FI115214B - Process for Preparation of Oligonucleotide Analogs and Their Use - Google Patents

Abstract

The invention relates to compounds of the formula I <IMAGE> in which R<1> is H, alkyl, acyl, aryl or a phosphate residue; R<2> is H, OH, alkoxy, NH2 or halogen; B is a base customary in nucleotide chemistry; a is O or CH2; n is an integer from 1 to 100; W is O, S or Se; V is O, S or NH; Y is O, S, NH or CH2; Y' is O, S, NH or alkylene; X is OH or SH; U is OH, SH, SeH, alkyl, aryl or amine, and Z is OH, SH, SeH, an optionally substituted radical from the series comprising alkyl, aryl, heteroaryl, alkoxy or amino or a group which favours intracellular uptake or as labelling of a DNA probe or which on hybridisation interacts with the target nucleic acid, where if Z is OH, SH, CH3 or OC2H5, at least one of the groups X, Y, Y', V, W is not equal to OH or O, or R<1> is not equal to H; process for their preparation and their use as inhibitors of gene expression, as probes for detecting nucleic acids and as aids in molecular biology.

Description

115214

Method for the preparation of oligonucleotide analogs and their use

The present invention relates to a process for the preparation of novel ol-5 gonucleotide analogues having valuable physical, biological and pharmacological properties. Their use is based on their use as gene expression inhibitors (non-information oligonucleotides, ribozymes, information oligonucleotides and triplex oligonucleotides), as probes for nucleic acids and as tools in molecular biology. Oligonucleotides are increasingly used as inhibitors of gene expression (G. Zon, Pharmaceutical Research 5: 539 (1988); JS Cohen, "Topics in Molecular and Structural Biology", 12, Macmillan 15 Press, 1989; C. Helene and JJ Toulme, Biochemica). et al., Biophysica Acta 99: 1049 (1990); E. Uhlmann and A. Peyman,

Chemical Reviews 90 (1990) 543). Non-oligonucleotides are nucleic acid fragments whose base sequence is complementary to the base sequence of the mRNA to be inhibited. These target mRNAs may be of cellular or viral origin or other pathogenic origin. Suitable cell wall sequences include, for example, sequences of receptors, enzymes, immune regulators, ion channels, or tumor factors. Inhibition of viral proliferation using non-known oligonucleotides has been described, for example, for RSV «·% (Rous sarcoma virus), HSV-1 and -2 (herpes simplex virus types I and II), HIV ( human immunodeficiency virus) and influenza viruses. Oligonucleotides complementary to viral nucleic acid are used. In contrast, information oligonucleotides are designed to sequester (e.g., capture), for example, nucleic acid binding proteins or enzymes that process nucleic acids and thereby inhibit their biological activity (Helene, 1990). Examples of viral targets include reverse transcriptase, DNA polymerase, and transactivator proteins. The triplex-forming oligonucleotides generally target DNA and, once bound, form a triple helix structure. While the use of non-information oligonucleotides generally prevents mRNA processing (splicing, etc.) or its translation into protein, triplexing oligonucleotides inhibit DNA copy or translation (Helene et al., 1990, Uhlmann and Peyman, 1990). . However, it is also possible to bind single-stranded nucleic acids during the first hybridization of en-10 to a non-information oligonucleotide to form a double strand, which then forms a triplex structure with the triplexing oligonucleotide in the second hybridization. The non-information and triplex binding sites can then be located either on two separate oligonucleotides or on a single oligonucleotide. The next use of synthetic oligonucleotides is the so-called ribozymes, which destroy the target RNA by their ribonuclease activity (JJ Rossi and N. Sarver, Tibtech 8 (1990) 17). 9. DNA diagnostics utilize 20 appropriately labeled nucleic acid fragments called specific nucleic acid to be probed into a specific hybrid foundation, whereby the specific formation of the new double-strand is monitored by the use of a label: * ·, · which is preferably non-radioactive. : V. Diseases caused by pathogens.

• ·

For most of the uses mentioned, oligonucleotides, in their naturally occurring form, are unsuitable or totally unsuitable. They must be chemically modified to meet specific requirements. Thus, oligonucleotides that can be used in biological systems, for example, to inhibit viral proliferation, must meet the following requirements: III. serum and also intracellularly, sufficient stability.

