br/> Various processes based on the solid-phase method have<br/>= been described, which processes, however, are all<br/>laborious and often cannot be used for preparing<br/>derivatives such as phosphate esters or alkylphosphonates<br/>(R. Eritja et al., Tetrahedron Lett. 32, 1511 (1991); P.<br/> Kumar et al., Tetrahedron Lett. 32, 967 (1991); W. T.<br/><br/>~~~~8 1~<br/>- 14 -<br/> Markiewicz and T.K. Wyrzykiewicz, Phosphorus, Sulfur and<br/>Silicon 51/52, 374 (1990); E. Felder et al., Tetrahedron<br/>Lett. 25, 3967 (1984); R. Lohrmann and J. Ruth, DNA 3,<br/>122 (1984)).<br/> The invention therefore relates to a process for prepar-<br/>ing oligonucleotide analogs of the formula I, where<br/>a) a nucleotide unit with a 3'(2')-terminal phosphorus-<br/>(V) grouping and a free 5'-hydroxyl or mercapto<br/>group is reacted with a further nucleotide unit with<br/>a phosphorus(III) or phosphorus(V) grouping in the<br/>3' position, or its activated derivatives,<br/>or<br/>b) the oligonucleotide analog is constructed with<br/>fragments in a similar manner,<br/>and protective groups, which have been temporarily<br/>introduced in the oligonucleotides obtained<br/>according to (a) or (b) in order to protect other<br/>functions, are removed and the oligonucleotide<br/>analogs of the formula I thus obtained are, where<br/>appropriate, converted into their physiologically<br/>tolerated salt.<br/> Employed as starting component for the solid-phase<br/>synthesis is a solid support of the formula IV<br/> D-X' -CH2CH2-S ( O ) X-CH2CHZ-A-T ( IV ) ,<br/>where<br/> A is a linker arm, which, for example, is a residue of a<br/>dicarboxylic acid, a diol, an alkylamine, a dicarboxylic<br/>acid monoalkylamide, an acid amide or a phosphate of the<br/>formula<br/>0<br/>- 0 - P - 0 -<br/>OR<br/><br/> 2087818<br/>- 15 -<br/>where R is = hydrogen or C1-C6-alkyl which is optionally<br/>substituted by -CN, preferably methyl or 2-cyanoethyl, T<br/>is a solid support, for example of materials such as CPG<br/>(controlled pore glass), silica gel or an organic resin<br/>such as polystyrene (PS) or a graft copolymer of PS and<br/>polyethylene glycol (POE), which is modified in the side<br/>chain by functional groups such as hydroxyl, amino,<br/>halogen or COOH,<br/> D is a protective group which can be removed without<br/>cleaving the linker arm A and the X'-CHZCHZ-S(O)1-CH2CH2-<br/>radical (see Bioorg. Chem. 14 (1986) 274-325), such as 4-<br/>methoxytetrahydropyranyl and dimethoxytrityl, preferably<br/>dimethoxytrityl, x is an integer zero, 1 or 2 and X' is<br/>oxy or thio.<br/> The linker arm A, which connects the solid support T to<br/>the sulfur-containing radical by a chemical bond (amide,<br/>ester inter alia) (Damka et al., Nucleic Acids Res. 18,<br/>3813 (1990)), is preferably a succinic acid residue<br/>(O-C(O)-CHZCHZ-C(o)-), an oxalic acid residue<br/>(0-C(0)-C(0)-), an alkylamine, preferably LCAA (long<br/>chain alkylamine), or polyethylene glycol. A succinic<br/>acid residue is particularly preferred. In particular<br/>cases, for example in combination with substituents which<br/>do not withstand lengthy treatment with ammonia, more<br/>labile linkers such as the oxalyl linker are advantage-<br/>ous. The preparation of solid supports of the formulae IV<br/>a-c is described in Example 1.<br/><br/>2087818<br/>- 16 -<br/> Trager D X' x A-T<br/> IVa DMTr 0 2 OEt<br/>-O-C-(CH2)2-C-N-(CH2)3-Si-CPG<br/>0 OH OEt<br/> IVb DMTr 0 2-O-C-(CH2)2-C-N - TentaGel<br/>O OH<br/>Trager D X' x A-T<br/> IVc DMTr 0 0 0<br/>I I<br/> -O-P-O - TentaGel<br/>OCH2-CH2-CN<br/> The solid-phase synthesis can take place according to the<br/>phosphate triester method, the H-phosphonate method or<br/>the phosphoramidite method, preferably according to the<br/>phosphoramidite method (E. Sonveaux, Bioorg. Chem. 14,<br/>274 (1986)). The protective group D is always first of<br/>all removed from the support of the formula IV, prefer-<br/>ably by an acid, for example trichloroacetic acid in<br/>methylene chloride. In the case of the phosphoramidite<br/>method, the support of the formula IV' thus obtained<br/>' HX' -CHZ-CHZ-S ( O ) X CHZCH2-A-T ( IV' ) ,<br/>where x, X', A and T have the abovementioned meaning, is<br/>condensed in the presence of a weak acid such as<br/>tetrazole with a nucleoside phosphoramidite of the<br/>formula V R--V B '<br/> Q<br/>Y R2 R5 (V)<br/>I<br/> Z P--N~<br/>where<br/><br/>- 17 -<br/> R is a protective group which can be removed under<br/>mild conditions, such as 4-methoxytetrahydropyranyl<br/>or dimethoxytrityl,<br/> RZ' is hydrogen, C,-C18-alkoxy, halogen or a protected<br/>hydroxyl or amino group and<br/> R5 and R6 independently of each other are C1-C12-alkyl, or<br/>both residues together form a 5 to 6-membered ring,<br/>Y" is oxy, thio or (CHZ)m, and<br/> a, m, V and Z have the abovementioned meaning.<br/> Subsequently, the support thus obtained is oxidized in a<br/>manner known per se with iodine water (W = 0) or with<br/>TETD (tetraethylthiuram disulfide) or elemental sulfur<br/>(W = S) or with selenium (W = Se) to form the derivatized<br/>support of the formula VII<br/> R--V B<br/>0<br/>(VII)<br/>Y RZ<br/>= I<br/>Z-P-X'-CH2CH2-SO2-CH2-CH2-A-T<br/>II<br/> where W<br/> R, V, B' , R2' , Z, X', W, Y", A and T have the<br/>abovementioned meaning. Supports of the formula VIIa<br/>R-V B'<br/> 0 = (VIIa)<br/>O 0 0 OEI<br/> I ti ~~ I<br/>Z-IPI-O-(CH2)2-S02-(CH2)2-0-C-(CH2)2-C-N-(CH2)3-SI-CPO<br/> w H I<br/> OEf<br/>are preferably prepared.<br/> The phosphoramidite of the formula V can be obtained, for<br/>example, from the bisamidite of the formula VI<br/><br/>2087818<br/>R-V 18 - B<br/> a<br/>(VI)<br/>R7 Y R2 Rs<br/> where R~N-P-NCR<br/>6<br/> R7 and RB are identical to R5 and R6 and<br/>a, R, V, B' , R", Y' ', Rs and R6 have the abovementioned<br/>meaning, by reaction with the corresponding alcohol or<br/>thioalcohol using tetrazole catalysis (Example 2, Method<br/>A), if Z is = alkoxy or alkylmercapto (J.E. Marugg et<br/>al., Tetrahedron Lett. 27, 2271 (1986). Preferred bis-<br/>amidites are those of the formula VIa<br/> DMTr--O B'<br/>0<br/> VIa)<br/> R 0 RS<br/>R'~N-P-N< R<br/> 6<br/> In this way the amidites of the formulae VIII a-m were<br/>prepared, for example,<br/> DIdT r--0 B'<br/>(VIII)<br/>0 Rs<br/> Z-P-NC<br/> R6<br/>where<br/> R5 and R6 have the abovementioned meaning, Z has the<br/>meaning of<br/>a) O-CHZCH3,<br/>b) O-i-C3Hõ<br/>c ) O-n-C6H13<br/>d) O-n-C1eH371<br/> N<br/>e) 0-(CH2)3<br/>f ) 0-(CH2)2 / NOi<br/><br/>2087818<br/>- 19 -<br/>g-k) a residue of the formula III (R11 = H), where in the<br/>case of<br/>g) p = 3 andq=0,<br/>h) p= 4 and q= 9,<br/>i) p = 5 and q = 4 and in the case of<br/>k) p= B and q= 13,<br/>rr;<br/>p) CH3 ~C Hs)4-0-<br/>I<br/> m<br/> N~<br/>_<br/>and B' is<br/>Cyti'B in the case of a), c) and d),<br/>Thy in the case of b) and p) and<br/>CytBZ in the case of e) - k) and m).<br/> An alternative method for loading the support is the<br/>reaction of the phosphitylation reagent of the formula IX<br/>R9<br/> Zõ - P< 10 (IX),<br/>R<br/>where<br/>R9 and R20 are, independently of each other, Cl, or Z",<br/>where Z" is = Z, with the proviso that hydroxyl, mer-<br/>capto and SeH must be present as protected derivatives,<br/>R5 R7<br/> -NC R g , -NC R s<br/>for example as O-CHZCHZ-CN, O-CH3, S-CHZCH2CN,<br/>X' -CH2CH2-S ( O ) x,-CHZCHZ-X' -D<br/> or C,<br/>5-CN2 o C 1<br/>preferably as X' -CH2CH2-S ( 0) X,-CH2CH2-X' -DMTr, where x' is<br/>an integer zero or 1, in particular as O-CHZCH2-S-CH2CH2-<br/>0-DMTr, and R5, R6, R7, Re, X', DMTr and D have the above-<br/>mentioned meaning, with a nucleoside with a free 3'(2')-<br/>,t<br/><br/>~<br/>2~~3~~'818<br/>- 20 -<br/>group of the formula X<br/>R-V<br/> a<br/>(X)<br/>2,<br/> R<br/>where<br/> Y"' is oxy or thio and V, B' and R have the abovemen-<br/>tioned meaning, and subsequent condensation of the<br/>compound thus obtained onto the support of the formula<br/>IV' ir. the presence of a condensing agent, such as<br/>tetrazole (for R9, R10 = NR5R6 or NR'RB) or diisopropylamine<br/>(for R9, R10 = Cl); this often represents the quicker<br/> method (Example 3, method B). Subsequent oxidation with<br/>iodine water or sulfur or selenium then leads to the<br/>compound of the formula VIIa. The protective group R can<br/>now be removed and the oligonucleotide synthesis con-<br/>tinued in a known manner. At the end of the synthesis,<br/>the protective groups are removed in a known manner from<br/>the support-bound oligonucleotide analog thus obtained,<br/>and the oligonucleotide analog of the formula I according<br/>to the invention is then cleaved off the support.