2. They must be constructed in such a way that they can penetrate the cell and nuclear membrane.

3. Under physiological conditions, they must bind in a base-specific manner to obtain an inhibitory effect on their target nucleic acid.

For DNA probes, these requirements are not uncompromising; however, such oligonucleotides must be derivatized such that detection, for example by fluorescence, chemiluminescence, colorimetry, or specific staining, is possible (Beck and Köster, Anal. Chem. 62, 2258 (1990)). Chemical alteration of oligonucleotides is most often accomplished by altering the phosphate backbone, ribose unit or nucleobases accordingly (Cohen, 1989; Uhlmann and Peyman, 1990). Another commonly used method is the preparation of oligonucleotide 5'-conjugates by altering the 5'-hydroxyl group with the corresponding phospho-20-ylylation reagents. Oligonucleotides modified only at the 5 'end have the disadvantage that they degrade in serum. In contrast, when altering all phosphate residues between nucleotides, the properties of the oligonucleotides often change dramatically. For example, methyl · ·. ··. The solubility of the lifophosphonate oligonucleotides in the aqueous medium is decreased and the hybridization ability is reduced. Phosphorous- \.! thioate oligonucleotides act nonspecifically, whereas, for example, homo-oligomers also act against viruses.

The function is thus to provide oligonucleotide analogues with specific activity, higher serum stability and good solubility.

The invention relates to a process for the preparation of oligonucleotide analog 35 having the formula I: 4

1152U

ήί r * (ΐ) U-P-V - 1 8 - "-Ή • W, Y 'R *

2-P-X

II

w

and the physiologically acceptable salts thereof, wherein the formula I

R1 is hydrogen, C1-8 alkyl, C2-18 alkenyl, C2-18 alkynyl, C2-18 alkylcarbonyl, C3-19 alkenylcarbonyl, C3-19 alkynylcarbonyl, C6-20 aryl, C6 -14-aryl-C 1-8 -alkyl, or a residue of formula II: z - p - r (H) en 10 w - · · · · · · · · * R 2 is hydrogen, hydroxyl, C alkoxy, halogen,? -linked or NH 2; »F» V * B is a base common in nucleotide chemistry; 15a is oxy or methylene; ··· n is an integer from 1 to 100; · · · »W is oxo, thioxo or selenoxo; V is oxy, sulfandiyl or imino; '· Y is oxy, sulfandiyl, imino or methylene; * * 20 Y 'is oxy, sulfandiyl, imino, (CH 2) m or V (CH 2) m /:; : where • 11 · »• 1 5 115 2 Ui m is an integer from 1 to 18; X is hydroxyl or mercapto; U is hydroxyl, mercapto, SeH, C 1-8 alkoxy, C 1-8 alkyl, C 6-20 aryl, C 6-14 arylC 1-8 alkyl, NHR 3, NR 3 R 4 or a residue of formula III: (OCH 2 CH 2) p O (CH 2) q C tb R 11 (III) wherein: R is C 1-8 alkyl, C 6-20 aryl, C 6-14 arylC 1-8 alkyl, 2- (CH 2) c - [NH (CH 2) c] d -NR 12 R 12 wherein c is an integer from 2 to 6 and d is an integer from 0 to 6, and R 12 are each independently hydrogen or C 1-6 alkyl or C 1-4 alkoxy C 1-6 alkyl; R 4 is C 1-8 alkyl, C 6-20 aryl or C 6-10 arylC 1-8 alkyl, or in the case of NR 3 R 4 taken together with R 3 and the nitrogen atom to which they are attached form a 5-6 membered heteroaryl. a cyclic ring which may additionally contain another heteroatom from the group O, S, N, p is an integer from 1 to 100, q is an integer from 0 to 22, R 11 is hydrogen or a functional group; Z = Z 'mercapto, C 1 -C 22 -alkoxy, C 1 -C 14 -alkyl, C 6 -C 14 -arylC 1 -C 8 -alkoxy, wherein aryl is also heteroaryl and aryl is optionally substituted with one, two or three identical or different residues from group 25, carboxyl, amino, nitro, C 1-4 alkylamino, C 1-6 alkoxy, hydroxyl, halogen and cyano, or a residue of formula III and wherein the parentheses indicate that R 2 and the adjacent phosphoryl residue may be 2 'and 3'; position or vice versa 3 'and 2' position, * ;; Wherein each nucleotide may be in the D- or L-? · * Configuration and the base B may be in the a- or β-position provided that when Z is mercapto, C 1-8 alkyl or ethoxy, at least one of X, Y, Y ', V, W are not hydroxyl, thio, oxy or oxo, or R 1 is not hydrogen.

35 6

11S2U

B is a base customary in nucleotide chemistry, for example, a natural base such as adenine, cytosine, guanine or thymine, or a non-natural base such as purine, 2,6-diaminopurine, 7-deaza-adenine, 7-deazaguanine, 5 N4N4-etanosytosine, N6N6 -etano-2,6-diaminopurine, pseudo-isocytosine.

n is preferably from 10 to 40.

m is preferably 1-6.

U is hydroxyl, mercapto, SeH, C 1-6 alkoxy, preferably C 1-6 alkoxy, C 1-8 alkyl, preferably C 1-6 alkyl, C 6-20 aryl, C 6-14 arylC -8-alkyl, NHR3, NR3R4 or a residue of formula III: (OCH2CH2) PO (CH2) qC1R11 (III) wherein: R3 is C1-8 alkyl, preferably C1-8 alkyl, C6-20 ~ 15 aryl, C 8 -U-aryl-C 1-8 alkyl, - (CH 2) c - [NH (CH 2) c] d -NR 12 R 12 where c is an integer from 2 to 6 and d is an integer from 0 to 6, and R 12 each independently is hydrogen or C 1-6 alkyl or C 1-4 alkoxyC 1-6 alkyl, preferably methoxyethyl; R 4 is C 1-8 alkyl, preferably C 1-8 alkyl, and particularly preferably C 1-4 alkyl, C 6-20 aryl or C 6-10 aryl.

C 1 -C 8 -alkyl, or, in the case of NR R, together with R 3 and the nitrogen atom which binds them, forms a 5-6 membered heterocyclic ring which may additionally contain another • 25 heteroatoms from the group O, S, N .

p is preferably from 3 to 20 and particularly preferably from 3 to 8.

• «* ..! q is preferably 0 to 15.

R 1 is hydrogen or a functional group such as hydroxyl, amino, NHR 13, COOH, CONH 2, COOR 12 or halogen, wherein R 3 is C 1-4 alkyl, preferably methyl; Z = Z 'mercapto, C 1 -C 22 alkoxy, preferably C 6 -C 18 alkoxy,?

C 1 -C 18 alkyl, preferably C 1 -C 8 alkyl, C 6 -C 14 aryl-C 1 -C 5 alkoxy, preferably C 6 -C 10 aryl C 1 -C 4 alkoxy, wherein also denotes heteroaryl and aryl is optionally substituted with one, two or three moieties of the same or different moieties from carboxyl, amino, nitro, C 1-4 alkylamino, C 1-6 alkoxy, hydroxyl, halogen and cyano, or a radical of formula III and wherein the ka-5 brackets mean that R 2 and the adjacent phosphoryl residue may be in the 2 'and 3' positions or vice versa in the 3 'and 2' positions,

Preference is given to oligonucleotide dianalogues of formula I and their physiologically acceptable salts wherein the base B is in the β position, the nucleotides are in the D configuration, R2 is in the 2 'position, and a is branched.

Especially preferred are oligonucleotide analogs of formula I wherein R 1 is hydrogen, C 1-6 alkyl, especially methyl, or the residue of formula II; R 2 is hydrogen or hydroxyl, especially hydrogen; n is an integer from 10 to 40, in particular 12 to 30; m is an integer from 1 to 6, especially 1; U is hydroxyl, mercapto, C 1-6 alkoxy, C 1-6 alkyl, NR 3 R 4 or NHR 3, especially hydroxyl or C 1-6 alkyl, where 3 R is C 1-8 alkyl, preferably C 1-4 alkyl, or methoxyethyl, and B, W, V, Y, Y ', X and Z are as defined above.