<br/> If the synthesis was concluded in the last cycle with a<br/>unit of the formula V, an oligonucleotide analog of the<br/>formula I(Rl = H) is obtained with a 5'-hydroxyl group<br/>and a phosphorus-containing conjugation at the 3'-end.<br/>If, on the other hand, a phosphorylating reagent, for<br/>example of the formula IX, where R9 is = Z", is employed<br/> in the last condensation step, an oligonucleotide analog<br/>of the formula I with R' = formula II, which possesses a<br/>phosphate-containing substitution at both the 3'- and 5'-<br/>ends, then results from the synthesis.<br/> The preparation of oligonucleotides with a 3'-terminal<br/>phosphoramidate group is, for example, possible by<br/>reaction of the support of the formula IV' (x =<br/><br/>2087818<br/>- 21 -<br/>0) with the monomeric methoxyphosphoramidite of the<br/>formula V (Z = O-CH3) in the presence of tetrazole, if the<br/>oxidation is carried out, as described in Jager et al.<br/>(Biochemistry 27, 7237 (1988), with iodinelH2NR3 or<br/> HNR3R", where R3 and R have the abovementioned meaning.<br/>In certain cases (Z=NHR3, NR3R' , 0, S or Se) the<br/>introduction of the group Z can also take place by the H-<br/>phosphonate method, in which a nucleoside H-phosphonate<br/>of the formula XI<br/> R - V<br/>, . R2=<br/>(XI)<br/> II<br/>where R, V, a, B', Y', X' and W have the abovementioned<br/>meaning, is initially reacted with a support of the<br/>= formula IV' in the presence of a condensing agent such as<br/>pivaloyl or adamantoyl chloride and a base such as<br/>pyridine. The H-phosphonate diester formed, of the<br/>formula VII' R - V<br/>a<br/> B'<br/>(VII')<br/>RZ<br/> Yi<br/> H<br/>W P ~X ' -CH2CHZ-S02-CHZCH2-A-T<br/>is then subjected to an oxidative phosphoramidation (B.<br/>Froehler, Tetrahedron Lett. 27, 5575 (1986)) or to<br/> oxidation with iodine water, sulfur or selenium. In this<br/>way an oligonucleotide with a 3'-terminal cholesteryl<br/>group can be prepared starting from, for example, VII' (x<br/>= 0), with a cholesteryloxycarbonyl-aminoalkylamine in<br/>the presence of carbon tetrachloride. By oxidative<br/>amidation with 2-methoxyethylamine, oligonucleotides with<br/>a 3'-O-(2'-methoxyethyl)-phosphoramidate residue are<br/><br/>208ti 818<br/>22 -<br/>obtained, for example. Subsequent chain construction<br/>takes place in a known manner according to the phosphor-<br/>amidite, H-phosphonate or triester methods.<br/> The preparation of oligonucleotide analogs of the formula<br/> I is also possible using the triester method, where the<br/>hydroxyl group of the support of the formula IV' is<br/>reacted with a protected phosphate diester of the formula<br/>XII<br/> R - V<br/> R2.<br/>Y' (XII)<br/>M=P1-X'0<br/> where R, V, a, B', R2, Y', Z, W and X' have the abovemen-<br/>tioned meaning, in the presence of a condensing agent.<br/>Preferred condensation reagents are arylsulfonyl chlor-<br/>ides such as mesitylenesulfonyl chloride,<br/> 2,4,6-triisopropylbenzenesulfonyl chloride or 8-quino-<br/>linesulfonyl chloride in the presence of nucleophilic<br/>catalysts such as imidazole, triazole, tetrazole or their<br/>substituted derivatives such as N-methylimidazole, 3-<br/>nitrotriazole or 5-(p-nitrophenyl)-tetrazole. Particular-<br/>ly preferred condensing agents are 4-substituted deriva-<br/>tives of pyridine-N-oxide or quinoline-N-oxide (Efimov et<br/>al., Nucleic Acids Research 13 (1985) 3651). Compared<br/>with the H-phosphonate and phosphoramidite processes, the<br/>triester process has the advantage that no additional<br/>oxidation step is required.<br/> If the oligonucleotide synthesis is carried out with a<br/>thio (x = 0) or sulfinyl (x = 1) support of the formula<br/>IV', these groups are then at the end oxidized to the<br/>sulfonyl radical in a manner known per se [Funakoshi et<br/> al., Proc. Natl. Acad. Sci. 88 (1991), 6982], in order to<br/>ensure ready cleavage with bases, preferably ammonia.<br/><br/>2037318<br/>- 23 -<br/> The nature of the amino-protective groups of the bases B'<br/>and the constitution of the linker arm A depend, in the<br/>individual case, on the nature of the substituent Z,<br/>since the latter must be removable without difficulty<br/>once synthesis has been completed. For example, in<br/>preparing an oligonucleotide 3'-phosphate isopropyl ester<br/>(Z = O-i-C,H,), Benzoyl (Bz) protective groups can be used<br/>for B = Ade and Cyt and isobutyryl (i-Bu) protective<br/>groups for B = Gua. On the other hand, to synthesize an<br/>oligonucleotide 3'-methylphosphonate ester (Z = CH3) or<br/>ethyl ester (Z = O-C2H5 ), the more labile phenoxyacetyl<br/>(PAC) and isobutyryl protective groups are used for B<br/>Ade and Gua, and for B = Cyt, respectively.<br/> Many conjugates possess additional functional groups,<br/>which must be protected in a suitable manner before<br/>incorporation into the monomeric units of the formula V.<br/>For example, the carboxyl group of fluorescein must be<br/>protected as an alkyl ester. In psoralen, the amide group<br/>can be present as a N-Fmoc (fluorenylmethoxycarbonyl)-<br/> protected compound. Hydroxyl groups can be protected from<br/>side reactions by acylation or silylation (t-butyldi-<br/>methylsilyl). Amino groups can also be present in the<br/>trifluoroacetyl-protected form. In exceptional cases, the<br/>conjugates may be so unstable that they would be decom-<br/>posed under the conditions of protective-group removal<br/>during the oligonucleotide synthesis. In such cases it is<br/>convenient to incorporate only one linker arm with a<br/>functional group, for example Z = HN-(CH2),-NH-Fmoc, where<br/>x is an integer from 2-12, preferably 4-6, in the monomer<br/>of the formula V. After incorporation into the oligonuc-<br/>leotide and removal of the protective groups, preferably<br/>with ammonia, the free amino group may be coupled to<br/>active esters. The base-labile acridinium ester, for<br/>example, was prepared in this way.<br/> Characterization of the synthesized oligonucleotide<br/>derivatives takes place by electro-spray ionization mass<br/><br/>24 -<br/>spectrometry (Stults and Masters, Rapid Commun. Mass.<br/>Spectr. 5 (1991) 350).<br/> The oligonucleotide analogs of the formula I according to<br/>the invention were tested for their stability in serum<br/>and toward known exonucleases.<br/> It was found, surprisingly, that, in comparison with the<br/>unmodified oligonucleotides, all oligonucleotide analogs<br/>of the formula I possess markedly increased stability<br/>toward the serum nucleases, while their hybridization<br/>behavior is only slightly affected.<br/> While unmodified oligonucleotides have a half life of<br/>about two hours in fetal calf serum, all oligonucleotide<br/>analogs of the formula I are satisfactorily stable for<br/>about 16 hours. In addition, the oligonucleotide analogs<br/>of the formula I are stable toward snake venom phospho-<br/>diesterase, whereas only those where R' is not hydrogen<br/>are resistant to spleen phosphodiesterase. Unmodified<br/>oligonucleotides are degraded exonucleolytically from the<br/>3'-end by snake venom phosphodiesterase and from the<br/>5'-end by spleen phosphodiesterase.<br/> With complementary single-stranded nucleotide sequences,<br/>the oligonucleotide analogs of the formula I form stable,<br/>double-stranded hybrids due to Watson-Crick base pairing,<br/>while they form triple helical structures with double-<br/>stranded nucleic acids due to Hoogsteen base pairing.<br/>In this way, the regulation or suppression of biological<br/>functions of nucleic acids is possible using the<br/>oligonucleotide analogs according to the invention, for<br/>example suppression of the expression of cellular genes<br/> as well as of oncogenes or of viral genome functions.<br/>Oligonucleotide analogs of the formula I may therefore be<br/>used as medicaments for the therapy or prophylaxis of<br/>viral infections or cancers.<br/><br/> - 25 -<br/> The activity of the oligonucleotides according to the<br/>invention was determined on the basis of the inhibition<br/>of HSV-1 viral replication. By way of example, the<br/>following oligonucleotides of the formula 1 were found to<br/>be active against HSV-1:<br/> Sequence Points of attack in HSV-1<br/>5' GGG GCG GGG CTC CAT GGG GG IE 110 (start)<br/>5' CCG GAA AAC ATC GCG GTT GT UL 30 (middle)<br/>5' GGT GCT GGT GCT GGA CGA CA UL 48 (middle)<br/>5' GGC CCT GCT GTT CCG TGG CG UL 52 (middle)<br/>5' CGT CCA TGT CGG CAA ACA GCT UL 48 (start)<br/>5' GAC GTT CCT CCT GCG GGA AG IE4/5 (splice site)<br/>In their natural form, i.e. without 3'-derivatization,<br/>the selected sequences are inactive toward HSV-1 in cell<br/> culture, probably since they are subject to rapid degra-<br/>dation in serum or have insufficient cell penetration. On<br/>the other hand, the 3'-derivatized oligonucleotides of<br/>the formula I inhibit HSV-1 replication to differing<br/>extents. The following served as control sequences with<br/>the appropriate chemical derivatization but with no<br/>antiviral activity:<br/>5' CCA GGG TAC AGG TGG CCG GC control<br/>5' GAC TAA TCG GGA ATG TTA AG control<br/> An oligonucleotide of the formula I modified with<br/>psoralen at the 3'-end (Example 4s) recognizes the IE4/5<br/>region of HSV-2 and inhibits the replication of HSV-2.<br/>The anti-viral activity of the psoralen conjugates may be<br/>significantly increased by irradiation with UV light. The<br/>HSV-1/2 genome, with its 160,000 bases, naturally offers<br/> innumerable alternative target sequences of diverse<br/>efficiency for inhibiting viral replication. By varying<br/>the nucleotide sequences, the therapeutic principle may<br/>be applied to any other viruses, bacteria or other<br/>pathogens. The sole prerequisite for transfer to other<br/><br/>2087818<br/>- 26 -<br/>pathogens is that the genes which are essential for the<br/>life cycle of these pathogens are known. The sequences of<br/>these genes are deposited in great variety in the so-<br/>called gene databases. This is also the case for<br/>oncogenes and other cellular genes whose function is to<br/>be suppressed. Examples of other cellular genes are those<br/>which encode enzymes, receptors, ion channels, immunomo-<br/>dulators, growth factors and other regulatory proteins.<br/>Examples of oncogenes are abl, neu, myc, myb, ras, fos,<br/> mos, erbB, ets, jun, p53, src and rel.<br/> Antisense and triplex-forming oligonucleotide sequences<br/>are, for example, known as inhibitors of the cyclic AMP-<br/>dependent protein kinase (L. Sheffield, Exp. Cell Res.<br/>192 (1991) 307), the strychnine-sensitive glycine recep-<br/>tor (Akagi et al., Proc. Natl. Acad. Sci. USA 86 (1989),<br/>86, 8103), the chloride channel (Sorscher et al., Proc.<br/>Natl. Acad. Sci. USA 88 (1991), 7759), Interleukin-6<br/>(Levy et al., J. Clin. Invest. 88 (1991), 696), the basic<br/>fibroblast growth factor (Becker et al., EMBO J. 8<br/> (1989), 3685) and the c-myc oncogene (Postel et al.,<br/>Proc. Natl. Acad. Sci. USA 88 (1991), 8227). The follow-<br/>ing further examples of sequences of other target mole-<br/>cules are intended to illustrate the broad applicability<br/>of the oligonucleotides according to the invention.<br/> a) Antisense oligonucleotides against HIV-1:<br/>5' ACA CCC AAT TCT GAA AAT GG 3' (splice site)<br/>5' AGG TCC CTG TTC GGG CGC CA 3' (primer binding<br/>site)<br/>b) EGF receptor (epidermal growth factor receptor)<br/>= 30 5' GGG ACT CCG GCG CAG CGC 3' (5' untranslated)<br/>5' GGC AAA CTT TCT TTT CCT CC 3' (aminoterminal)<br/>c) p53 tumor suppressor<br/>5' GGG AAG GAG GAG GAT GAG G 3' (5'-noncoding)<br/><br/>}n 1<br/>~0 ~ 1<br/>- 27 -<br/>5' GGC AGT CAT CCA GCT TCG GAG 3' (start of trans-<br/>lation)<br/>d) c-fos oncogene<br/>5' CCC GAG AAC ATC ATG GTC GAA G 3' (start of trans-<br/>5 lation)<br/>5' GGG GAA AGC CCG GCA AGG GG 3' (5'-noncoding)<br/>e) ELAM-1 (endothelial leucocyte adhesion molecule)<br/>5' ACT GCT GCC TCT TGT CTC AGG 3' (5'-noncoding)<br/>5' CAA TCA ATG ACT TCA AGA GTT C 3' (start of trans-<br/>10 lation)<br/>f) ICAM-1 (intracellular adhesion molecule)<br/>5' CTC CCC CAC CAC TTC CCC TC 3' (3'-untranslated)<br/>5' GCT GGG AGC CAT AGC GAG G 3' (start of trans-<br/>lation)<br/>g) BCR-ABL (Philadelphia chromosome translocation)<br/>5' GCT GAA GGG CTT CTT CCT TAT TG 3' (BCR-ABL<br/>breakpoint)<br/> Compared to the oligonucleotide derivatives with a 3'-<br/>hydroxyl group, known from the literature, DNA probes<br/>which comprise oligonucleotide analogs of the formula I<br/>on the one hand offer the advantage of increased nuclease<br/>stability and on the other permit the acceptance of<br/>identical or different marker molecules at both ends of<br/>the oligonucleotide. it is of advantage that different<br/>marker groupings can be selectively activated within one<br/>oligonucleotide (double labeling). The bifunctional<br/>derivatization can also be used to introduce a label at<br/>the one end and an additional function (for example an<br/>affinity label) at the other end. For this purpose,<br/>biotin, which recognizes avidin or streptavidin, can, for<br/>example, be incorporated at the 3'-end of the<br/>oligonucleotide, while an acridinium ester chemi-<br/>luminescence label can be attached to the 5'-end via an<br/><br/>2 0N 8 18<br/>- 28 -<br/>alkylamino linker.<br/> In addition, the penetration behavior of the<br/>oligonucleotide analogs according to the invention is in<br/>many cases more favorable than in the case of unmodified<br/>oligonucleotides, in particular if lipophilic radicals<br/>are introduced. The increased stability of the oligonuc-<br/>leotides and their improved cell penetration are<br/>expressed in the form of a higher biological activity as<br/>compared with the unmodified oligonucleotides.<br/> The previously mentioned diagnostic, prophylactic and<br/>therapeutic applications of the oligonucleotide analogs<br/>according to the invention are only a selection of<br/>representative examples, and the use of the analogs is<br/>therefore not limited to them. In addition, the<br/>oligonucleotide analogs according to the invention may,<br/>for example, be employed as aids in biotechnology and<br/>molecular biology.<br/> The invention relates furthermore to pharmaceutical<br/>preparations which contain an effective amount of one or<br/>more compounds of the formula I or their physiologically<br/>tolerated salts, where appropriate together with physio-<br/>logically tolerated adjuvants and/or excipients, and/or<br/>other known active substances, as well as a process for<br/>preparing these preparations, wherein the active sub-<br/>stance, together with the excipient and possibly further<br/>adjuvants, additives or active substances, is converted<br/>into a suitable presentation. Administration preferably<br/>takes place intravenously, topically or intranasally.<br/>Example 1: Preparation of a support of the formula IV<br/> a) Preparation of the support of the formula IVa by<br/>reacting aminopropyl-CPG with the succinate of bis-<br/>hydroxyethyl sulfone dimethoxytrityl ether<br/><br/>~~37818<br/>- 29 -<br/>4.56 g of the dimethoxytrityl (DMTr) monoether of bis-(2-<br/>hydroxyethyl) sulfone (10 mmol) are dried by twice being<br/>taken up and concentrated in abs. pyridine, and are<br/>dissolved in 25 ml of abs. pyridine, then 1.78 g<br/>(14 mmol) of DMAP (dimethylaminopyridine) and 1.4 g of<br/>succinic anhydride (14 mmol) are added and this mixture<br/>is stirred at room temperature for 3 hours. After the<br/>reaction is complete, the mixture is concentrated, the<br/>residue is taken up and concentrated three times in<br/>toluene to remove the pyridine, and then taken up<br/>in 220 ml of methylene chloride. The organic phase is<br/>washed with 10% strength citric acid (110 ml) and 3 times<br/>with 110 ml of water, dried over sodium sulfate and<br/>concentrated. The resulting solid residue is dried in<br/>vacuo (5.64 g). 