Particularly preferred are oligonucleotide analogs of formula I wherein V, Y 'and Y are each oxy.

• ·

Further particularly preferred are the compounds of formula I

• t I

gonucleotide analogs wherein V, Y, Y 'and H are oxy or

, respectively, oxo. Particularly preferred are those of formula I

Oligonucleotide analogs of V, Y, Y ', W and U according to;

* · Are oxy, oxo or hydroxyl, respectively. Further preferred are oligonucleotide analogs of formula I wherein R 1 is hydrogen. Especially preferred are the oligonucleotide analogs of formula I wherein U, V, W, X, Y 'and Y are oxy, oxo or hydroxyl, respectively, and R 1 is hydrogen.

* · 8 7 7 ^ ° 1 *

Repeated residues such as R 2, B, a, W, V, Y, U, R 3, R 4, p, q and Z may independently have the same or different meanings, e.g., V is each independently oxy, sulfandiyl or imino. Halogen is preferably fluorine, chlorine or bromine. Heteroaryl refers to a monocyclic or bicyclic C3-8 heteroaromatic moiety containing one or two N atoms and / or an S or O atom in its ring system.

As examples of NR3R4, where R3 and R4 together with the nitrogen atom to which they are attached form a 5-6 membered heterocyclic ring containing an additional heteroatom, a morpholinyl and imidazolidinyl radical.

The invention is not limited to α- and β-D- or L-ribofuranosides, α- and β-D- or L-deoxyribofuranosides and corresponding carbon cyclic five (member) ring analogs, but also to oligonucleotide analogs based on other sugar moieties, e.g. enlarged and truncated sugars, acyclic sugars or other suitable derivatives of sugars.

The synthesis of the oligonucleotide analogs of formula I is similar to the synthesis of biological oligonucleotides in solution or preferably in solid phase, optionally with the aid of an automated synthesis device.

• »

However, solid phase synthesis for oligonucleotides having a phosphate or phosphate ester residue at the 3 'end is not possible using conventional phosphoramidite chemistry according to Caruthers (MD Matteucci and MH Ca- · · 30 ruthers, J.Am.Chem 103, 3185 (1981), since the first nucleotide moiety is bound to a solid support via a 3'-hydroxyl group, so that these syntheses always result in oligonucleotides having a 3'-hydroxyl group. Various solid-state methods have been described, however, which are »» »« · 9 1? C 9 1.

all laborious and often unable to prepare derivatives such as phosphate esters or alkyl phosphonates (R. Eritja et al., Tetrahedron Lett. 32 (1991) 1511; P. Kumar et al., Tetrahedron Lett. 32 (1991) 967; 5 WT Markiewcz and TK Wyrzykiewicz, Phosphorus, Sulfur and Silicon, 51/52, 374 (1990); Felder, E. et al., Tetrahedron

Lett. 25: 3967 (1984); R. Lohrmann and J. Ruth DNA 3 (122) (1984).

The process of the invention is characterized in that a) a nucleotide unit having a 3 '(2') main phosphorus (V) group and a free 5'-hydroxyl or mercapto group is reacted with a phosphorus at the 3 'end III) or a phosphorus (V) group, or with activated derivatives thereof, or b) the oligonucleotide analog is constructed in the same manner, the oligonucleotides obtained according to (a) or (b) being temporarily cleaved to protect other functions; the oligo-gonucleotide analogs of formula I thus obtained may be converted into their physiologically acceptable salt.

The solid support of formula IV: D-X'-CH2CH2-S (O) x-CH2CH2-AT (IV), t · · · is used as starting material in the solid phase synthesis, where A is a connecting arm which is, for example, a dicarboxyl radical. , sylic acid, diol, alkylamine, dicarboxylic acid, · pomonoalkylamide, acid amide or phosphate of the formula:

* I

»»

»* I

10

115 2U

0 «- O - P - O -

OR

wherein R is hydrogen or C 1-6 alkyl optionally substituted with -CN, and is preferably methyl or 2-cyanoethyl, T is a solid support, for example, from materials such as CPG (Controlled Pore Glass). , Kiesel gel, or an organic resin such as polystyrene (PS) or graft copolymer (POE) of PS and polyethylene glycol modified with functional groups such as hydroxyl, amino, halogen or COOH, D is a protecting group which can be removed without cleavage -A and X'-CH 2 CH 2 -S (O) x -CH 2 CH 2 (see Bio-orq. Chem. 14, 274-325 (1986)), such as 4-methoxytetrahydro-pyranoyl and dimethoxytrityl, preferably dimethoxytriyl, x is an integer of 0, 1 or 2, and X 'is oxy or sulfandiyl.