1.67 g (3 mmol) of this succinate are<br/>taken up and concentrated twice in abs. pyridine and<br/>dissolved in a mixture of 0.65 ml of abs. pyridine and<br/>6 ml of tetrahydrofuran (THF). A solution of 420 mg<br/>(3 mmol) of p-nitrophenol and 687 mg of DCC (dicyclo-<br/>hexylcarbodiimide, 3.3 mmol) in 2.1 ml of abs. THF is<br/>then added and the mixture is stirred at room temperature<br/>for two hours. Once the reaction is complete, the<br/>precipitated dicyclohexylurea is removed by<br/>centrifugation. The sediment is suspended in 1 ml of abs.<br/>ether and centrifuged once again. 1.5 g of the<br/>aminopropyl-CPG support from Fluka (500 A, 100 mol/g of<br/>amino group) are suspended in a mixture of 1.8 ml of abs.<br/>DMF and 350 pl of triethylamine, and the combined<br/>solutions of the nitrophenyl succinate ester, which have<br/> been decanted from the sediment, are added, and the<br/>mixture shaken at room temperature for 16 hours. The<br/>solid support is separated off and shaken at room<br/>temperature for one hour with 3 ml of capping reagent<br/>(acetic anhydride/2,6-lutidine/DMAP; each 0.25 M in THF)<br/>to block reactive groups. The derivatized CPG support is<br/>then filtered off with suction, washed with methanol,<br/>THF, methylene chloride and ether and subsequently dried<br/>in vacuo at 40 C. The loading of the support of the<br/><br/> - 30 -<br/>formula IVa with dimethoxytrityl-containing component is<br/>38 mol/g.<br/>b) Preparation of the support of the formula IVb by<br/>reacting TentaGel ( = registered trademark of the Rapp<br/>company, TUbingen) with the succinate of the bishydroxy<br/>ethyl sulfone dimethoxytrityl ether.<br/>100 mg of the amino form of the TentaGel resin, a PS/POE<br/>copolymer with 250 pmol/g amino group, are suspended in<br/>a mixture of 360 pl of DMF and 70 pl of triethylamine,<br/>and 400 mol of the p-nitrophenyl succinate ester (prepa-<br/>ration see Ex. la) are added and the mixture is shaken at<br/>room temperature for 16 hours. The subsequent workup is<br/>as described in Ex. la). The loading of the TentaGel<br/>,". resin of the formula IVb with dimethoxytrityl-containing<br/>component is 98 mol/g.<br/>c) Preparation of the support IVc by reacting TentaGel<br/>(hydroxy form) with the phosphitylating reagent of the<br/>formula IX ( Z''-DMTr-O-CH2CHZ-S-CHzCH2-O-; R9 = N( i-C3H, ) z;<br/>R10 = 0-CH2CHZCN ) .<br/> 50 mg of the hydroxy form of the TentaGel resin with<br/>500 pmol/g hydroxyl group are reacted in acetonitrile at<br/>22 C with 10 equivalents of the phosphitylating reagent<br/>of the formula IX ( Z"= DMTr-O-CH2CH2-S-CH2CH2-O-;<br/>R9 = N( i-C3H, ) 2; R10 = O-CH2CH,CN ) in the presence of 25<br/> equivalents of, tetrazole. After oxidizing with iodine<br/>water (1.3 g of iodine in THF/water/pyridine;<br/>70:20:5=v:v:v), working up is carried out as described in<br/>Example la. The loading of the support of the formula IVc<br/>with dimethoxytrityl-containing component is 247 mol/g.<br/> Example 2: Preparation of protected nucleoside 3'-phos-<br/>phoramidites of the formula VIII<br/><br/>31 20fl71818<br/>- -<br/>a) Preparation of VI I Ia (B' = CytiB , Z=0-CH2CH37<br/>R5=R6=i-C3H, )<br/> 2 mmol of the nucleoside 3'-phosphorobisamidite of the<br/>formula VI ( B' -Cytien, R5=R6=R'=R8=i-C,H, ) are taken up and<br/>concentrated twice in 20 ml of abs. acetonitrile and then<br/>' dissolved in 20 ml of abs. acetonitrile. A solution of 2.4 mmol of ethanol <br/>and 1.2 mmol of sublimed tetrazole<br/>in 5 ml of abs. acetonitrile is then added dropwise over<br/>a period of 15 minutes. After stirring has been continued<br/>for a further 2.5 hours, the mixture is diluted with<br/>75 ml of methylene chloride, and the organic phase is<br/>extracted with 50 ml of 5% strength sodium bicarbonate<br/>solution. The aqueous solution is washed twice with 50 ml<br/>of methylene chloride, the combined organic phases are<br/>dried over sodium sulfate and concentrated in vacuo. The<br/>residue is purified by column chromatography on silica<br/>gel with methylene chloride/n-heptane/triethylamine<br/>(45:45:10;v:v:v). 0.7 g of the required diastereomeric<br/>substance is obtained as a compound which is pure by<br/>thin-layer chromatography. (31P-NMR o=146.7, 147.5 ppm).<br/>Traces of the corresponding bis-ethyl phosphite are<br/>isolated as byproduct (31P-NMR o=139.3 ppm).<br/> b) Preparation of VIIib (B'= Thy, Z = O-i-C,Hõ<br/>R5=R6=i-C3H7)<br/> The preparation takes place by phosphitylation of the 5'-<br/>0-dimethoxytritylthymidine of the formula X (B' = Thy<br/>position); R = DMTr, V = 0, a=0, Y"=O; 2mmol) with the<br/>bisamidite of the formula IX (Z" = O-i-C3Hõ<br/>R'=R10=N ( i-C,H, ) Z; 4 mmol) in the presence of tetrazole<br/> (0.5 mmol) in 10 ml of abs. methylene chloride. The<br/>mixture is worked up as in Example 2a. ("P-NMR<br/>a=145.04 ppm, 145.66 ppm).<br/>c) Preparation of VIIic (B'= CytiB , Z O-n-C6H131<br/>RS=R6=1-C3H7)<br/><br/> 2087818<br/>- 32 -<br/> In an analogous manner to Example 2a from the bisamidite<br/>of the formula VIa (B'=CytiBn, R5=R6=R'=RB=i-C,H,) by<br/>reaction with one equivalent of n-hexanol with tetrazole<br/>catalysis. (31P-NMR 148.1 ppm, 148.5 ppm).<br/>d) Preparation of VIII d(B'= Cyti$ , Z = O-n-C16H37<br/>R5=R6=i-C3H7)<br/> In an analogous manner to Example 2a from the bisamidite<br/>of the formula VIa (B'=CytiH , R5=R6=R'=RB=i-C3H,) by<br/>reaction with one equivalent of n-octadecanol with<br/>tetrazole catalysis. (31P-NMR 147.2 ppm, 147.9 ppm).<br/>e) Preparation of VIIIe (B'= CytBZ, Z = 3-pyridylpropan-<br/>3-oxy, R5=R6=R7 =RB=i-C3H, )<br/> In an analogous manner to Example 2a from the bisamidite<br/>of the formula VIa (B'= Cytaz, R5=R6=R'=RB=i-C,Hõ R2" = H)<br/>by reaction with one equivalent of 3-pyridine(propan-3-<br/>ol) with tetrazole catalysis. In this case it was<br/>possible to separate the two diastereomers by column<br/>chromatography. (31P-NMR diastereomer 1: 147.7 ppm,<br/>diastereomer 2: 148.2 ppm)<br/>f) Preparation of VIIif (B' = CytBZ, Z= p-nitro-<br/>phenylethyl-2-oxy, R5=R6=i-C,H,)<br/> In an analogous manner to Example 2a from the bisamidite<br/>of the formula VIa (B'= CytBz, R5=R6=R7 =RH=i-C3H7) by<br/>reaction with one equivalent of p-nitrophenylethan-2-ol<br/>with tetrazole catalysis. (31P-NMR 148.1 ppm, 148.6 ppm).<br/>g) Preparation of VI I Ig ( B' = CytBZ, Z=- ( OCH2CH2 ) 30CH31<br/>R5=R6=i.-C 3H,)<br/> In an analogous manner to Example 2a from the bisamidite<br/>of the formula VIa (B'= CytBz, R5=R6=R'=RB=i-C3H7) by<br/><br/>2037818<br/>- 33 -<br/>reaction with one equivalent of triethylene glycol<br/>monomethyl ether with tetrazole catalysis. (31P-NMR<br/>148.5 ppm, 148.9 ppm).<br/>h) Preparation of VI I I h (B' =Cytez, Z= -(OCH2CH2 ),O ( CHa ) 9CH3,<br/> R5=R6=i-C,H, )<br/> In an analogous manner to Example 2a from the bisama.dite<br/>of the formula VIa (B'= CytBZ, R5=R6=R7 =R8=i-C,H7) by<br/>reaction with one equivalent of tetraethylene glycol<br/>monodecyl ether with tetrazole catalysis. (31P-NMR<br/>148.4 ppm, 148.8 ppm).<br/>i) Preparation of VIIIi (B'=Cyt$z, Z(OCHZCHZ)50(CH2),CH3,<br/>R5=R6=i-CsH7)<br/> In an analogous manner to Example 2a from the bisamidite<br/>of the formula VIa (B'= CytBz, R5=R6=R7 =R3=i-C3H7) by<br/>reaction with one equivalent of pentaethylene glycol<br/>monopentyl ether with tetrazole catalysis.<br/>(31P-NMR 148.4 ppm, 148.9 ppm).<br/>k) Preparation of VI I I k( B' =CytHZ, Z=-(OCHZCHZ ) BO ( CHZ )13CH3,<br/>R5=R6=i-C3H7)<br/> in an analogous manner to Example 2a from the bisamidite<br/>of the formula VIa (B'= Cytez, R5=R6=R7=R8=i-C3H7) by<br/>reaction with one equivalent of octaethylene glycol<br/>monotetradecyl ether with tetrazole catalysis (31P-NMR<br/>148.4 ppm, 148.8 ppm).<br/>1) Preparation of VIIip (B' = Thy, Z CH3, R5=R6=i-C3H,)<br/>In an analogous manner to Example 2b from 5'-O-<br/>dimethoxytritylthymidine by phosphitylation with the<br/>reagent of the formula IX ( Z' CH3, R9 = Cl, R'o =<br/>N(i-C3H7)2, where, instead of tetrazole, catalysis is<br/> effected with two equivalents of diisopropylethylamine.<br/><br/>~ 000a 818<br/>- 34 -<br/>(31P-NMR 120.6 ppm, 121.0 ppm).<br/>m) Preparation of VIIim (B'=CytBZ, Z = acridine-9-(butyl-<br/>4-oxy ) -, R5=R6=i-C3H7)<br/> In an analogous manner to Example 2a from the bisamidite<br/>of the formula VIa (B' = CytsZ, R5=R6=R7 =R8=i-C3H7) by<br/>reaction with one equivalent of 9-(4-hydroxybutyl)-<br/>acridine with tetrazole catalysis.<br/>(31P-NMR 146.7 ppm, 147.4 ppm).<br/> Example 3: Preparation of the support-bound nucleotide of<br/>the formula VII<br/>a) Method A: Preparation of a support of the formula<br/>VIIa-1 by coupling the nucleoside 3'-phosphoramidite of<br/>the formula VIIib<br/> 7.5 mg of the support from Example la, to which is bound<br/>0.2 mol of the bishydroxyethyl sulfone dimethoxytrityl<br/>ether, are treated with 3% strength trichloroacetic acid,<br/>thereby removing the DMTr protective group, washed with<br/>acetonitrile, and subsequently reacted with 2 pmol of the<br/>nucleoside 3'-phosphoramidite of the formula VIIib (B' _<br/> Thy, Z=O-i-C3Hõ R5=R6=i-C3H7) in the presence of tetrazole<br/>(10 mol) in acetonitrile. The reaction time is 2.5<br/>minutes. Oxidation with iodine (for W=O; 1.3 g of iodine<br/>in THF/water/pyridine; 70:20:5=v:v:v) then takes place.<br/>b) Method B: Preparation of a support of the formula<br/> VIIa-2 by reaction via the phosphitylation reagent of the<br/>formula IX<br/> The phosphitylation reagent of the formula IX (Z "= n-<br/>octyl, R9=R10=C1; 1 equivalent) is reacted in the presence<br/>of 1.2 equivalents of diisopropylethylamine (DIPEA) in<br/>abs. acetonitrile or methylene chloride with a nucleoside<br/>of the formula X (1 equivalent of 5'-O-dimethoxytrityl-<br/><br/>2 08<br/>35 -<br/>thymidine, B' = p-position, Y"'= 0,) at -78 C to form<br/>the corresponding nucleoside-3'-0-n-octylphosphone<br/>monochloride. To remove the protective group D=DMTr, the<br/>= support of the formula IVa is treated as described in<br/> Method A, and then washed with acetonitrile and reacted<br/>with an excess of the nucleoside-3'-0-n-octylphosphone<br/>monochloride, prepared in situ, in the presence of DIPEA.<br/>After oxidation with iodine water, a support-bound<br/>nucleotide of the formula VIIa-2 is obtained, which is<br/> available for the subsequent oligonucleotide synthesis.<br/>Example 4: Preparation of oligonucleotides of the formula<br/>I (the monomer is in each case a,e-D-deoxyribonucleoside)<br/>a) Preparation of an oligonucleotide of the formula Ia<br/>(R'=R2=H, Z=O-i-C3Hõ a=U=V=W=X=Y=Y'=O, B=Thy, n=9)<br/> TpTpTpTpTpTpTpTpTpTp-(O-i-C3H,)<br/>0.2 mol of the support VIIa-1 (B' = Thy, W=O, Z=O-i-C,H,)<br/>from Example 3a is treated with the following reagents in<br/>turn:<br/>1. abs. acetonitrile<br/>2. 3% trichloroacetic acid in dichloromethane<br/>3. abs. acetonitrile<br/>4. 4 pmol of p-cyanoethyl 5'-O-dimethoxytrityl-<br/>thymidine-3'-phosphite-diisopropylamidite and<br/>pmo1 of tetrazole in 0.15 ml of abs.<br/>25 acetonitrile.<br/>5. Acetonitrile<br/>6. 20% acetic anhydride in THF with 40% lutidine and<br/>10% dimethylaminopyridine<br/>= 7. Acetonitrile<br/>8. Iodine (1.3 g in THF/water/pyridine;<br/>70:20:5=v:v:v)<br/><br/>- 36 -<br/> The steps 1 to 8, hereinafter termed one reaction cycle,<br/>are repeated 8 times to construct the decathymidylate<br/>derivative. After the synthesis has been completed,<br/>removal of the dimethoxytrityl group takes place as<br/>described in steps 1 to 3. The oligonucleotide is cleaved<br/>from the support, and the p-cyanoethyl groups are<br/>simultaneously eliminated, by treatment for 1.5 hours<br/>with ammonia. Since the oligonucleotide does not contain<br/>any amino-protective groups, no further treatment with<br/>ammonia is necessary. The resultant crude product of<br/>isopropyl decathymidylate 3'-phosphate is purified by<br/>polyacrylamide gel electrophoresis or HPLC.<br/>b) Preparation of an oligonucleotide of the formula lb<br/>(Rl = RZ = H, Z = O-i-C3Hõ a=U=V=W=X=Y=Y'=O)<br/>d(CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCpTp-O-i-C3H,)<br/>The synthesis takes place in an analogous manner to<br/>Example 4a, but with different nucleotide bases in the<br/>monomer. In synthesis steps 1 to 8, the monomer is<br/>generally employed as a,e-cyanoethyl 5'-O-dimethoxy-<br/> trityl-nucleoside-3'-phosphite-dialkylamide, where the<br/>amino group of adenine (Ade), cytosine (Cyt) or guanine<br/>(Gua) is provided with suitable protective groups. In<br/>this example, N6-benzoyl-Ade (AdeBZ) , N4 -benzoyl-Cyt (CytBz)<br/>and NZ-isobutyryl-Gua (GuaiB ) are used. Chain construction<br/>takes place as described in Example 4a, starting with the<br/>support of the formula VIIa-1 (B' = Thy, W = 0, Z = O-i-<br/>C,H,), and condensing on the corresponding monomers<br/>according to the above sequence. However, to remove the<br/>amino-protective groups, an additional treatment with<br/> ammonia (50 C for 16 hours) is carried out.<br/>c) Preparation of an oligonucleotide of the formula Ic ( Rl<br/>R2 = H, Z 0-CHZCH3, a U V W Y Y' = 0)<br/>rs d(CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-O-CHZCH,<br/><br/> - 37 -<br/> Starting with the support of the formula VIIa-3<br/>(B'=Cyt'B , W=O, Z=0-CZHS) , whose preparation takes place<br/>with the aid of the monomer of the formula VIIIa accord-<br/>ing to method A (Example 3a) , the synthesis is carried<br/>out in an analogous manner to Example 4b. However, the<br/>more labile amino-protective groups N6-phenoxyacetyl-Ade<br/>(AdePA ) , N -isobutyryl-Cyt ( CytiBn ) and NZ-phenoxyacetyl-<br/>Gua (GuaPAd), which are easier to cleave at the end of the<br/>synthesis, are advantageously used to prepare base-labile<br/> substitutions (as here for Z = O-C2H5) . Removal of the<br/>protective groups with ammonia then only takes 2 hours at<br/>50 C. If the product is treated with ammonia for a<br/>further 6 hours at 50 C, about 5 to 10 percent of the<br/>oligonucleotide-3'-phosphate is obtained as a byproduct<br/>as a result of cleavage of the ethyl phosphate ester.<br/>d) Preparation of an oligonucleotide of the formula Id (R1<br/>= R2 = H, Z = 0-(CHZ) 17CHõ a = U = V = X = Y = Y' = 0; W<br/>= 0, except for the last two 5'-terminal phosphorothioate<br/>= internucleotide bonds, where W = S (indicated as ps))<br/>d( CpsGpsTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-O- ( CH2 )17CH3 )<br/>Starting with the support of the formula VIIa-4 (B' _<br/>CytiB , W = 0, Z = O- (CH2 )õCH, ), whose preparation takes<br/>place with the aid of the monomer of the formula VIIId<br/>according to method A (Example 3a), the synthesis is<br/> carried out with the more labile protective groups in an<br/>analogous manner to that described in Example 4c. After<br/>coupling the penultimate nucleotide (G) and the last<br/>nucleotide (C), a TETD solution (0.4 M tetraethylthiuram<br/>disulfide in acetonitrile) is employed for the sulfur<br/>oxidation instead of iodine water. The protective groups<br/>are removed by treatment with ammonia for 2 hours. An<br/>oligonucleotide of the formula Id is obtained with two<br/>5'-terminal phosphorothioate internucleotide bonds and a<br/>3'-O-n-octadecyl phosphate ester residue.<br/><br/>- 38 -<br/>e) Preparation of an oligonucleotide of the formula Ie (R'<br/>= R 2 = H, Z = CHõ a- V = W = X- Y = Y' = 0; U = 0,<br/>except for the two 5'-terminal methylphosphonate inter-<br/>nucleotide bonds, where U = CH3 (indicated as põe))<br/>d ( Cpr,eGp,,aTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCpTpM, )<br/>Starting with the support of the formula VIIa-5 (B' _<br/>Thy, W = 0, Z = CH3)1 whose preparation takes place with<br/>the aid of the monomer of the formula VIIIp according to<br/>method A (Example 3a), the synthesis is carried out in an<br/> analogous manner to Example 4c. Instead of the normal<br/>cyanoethyl-protected monomers (formula VIIi,<br/>Z = OCH2CH2CN), the corresponding methylphosphonamidites<br/>(formula VIII, Z = CH3 ) are employed for coupling the last<br/>two nucleotide units (G and C). Cleavage from the support<br/> with conc. ammonia (1.5 hours at room temperature) is<br/>followed by a 6-hour treatment with ethylenediamine/<br/>ethanol/water (5:4:1; v:v:v) to liberate the amino groups<br/>of the bases. The result is an oligonucleotide-3'-methyl-<br/>phosphonate with two 5'-terminal methylphosphonate<br/>internucleotide bonds of the formula Ie.