• · ·. ·. : Compound Arm A, which binds with a chemical bond * «· • ·. ·. : (i. e., amide, ester) solid support T to sulfur-containing 20 (Damka et al., Nucl. Acids Res. 18: 3813 (1990)), preferably has succinic acid residue (OC (O) -CH 2 CH 2 -). C (O) -),

t <I

oxalic acid residue (O-C (O) -C (O) -), alkylamine, preferably LCAA (Long Chain Alkyl Amin), or polyethylene glycol. Particularly preferred is the marine 25 succinic acid residue. In certain cases, such as sub-

* I I

in conjunction with substituents that do not withstand prolonged ammo, · [; niak treatment, the most labile linkers are inexpensive, * - *! ten oxalyl linker. Fixed • · in the formulas IV a - c. The preparation of the carriers is described in Example 1.

30

»'* I

11

1152 U

"--------- I" ..... i

Kan- D X 'X A-T

ora ______________ IVa DMTr 0 2 OEt -0-C- (CH2) 2-C-N- (CH2) 3-Si-CPG 0 OH OEt IVb DMTr O 2 -0-C- (CH9) rC-N - TentaGel

II II I

0 OH

-------

IVc DMTr O 0 O

II

-0-P-0 - TentaGel och2-ch2-cn == B3Ssss == l = s = s === sis = ssLassisssssa ^^ BnaMBaBaM0S9snMMenB ===: = B '

Solid phase synthesis can be carried out by the phosphate-rester method, the H-phosphonate method and the phosphoramidite method, preferably the phosphoramidite method (E. Sonveaux, Bioorq.Chem. 14 (274) 1986). I do not always cleave protecting group D, preferably with acid, for example trichloroacetic acid in methylene chloride, from the support of formula IV. In the case of the phosphorus · · · ',' ·· 10 amidite process, the thus obtained carrier of the formula IV: HX'-CH 2 -CH 2 -S (O) x -CH 2 CH 2 -AT (IV), where x, X ', A and T, as defined above, are condensed into nucleoside phosphoramidite of the formula:

• f t 'IM

115214 12 R-V — i B 'H - Y R2 p s

I / R

ZP-XRe wherein B 'is B, wherein any NH 2 groups present in B may be protected, 5 R is a protecting group which may be removed under gentle conditions such as 4-methoxytetrahydropyranoyl or dimethoxytrityl, R 2' is hydrogen, C 1-18 ~ alkoxy, halogen, or a protected hydroxyl or amino function, and R 5 and R 6 are each independently C 1 -C 12 alkyl or both moieties form a 5-6 membered ring, Y 1 is oxy, sulfandiyl or (CH 2) m, and a , m, V, and Z are defined as above, 1t; 15 in the presence of a weak acid such as tetrazole. Then, 1 ·, · the thus obtained support is oxidized in a manner known per se * with iodine water (W = O) or TETD (tetraethylthiour -, ·, · 1 ·, fidi) or elemental sulfur (W = S) or selenium (W = Se) • », 44 to a carrier derivative of formula VII: 20 * ♦» RV—, B 'id? 1¾ ·; · y rj

Z-P-X '- CH 3 CH 3 -SO 3 -CH 3 -CH 3 -A-T

. B

*: 1 1: w • »t» 1 t »111 1 · 1» 13 115214 where R, V, B ', R 2', Z, X ', W, Y ", A and T are defined as above. carrier of formula Vila: 5 RV— | B '(VM.) lp 0 oo

1 II 1 'I

* -P-0- (CH) - S0, - (CH,), - 0-C (CH,), - C M (CH,), - $ l-CP6

H H I

W 0 (1

The phosphoramidite of Formula V may be obtained, for example, from bisamidite of Formula VI: 10 - 7 Y "R. R» N * · · * · v wherein ·. ': R7 and R8 are the same as R5 and R6 and a, R, V, B ', R 2', Y ", R 5 and R 6 are defined as: *: 15, by reaction with the corresponding alcohol or thio: * · *: alcohol catalyzed by tetrazole (Example 2, Method A) when Z = alkoxy or alkyl mercapto (JE Marugg et al., Tetrahedron Lett. 27, 2271 (1986)). Preferred bisamidites are those having the formula Via: 20 * »» *. I «115214 14 DMT r-0-1 B 1

7 0 S

R \ I / R

> NPN <R&X XR «In this way, for example, amidites of Formulas VIIIa-m were prepared: DMT r-0-IB 'H (Vili) 0 p5 1 / R ZPN <XR» wherein R5 and R6 are as defined above and Z is : 10 a) O-CH 2 CH 3, b) O-1-C 3 H 7, • ·. c) 0-n-C 6 H 13, • * * * 'd) 0-n-C 6 H 37,

* · / —N

e) «» ·: T: f) o- (CH 1) f - (^) ~ NO 3, 15 g - k) a residue of formula III (R 11 = H), where:; in * ··· g) p = 3 and q = 0, h) p = 4 and q = 9, * »i) p = 5 and q = 4, and in 20 k) p = 8 and q = 13, p) ch3 t 1 115214 15 (CH2) 4-0- m) Λ Λ and B 'are at 5 a), c) and d) Cyt1_Bu, at b) and p) Thy, and at e) - k) and m) CytBz.

An alternative method of preparing the support is to use a phosphitylation reagent of formula IX: 10 R 9 z "- p <" "<'*>.

wherein R 9 and R 10 are each independently Cl, or Z ", where Z" = Z, provided that the mercapto is necessarily in the form of a protected derivative, e.g.

• 1 I

• »• * * / R5 a *: -N <, -N <^ • ·

V: O-CH 2 CH 2 CN, O-CH 3, S-CH 2 CH 2 CN, X'-CH 2 CH 2 -S (O) x'-CH 2 CH 2 -X'-D

Or c, v ... · 's ~ CH> ~ yJ / -c 1 • · preferably X' -CH 2 CH 2 -S (O) x> -CH 2 CH 2 -X '-DMTr, where x' is co-: 4 : an integer of 0 or 1, in particular 0-CH 2 CH 2 -S-CH 2 CH 2 O-DMTr, and 115214 16 R 5, R 6, R 7, R 8, X ', DMTr and D are defined as above, reacting with a free 3' nucleoside (2 ') - function of the formula X: RV-B' (Y)> Y1> · where Y "'is oxy or sulfandiyl and V, B' and R are as defined above, followed by condensing compound 10 to a carrier of formula IV in the presence of a condensing agent such as tetrazole (when R 9, R 10 = NR 5 R 6 or NR R) or diisopropylamine (when R, R = Cl) is often a faster method (Example 3, Method B) The oxidation using iodine water or sulfur or selenium then yields the compound of formula Vila, and now the protecting group R can be removed and further synthesized by oligonucleotide: * ·. · In known manner. ··, the protecting groups are removed in a known manner, and then the oligonucleotide analog of formula I of the invention is cleaved from the carrier.

• · e * If the synthesis were terminated in the final round by a moiety of formula V, an oligonucleotide analog of formula I (R1 = H) having a 5'-hydroxyl group and a phosphorus-containing conjugation at the 3'-end would be obtained.

Conversely, if a phosphorylation reagent is used in the final condensation step, for example, a formula of formula IX wherein R 9 = Z ", the synthesis results in an oligonucleotide analog of formula I wherein R 1 = formula II and '- that at the 5' end there is a phosphate-containing substitution.

For example, oligonucleotides having a phosphoramidate group at the 3'-end can be prepared, for example, by reacting a carrier of formula IV (x = 0) with monomeric methoxyphosphoramidite of formula V (Z = 0-CH3) in the presence of tetrazole. Jäger et al. (Biochemistry 27, 7237 (1988)) with iodine / H2NR3 or HNR3R4, wherein R3 and R410 are as defined above.