<br/>f) Preparation of an oligonucleotide of the formula If (R1<br/>= R2 = H, Z CH31 X S, a U V W Y Y' = 0)<br/>d(CpGpTPCpCpApTpGpTpCpGpGpCpApApApCpApGpCp(s)M )<br/>Starting with the support of the formula IVa from<br/> Example la, the methylphosphonamidite of the formula VIII<br/>(Z = CH3, B' = CytiB , R. = R6 = i-C,H, ) is coupled in the<br/>first reaction cycle as described in Example 3a. Oxida-<br/>tion is carried out with TETD. Further synthesis is as<br/>described in Example 3c. After removal of the protective<br/>groups in an analogous manner to Example 3e, an oligonuc-<br/>leotide-3'-methylphosphonothioate of the formula If is<br/>obtained.<br/>L .. , . . . . . .. . . .... . . . . ~ . .. . . .. . ' .. ... . - . . . . .<br/><br/> 39 - 2087818<br/>-<br/>g) Preparation of an oligonucleotide of the formula Ig (R1<br/>= RZ = H, Z= X = S, a = U = V = W = Y = Y' = 0)<br/>d(CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp(s)s)<br/>In analogy with the synthesis described in Example 4b,<br/> starting with the support of the formula IVa from Example<br/>la, with the difference, however, that in the first<br/>condensation step a nucleoside-3'-phosphoramidite of the<br/>formula VIII (Z = 2,4-dichlorothiobenzyl; R5 = R6 = ethyl)<br/>is employed instead of the methylphosphonamidite. Once<br/>again the introduction of the second S atom takes place<br/>by oxidation with TETD (0.4 M in acetonitrile). Cleavage<br/>of the dichlorobenzylthio group takes place in a known<br/>manner with thiophenol/triethylamine. After removal of<br/>the protective groups with conc. ammonia, an oligonucleo-<br/>tide-3'-phosphorodithioate of the formula Ig is obtained.<br/>h) Preparation of an oligonucleotide of the formula Ih (R'<br/>= R2 = H, Z= p-nitrophenylethyl-2-oxy, a = U= V= W= X<br/>= Y = Y' = O)<br/>d(CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-O-CHZCH2-( O,-<br/> NOZ) ~<br/>Starting with the support of the formula VIIa-6 (B' _<br/>CytBZ, W = 0, Z= p-nitrophenylethyl-2-oxy), whose prepa-<br/>ration takes place with the aid of the monomer of the<br/>formula VIIIf according to method A (Example 3a), the<br/> synthesis is carried out in an analogous manner to<br/>Example 4b. After removal of the protective groups by<br/>10-hour treatment with ammonia at 55 C, an oligonucleo-<br/>tide-3'-O-(p-nitrophenylethyl) phosphate of the<br/>formula Ih is obtained.<br/> i) Preparation of an oligonucleotide of the formula Ii (R'<br/>RZ = H, Z 3-pyridylpropan-3-oxy, a U V W X<br/>Y = Y' = 0)<br/><br/> Z087818<br/>- 40 -<br/>d(CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-O-(CHZ)3s~<br/>Starting with the support of the formula VIIa-7 (B'<br/>CytiB , W = 0, Z=-0-(CHZ),00 ), which was prepared with<br/>the aid of the amidite of the formula VIIIe as described<br/> in Example 3a, the oligonucleotide synthesis takes place<br/>in analogy with Example 4c.<br/>k) Preparation of an oligonucleotide of the formula Ik (R'<br/>= R2 = H, Z=-0-(CHZCH2O)3CH3, a = U = V = W = X = Y= Y'<br/>= O)<br/>d(GpApGpGpApCpGpTpTpCpCpTpCpCpTpGpCpGpGpGpApApGpGpCp-O-<br/>( CHZCH2O ) 3CH3 )<br/> Starting with the support of formula VIIa-8 (B' = CytBZ,<br/>W = 0, Z=-0-(CH2CH2O),CH3, which was prepared with the<br/>aid of the amidite of the formula VIIig as described in<br/> Example 3a, the oligonucleotide synthesis corresponding<br/>to the above sequence takes place in analogy with Example<br/>4b.<br/>1) Preparation of an oligonucleotide of the formula I1 (Rl<br/>= RZ = H, Z = -0-( CHZCH2O ) 5( CH2 ) 4CHõ a = U = V = W = X = Y<br/>= Y' = 0)<br/>d(CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-0-(CH2CH2O)5-<br/>( CHz ) aCHa )<br/> Starting with the support of the formula VIIa-9 (B' _<br/>CytBz, W= 0, Z= -O-( CH2CH2O ) 5( CHZ ) 4CHõ which was prepared<br/> with the aid of the amidite of the formula VIIIi as<br/>described in Example 3a, the oligonucleotide synthesis<br/>takes place in analogy with Example 4b.<br/>m) Preparation of an oligonucleotide of the formula Im (R'<br/>= R2 = H, Z = -0- ( CH2CHa0 ) e ( CHa ) iaCHa , a = U = V = W = X =<br/>{<br/><br/>2037818<br/>- 41 -<br/> Y = Y' = 0)<br/>d(CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-O-(CHZCH2O)e-<br/>(CHz) 13CH3)<br/> Starting with the support of the formula VIIa-10 (B' _<br/> CytBz, W = 0, Z= -O-( CHZCHZO ) B( CHZ )13CH3 ), which was pre-<br/>pared with the aid of the amidite of the formula VIIIk as<br/>described in Example 3a, the oligonucleotide synthesis<br/>takes place in analogy with Example 4b.<br/>n) Preparation of an oligonucleotide of the formula In<br/>(Rl = R2 = H, Z=-(CH3)N(CH2)ZN(CH3)Z, a = U = V= W = X<br/>Y = Y' = O)<br/> d(CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-N(CH3)(CH2)2N-<br/>(CH3)2<br/> Starting with the support of the formula IVc from Example<br/>lc, the oligonucleotide synthesis is carried out as<br/>described in Example 4a with the exception that a meth-<br/>oxyphosphoramidite of the formula VIII (B' = Cyt''B", Z=<br/>OCH3, R5 = R6 = N( i-C3H, ) 2 is employed for the first conden-<br/>sation reaction and the oxidative amidation takes place<br/> for two times 15 minutes with a 0.1 M iodine solution in<br/>THF/ N,N',N'-trimethylethylenediamine (2:1; v:v). After<br/>construction of the oligonucleotide sequence, the base-<br/>stable sulfide support is oxidized with NaIO4 in a manner<br/>known per se to the base-labile sulfone support. Cleavage<br/> from the support and removal of the protective groups<br/>(PAC for Ade and Gua; i-Bu for Cyt) is effected with t-<br/>butylamine/methanol (1:1, v:v) at 50 C for 16 hours. An<br/>oligonucleotide-3'-trimethylethylenediamine-phosphoramid-<br/>ate of the formula In is obtained.<br/>o) Preparation of an oligonucleotide of the formula Io (R1<br/>= R2 = H, Z=-HN(CHZ ) 20-CH3, a U V W X Y Y' _<br/>0)<br/><br/>- 42 -<br/>d(CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-HN(CH2)20-CH3)<br/>In analogy with Example 4n, the oxidative amidation with<br/>a 0.1 M iodine solution in THF/2-methoxy-ethylamine (2:1;<br/>v:v) takes place for two times 15 minutes. After removal<br/> of the protective groups, an oligonucleotide-3'-(2-<br/>methoxyethvl)-phosphoramidate of the formula Io is<br/>obtained.<br/>p) Preparation of an oligonucleotide of the formula Ip (R1<br/>=formula II, RZ=H, Z = S, a=U=V=W=X=Y=Y' _<br/> Z' = 0)<br/>d(psCpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCps)<br/>The synthesis is carried out as described in Example 4b,<br/>starting with the support of the formula IVa. However,<br/>after coupling the first unit (formula VIII; B' = CytHZ;<br/> Z = O-CHZCH2CN; R5 = R6 = N( i-C3H7 ) Z) oxidation is carried<br/>out with TETD. After removal of the DMTr protective group<br/>of the last base added, the free 5'-hydroxyl group is<br/>phosphitylated with the bis-cyanoethyloxy-phosphoramidite<br/>of the formula IX (R9 = N( i-C3H7 ) 2, Z" = R10 = OCH2CH2CN,<br/>and subsequently oxidized to the thiophosphate with TETD.<br/>The cyanoethyl-protective groups are eliminated during<br/>ammonia treatment. The result is an oligonucleotide-3'5'-<br/>bis-thiophosphate of the formula Ip.<br/>,f.<br/> q) Preparation of an oligonucleotide of the formula Iq (R1<br/>= formula II, R2 = H, Z= O-i-C3H7, a= U= V = W= X = Y<br/>= Y' = Z' = 0)<br/>d(i.-C3H7-O-pCpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCpTp-0-<br/>1-C3H7 )<br/> The synthesis is carried out as described in Example 4b.<br/> After removal of the DMTr protective group of the last<br/>base added, the free 5'-hydroxyl group is phosphitylated<br/>ti<br/><br/>2087818<br/>- 43 -<br/>with the cyanoethyloxy-i-propyloxy-phosphoramidite of the<br/>formula IX (R9 = N(i-C3H,)2, R10 = OCHZCH2CN, Z" = 0-i-C3Hõ<br/>and subsequently oxidized with iodine water. The result<br/>is an oligonucleotide-3'5'-bis-isopropyl phosphate ester<br/>of the formula Iq.<br/>r) Preparation of an oligonucleotide of the formula Ir (R1<br/>=formula II, RZ=H, Z=n-CBH,7, a=U=V=W=X=Y=<br/>Y' = Z' = 0)<br/> d(CH,(CHZ),-pCpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCpTp-<br/>(CH2)7CH3)<br/> Starting with the support of the formula VIIa-2 (B' _<br/>Thy, W = 0, Z=(CHZ)?CH3), whose preparation is described<br/>in Example 3b, the synthesis is carried out in analogy<br/>with Example 4c. After removal of the DMTr protective<br/> groupof the last base added, the free 5'-hydroxyl group<br/>is phosphitylated with n-octyldichlorophosphane of the<br/>formula IX (Z" =(CH2)7CH3, R9 = R10 = Cl) using DIPEA<br/>(diisopropylethylamine). After oxidation and hydrolysis,<br/>the oligonucleotide is cleaved from the support as in<br/> Example 4e. An oligonucleotide-3'5'-bis-(n-octylphosphon-<br/>ate) of the formula Ir is obtained.<br/>s) Preparation of an oligonucleotide of the formula Is<br/>(Rl=R2= H, Z" = "psoralen", a=U=V=W=X=Y=Y'<br/>0)<br/>d(GpGpCpGpCpCpCpGpGpCpCpTpGpCpGpApGpApApApGpCpGpCpGp-<br/>"psoralen )<br/> The synthesis takes place in analogy with Example 4c<br/>starting with the support of the formula VIIa-11 (B' _<br/>GuaPAQ, Z = "psoralen", W = 0), which was prepared in<br/> analogy with Example 3a from the monomer of the formula<br/>VI I I ( B' = GuaPA', Z- "psoralen", R5 = R6 = i-C3H, ), which<br/>had previously been obtained from the bisamidite VIa (B'<br/>= GuaPR , RS-R8 = i-C3H, ) by reaction with "psoralen"-H<br/><br/> - 44 -<br/>(U. Pieles and U. Englisch, Nucleic Acids Research (1989)<br/>17, 285.) in analogy with Example 2a. After removal of<br/>the protective groups with ammonia, an oligonucleotide of<br/>the formula Is is obtained, to which a "psoralen" phos-<br/>phate ester is bound at the 3'-end.<br/>t) Preparation of an oligonucleotide of the formula It (R'<br/>= R2 = H, Z = "biotin", a = U = V = W = X = Y = Y' = 0)<br/>d(GpGpCpGpCpCpCpGpGpCpCpTpGpCpGpApGpApApApGpCpGpCpGp-<br/>"biotin")<br/> The synthesis takes place in analogy with Example 4c<br/>starting with the support of the formula VIIa-12 (B'=<br/>GuaPA , Z="biotin", W = 0), which was prepared in<br/>analogy with Example 3a from the monomer of the formula<br/>VI I I (B' = GuaP7 , Z="biotin" , R5 = R6 = i-C3H, ), which<br/>had previously been obtained from the bisamidite VIa (B'<br/>= GuaPAQ, R5-R8 = i-C3H, ) by reaction with "biotin"-H<br/>(R. Pon, Tetrahedron Lett. (1991) 32, 1715) in analogy<br/>with Example 2a. After removal of the protective groups<br/>with ammonia, an oligonucleotide of the formula It is<br/>obtained, to which a "biotin" phosphate ester is bound at<br/>the 3'-end.<br/>u) Preparation of an oligonucleotide of the formula Iu (R'<br/>=R2=H, Z= "fluorescein", a=U=V=W=X=Y=Y' _<br/>0)<br/>d(GpGpCpGpCpCpCpGpGpCpCpTpGpCpGpApGpApApApGpCpGpCpGp-<br/>"fluorescein")<br/> The synthesis takes place in analogy with Example 4c,<br/>starting with the support of the formula VIIa-13 (B' _<br/>GuapAc, Z = "fluorescein", W = 0), which was prepared in<br/> analogy with Example 3a from the monomer of the formula<br/>VIII (B' = GuaPA', Z="fluorescein", RS = R6 = i-C,H,),<br/>which had previously been obtained from the bisamidite<br/><br/> 2087818<br/>- 45 -<br/> VIa (B' = GuaPAO, R5-R8 = i-C3H,) by reaction with "fluores-<br/>cein"-H (Schubert et al., Nucleic Acids Research (1991)<br/>18, 3427) in analogy with Example 2a. After removal of<br/>the protective groups with ammonia, an oligonucleotide of<br/>the formula Iu is obtained, to which a fluorescein"<br/>phosphate ester is bound at the 3'-end.<br/>v) Preparation of an oligonucleotide of the formula Iv (Rl<br/>R 2 = H, Z= acridin-9-yl-but-4-oxy, a = U = V = W = X<br/>Y = Y' = 0)<br/> d(CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-(acridin-9-yl-<br/>but-4-oxy))<br/> Starting with the support of the formula VIIa-14 (B' _<br/>CytBZ, W = 0, Z = acridin-9-yl-but-4-oxy), whose prepara-<br/>tion takes place using the monomer of the formula VIIIm<br/> in analogy with Example 3a, the oligonucleotide synthesis<br/>is carried out as described in Example 4b. After depro-<br/>tection, an oligonucleotide of the formula Iv is ob-<br/>tained, which contains an acridin-9-yl-but-4-yl phosphate<br/>ester at the 3'-end.<br/>w) Preparation of an oligonucleotide of the formula Iw (R1<br/>= R 2 = H, Z = HN ( CHZ ) 3NH ( CHZ ) qNH (CH2 ) ,NHZ , a = U = V = W =<br/>X = Y = Y' = O)<br/> d(CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-HN(CH2)3NH-<br/>( CHZ ) 4NH ( CH2 ) 3NH2 )<br/> The synthesis takes place in analogy with Example 4n,<br/>with the oxidative amidation being carried out with<br/>spermine. A capping reaction is then carried out with<br/>trifluoroacetic anhydride instead of acetic anhydride.<br/>After removing the protective groups, an oligonucleotide<br/> of the formula Iw is obtained, which contains a spermine-<br/>phosphoramidate residue at the 3'-end.<br/><br/>2087818<br/>- 46 -<br/>x) Preparation of an oligonucleotide of the formula Ix (R1<br/>R 2 = H, Z = aziridyl-N-ethyl-2-oxy, a = U = V = W = X-<br/>Y = Y' = O)<br/> CH2<br/>d(CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-O(CH2)ZN<br/> CH2<br/>~:.<br/> The synthesis takes place in analogy with Example 4c,<br/>starting with the support of the formula VIIa-15 (B'<br/>CytBZ, Z = aziridyl-N-ethyl-2-oxy, W = 0), which was<br/> prepared in analogy with Example 3a from the monomer of<br/>the formula VIII (B' = CytBz, Z = aziridyl-N-ethyl-2-oxy,<br/>RS = R6 = i-C3H, ), which had previously been obtained from<br/>the bisamidite of the formula VIa (B' = Cytex, R5-R = i-<br/>C3H7) by reaction with N-(2-hydroxyethyl)aziridine in<br/> analogy with Example 2a. After removal of the protective<br/>groups with ammonia, an oligonucleotide of the formula Ix<br/>is obtained, to which an aziridine-N-eth-2-yl phosphate<br/>ester is bound at the 3'-end.<br/>y-1) Preparation of an oligonucleotide of the formula<br/> Iy-1 (R1 = R2 = H, Z=-0-farnesyl, for ps is W S)<br/>5' CpSCpSGpSGpSApSApSApSApSCpSApSTpSCpSGpSCpSGpSGpSTp-<br/>STpSGpSTpS-0-farnesyl<br/>The synthesis takes place in analogy with Example 4d,<br/>starting with the support of the formula VIIa-16 (B' _<br/> Thy, Z = 0-farnesyl), which, in analogy with Example 3a,<br/>was prepared from the monomer of the formula VIII (B' _<br/>Thy, Z = 0-farnesyl, R5 = R6 = i-C3H,), which had previous-<br/>ly been prepared from the bisamidite VIa (B' = Thy, R5-RB<br/>= i-C3H,) by reaction with farnesol in analogy with<br/>Example 2b. In this case the oxidation is carried out on<br/>each occasion with TETD solution as described in Example<br/>4d. After removal of the protective groups with ammonia,<br/>an allophosphorothioate oligonucleotide of the formula<br/><br/>2 47 - 08 '7 8 ~.8<br/> Iy-1 is obtained, to which a farnesyl thiophosphate ester<br/>is bound at the 3'-end.<br/>y-2) Preparation of an oligonucleotide of the formula<br/>Iy-2 (R' = R 2 = H, Z = -0-phytyl for ps(s) is W = U= S)<br/> 5' CpS(S)CpS(S)GpGpApApApApCpApTpCpGpCpGGpTpTpS(S)Gp-<br/>S(S)Tp-0-phytyl3'<br/> The synthesis takes place in analogy with Example 4y-1,<br/>starting with the support of the formula VIIa-17 (B' _<br/>Thy, Z = 0-phytyl), which, in analogy with Example 3a,<br/> was prepared from the monomer of the formula VIII (B' _<br/>Thy, Z = 0-phytyl, RS = R6 = i-C3H7), which had previously<br/>been obtained from the bisamidite VIa (B' = Thy, R5-RB =<br/>i-C3H7 ) by reaction with phytol in analogy with Example<br/>2b. The nucleotides 2, 3, 19 and 20 (counting of the<br/> nucleotides corresponds to the direction of synthesis<br/>from 3' to 5') are [?] via the units of the formula VIII<br/>(Z = 2,4-dichlorothiobenzyl, R5, R6 = C2H5) . In the case of<br/>these nucleotides oxidation is carried out with TETD<br/>solution. In the other reaction cycles oxidation is with<br/>iodine water. After removal of the protective groups with<br/>ammonia, an oligonucleotide of the formula Iy-2 is<br/>obtained, which in each case has two phosphorodithioate<br/>internucleoside bonds 3'- and 5'-terminally, and to which<br/>is bound a farnesyl phosphate ester at the 3'-end.<br/>y-3) Preparation of an oligonucleotide of the formula<br/>Iy-3 (R' = R2 = H, Z="-0-cholesterol", for pMe is<br/>U = CH3)<br/> 5' CpMeCpMeGpGpApApApApCpApTpCpGpCpGpGpTpTpMeGpMeTp-"0-<br/>cholesterol"<br/> The synthesis takes place in analogy with Example 4y-1,<br/>starting with the support of the formula VIIa-18 (B' =<br/>Thy, z= 0-"cholesterol"), which, in analogy with<br/><br/> - 48 -<br/> Example 3a, was prepared from the monomer of the formula<br/>VIII (B' = Thy, Z = 0-"cholesterol", RS = R6 = i-C3H,),<br/>which had previously been obtained from the bisamidite<br/>VIa (B' = Thy, RS-Re = i-C3H,) by reaction with "cholester-<br/> ol" in analogy with Example 2b. The nucleotides 2, 3, 19<br/>and 20 are introduced, as described in Example 4e, via<br/>the methylphosphonamidites of the formula VIII (Z = CH,).<br/>In each case oxidation is with iodine water. After<br/>removal of the protective groups (cf. Example 4d), an<br/> oligonucleotide of the formula Iy-3 is obtained, which in<br/>each case has two methylphosphonate internucleoside bonds<br/>3'- and 5'-terminally, and to which a "cholesterol"<br/>phosphate ester is bound at the 3'-end.<br/>y-4) Preparation of an oligonucleotide of the formula<br/> Iy-4 (R' = Rz = H, Z=-0-testosterone, for pMe is<br/>U = CH3 )<br/> 5' CpMeCpMeGpMeGpMeApMeApMeApApCpApTpCpGpCpMeGpMeGpMe-<br/>TpMeTpMeGpMeTp-"testosterone"<br/>The synthesis takes place in analogy with Example 4y-3,<br/> starting with the support of the formula VIIa-19 (B' _<br/>Thy, Z = O-"testosterone"), which, in analogy with<br/>Example 3a, was prepared from the monomer of the<br/>formula VIII (B' = Thy, Z = O-"testosterone", R5 = R6 =<br/>i-C3H,), which had previously been obtained from the<br/> bisamidite VIa (B' = Thy, R5-R8 = i-C3H7) by reaction with<br/>"testosterone" in analogy with Example 2b. The nucleo-<br/>tides 2 to 7 and 15 to 20 are, as described in<br/>Example 4e, introduced via the methylphosphonamidites of<br/>the formula VIII (Z = CH3) . In each case oxidation is with<br/> iodine water. After removal of the protective groups, an<br/>oligonucleotide of the formula Ly-4 is obtained, which in<br/>each case has six methylphosphonate internucleoside bonds<br/>3'- and 5'-terminally, and to which a"testosterone"<br/>phosphate ester is bound at the 3'-end.<br/><br/>- 49 - ~~~7 81<br/>y-5) Preparation of an oligonucleotide of the formula<br/>Iy-5 (R' = R2 = H, Z=-0-vitamin-A, for pMe (S) is U<br/>=CH3andW=S, forpS is U-SandWa0)<br/> 5' CpMe(S)CpMe(S)GpMe(S)GpMe(S)ApMe(S)ApMe(S)ApSApSCpS<br/> ApSTpSCpSGpSCpMe(S)GpMe(S)GpMe(S)TpMe(S)TpMe(S)GpMe<br/>(S)Tp-O-"Vitamin A"<br/> The synthesis takes place in analogy with Example 4y-4 starting with<br/>the support of the formula VIIa-20 (B'-Thy, Z-O-"Vitamin A"), which,<br/>.;.<br/>= in analogy with Example 3a, was prepared from the monomer of the<br/>formula VIII (B'=Thy, Z=0-"Vitamin A", R5=R6=IC3H7), which had<br/>previously obtained from the bisamidite VIa (B'=Thy, R5-R8=i-C3H7) by<br/>reaction with "Vitamin A-alcohol" in analogy with Example 2b. The<br/>nucleotides 2 to 7 and 15 to 20 are introduced via the<br/>methylphosphoramidites of the formula VIII (Z=CH3) as described in<br/> Example 4e. Oxidation is with TETD as described in Example 4d. After<br/>removing the protective groups, an oligonucleotide of the formula Iy-5<br/>is obtained, which contains methylphosphonothioate and internally<br/>seven phosphorothioate internucleoside bonds. A "vitamin A" phosphate<br/>ester is additionally located at the 3'-end of this oligonucleotide.<br/>r'!<br/>y-6) Preparation of an oligonucleotide of the formula<br/>Iy-6 (R' = H, R2 = 0-CH3; R2 = H for T, Z=-0-vitamin<br/>E)<br/> 5' 2 '-O-CH3 (CpCpGpGpApApApApCpApUpCpGpCpGpGpUpUpGp) Tp-<br/>0-"vitamin E"<br/> The synthesis takes place in analogy with Example 4y-4,<br/>starting with the support of the formula VIIa-21 (B' _<br/>Thy, Z 0-"vitamin E"), which, in analogy with<br/>Example 3a, was prepared from the monomer of the formula<br/>VI I I ( B' = Thy, Z = 0-"vitamin E" , R5 = R6 = i-C3H,), which<br/> had previously been obtained from the bisamidite VIa (B'<br/>= Thy, R5-R8 = i-C3H7 ) by reaction with tocopherol in<br/>analogy with Example 2b. The nucleotides 2 to 20 are<br/>introduced via the 2'-0-methylribonucleoside-phosphor-<br/>amidites of the formula V (R = DMTr, R2 = O-CHA) .<br/> Oxidation is with iodine water, as described in<br/>Example 4a. After removing the labile phenoxyacetyl<br/>protective groups, a 2'-0-methyloligoribonucleotide of<br/>the formula Iy-6 is obtained, which contains a "viatamin<br/>E" phosphate ester at the 3'-end.<br/><br/> CA 02087818 2003-10-22<br/>-50-<br/>Example 5: Testing for nuclease stability<br/> nmol of the oligonucleotide under investigation are<br/>dissolved in 450 l of 20% strength fetal calf serum in<br/>RPMI medium and 50 ml of double-distilled water and<br/> 5 incubated at 37 C. 10 l samples, for gel<br/>electrophoresis, and 20 pl samples, for HPLC, are then<br/>removed immediately and after 1, 2, 4, 7 and 24 hours and<br/>in each case mixed with 5 or 10 pl of formamide,<br/>respectively, to stop the reaction, and then heated at<br/>10 95 C for 5 minutes. For the gel electrophoresis, the<br/>samples are loaded onto a 15% polyacrylamide gel (2%<br/>bis), which is then run for about 3,000 volt hours. The<br/>bands are visualized by silver staining. For the HPLC<br/>analysis, the samples are injected onto a Gen-Pak Fax'<br/> HPLC column (from Waters/Millipore) and chromatographed<br/>at 1 ml/min with 5 to 50% Buffer A in B (Buffer A: 10 mM<br/>sodium dihydrogen phosphate, 0.1 M NaCl in<br/>acetonitrile/water 1:4 (v:v) pH 6.8; Buffer B: as A, but<br/>1.5 M NaCl).<br/> Example 6: Anti-viral activity<br/> The anti-viral activity of the compounds according to the<br/>invention is examined in in vitro experiments. For this<br/>purpose, the compounds according to the invention are<br/>added in various dilutions to cell cultures of HeLa and<br/> Vero cells in microtiter plates. After 3 h, the cultures<br/>are infected with various viruses which are pathogenic<br/>for man (e.g.: herpes viruses HSV-1, HSV-21<br/>orthomyxoviruses influenza A2, picornaviruses rhinovirus<br/>2). 48 to 72 h after the infection, therapeutic success<br/>is determined on the basis of the cytopathic effect, as<br/>measured microscopically, and photometrically following<br/>uptake of neutral red (Finter's color test) (Finter, N.B.<br/>in "Interferons", N.B. Finter et al., North Holland<br/>Publishing Co., Amsterdam, 1966). The minimum concentra-<br/> tion at which half the infected cells show no cytopathic<br/><br/>2087818<br/>51 -<br/>effect is considered to be the minimum inhibitory con-<br/>centration (MIC).<br/> Example 7: Preparation of the phosphitylating reagent<br/>DMTr-O-CH2CH2-S-CH2CH2O-P- {OCHZCH2CN} {N ( i-C3H, ) 2} [ ? ] (claim<br/> 9; X' = 0, x' = zero, R9 = OCHZCHZCN, R10 = N( i-C3H, ) 2<br/> The bis-hydroxyethyl sulfide (3.05 g) is dissolved in<br/>75 ml of absolute pyridine and this solution is cooled to<br/>0 C. The dimethoxytrityl chloride (8.04 g) dissolved in<br/>60 ml of abs. pyridine is then added dropwise with<br/>stirring over a period one hour. After warming the<br/>reaction mixture at room temperature, this is stirred for<br/>a further 1.5 hours. 5 ml of water are added to the solu-<br/>tion which is then concentrated in vacuo. The residue is<br/>dissolved in 250 ml of methylene chloride. This solution<br/>is extracted three times with 125 ml of 0.1 M phosphate<br/>buffer, pH7, on each occasion, and the organic phase is<br/>dried over sodium sulfate and concentrated in vacuo. The<br/>crude product is chromatographed on a silica gel column<br/>using ethyl acetate/n-heptane/triethylamine (gradient<br/>6:14:1 to 2:2:1, v:v:v). 5.3 g of the bis-hydroxyethyl<br/>sulfide-mono-(dimethoxytrityl) ether DMTr-O-CH2CH2-S-<br/>CH2CHZOH (52%) are obtained.<br/> A solution of this dimethoxytrityl compound (1.06 g) and<br/>of tetrazole (88 mg) in 12.5 ml of absolute acetonitrile<br/>is slowly (20 min) added dropwise to a solution of<br/>cyanoethoxy-di-isopropylamino-phosphane (0.75 g) in abs.<br/>acetonitrile (20 ml). After a further 3 hours of reac-<br/>tion, the reaction solution is diluted with 95 ml of<br/>methylene chloride and washed with 5% strength sodium<br/>carbonate solution (65 ml). The organic phase is dried<br/>over sodium sulfate and concentrated in vacuo. The<br/>residue is purified by chromatography on a silica gel<br/>column with ethyl acetate /n-hexane /triethylamine (11:8:1,<br/>v:v:v). 1.25 g (80%) of the required phosphitylating<br/>reagent (31P-NMR: a 148 ppm [d], 99% of the total<br/><br/>- 52 -<br/>phosphorus content) are obtained.<br/> The compounds y-1 to y-6 described in Example 4 possess<br/>residues of the following definition:<br/> O'<br/>farnesyl<br/> O phytyl<br/>O<br/> "vitamin A"<br/>-'O H<br/>"vitamin E"<br/>O<br/> "cholesterol"<br/>O'<br/> O<br/>testosterone<br/>O<br/>