In certain cases (Z = NHR3, NR3R4, O, S or Se), the group Z may also be attached according to the H-phosphonate method, in which case the nucleoside H-phosphonate of formula XI: 15 R-V-1 ΊίΓ ....

r · *

II

Wherein R, V, a, B ', Y', X 'and W are as defined above, is reacted in the presence of a condensing agent such as pivaloyl or adamantoyl chloride and a base such as pyridine, with. The formed · * · '; H-phosphonate diester of formula VII ': · R · V-1 · · · · · · · · · · · · · ·:: / W * p *

h (· -CHjCHj-SO02-CHjCHj-A-T

115214 18 then undergoes oxidative phosphoramidation (B. Froehler, Tetrahedron Lett. 27, 5575 (1986)) or iodine, sulfur or selenium. For example, starting from VII '(x = 0), cholesteryloxycarbonyl-5-ylaminoalkylamine can be prepared in the presence of a tetrachlorocarbon using an oligonucleotide having a cholesteryl group at the 3' end. Oxidative amidation with 2-methoxyethylamine provides, for example, an oligonucleotide having a 3'-O- (2'-methoxyethyl) phosphoramidite residue. The upstream chain-linking is then carried out in a known manner by the phosphoramidite, H-phosphonate or triester method.

It is still possible to prepare oligonucleotide analogs of formula I according to the triester method by reacting the hydroxyl group of the carrier of formula IV with a protected phosphate diester of formula XII: R-V-1 ΊΩΤ ', v t r-i f. ·, Β. ti 115214 19 3651). The triester process has the advantage over the H-phosphonate and phosphoramidite process that no additional oxidation step is required.

If oligonucleotide synthesis is performed using a sulfandiyl (x = 0) or sulfinyl (x = 1) carrier of formula IV, these groups are finally oxidized in a manner known per se (Funakoshi et al., Proc.Natl.Acad.Sei.

88: 6982 (1991)) to provide a simple cleavage with bases, preferably ammonia.

The nature of the amino protecting groups of the bases B 'and the structure of the linker A are, in individual cases, determined by the nature of the substituent Z, since they must be readily cleaved upon completion of the synthesis. For example, the preparation of an oligonucleotide 3'-phosphate isopropyl ester (Z = O-1-C3H7) may be accomplished by the use of benzoyl (Bz-) when B =

Ade and Cyt, or isobutyryl (i-Bu-) when B = Gua, using protecting groups. In contrast, for the synthesis of an oligonucleotide 3'-methylphosphonate ester (Z = CH 3) or an ethyl ester (Z = O-C 2 H 5), preferably the more labile phenoxy-20acacetyl (PAC) when B = Ade and Gua, or isobutyryl when B = Cyt, protecting groups.

! § >> ί Many conjugates also contain functional groups which must be protected before construction in mono · ·: * ·, · meric constituents of formula V in a suitable manner. For example, the carboxyl group of fluorescein can be protected as an alkyl ester. In Psoralene, the amide group may be • 1 protected as an N-Fmoc (fluorenylmethoxycarbonyl) compound. The hydroxyl groups can be protected from side reactions by acylation or silylation (t-butyldimethyl-> ** silyl). Amino groups may also exist as trifluoroacetyl protected. In exceptional cases, conjugates may c. : be so unstable that they undergo immediate degradation under the conditions of protecting group cleavage of oligonucleotide synthesis. such

* In some cases it is preferable to include formula V

* * ► '• f 35 monomer only with a compound arm having

11521 A

One functional group, for example Z = HN- (CH 2) x -NH-Fmoc, where x is an integer from 2 to 12, preferably from 4 to 6. After incorporation into the oligonucleotide and removal of the protecting groups, preferably with ammonia, the free amino group 5 can be coupled to the active esters. For example, a base labile acridinium ester was prepared.

The synthesized oligonucleotide derivatives are characterized by electrospray ionization mass spectrometry (Stults and Masters, Rapid Commun.Mass.Spectr.

105: 350 (1991).

The stability of the oligonucleotide analogs of formula I according to the invention in serum and known exonucleases was tested.

Surprisingly, it was found that the stability of all oligonucleotide analogs of Formula I compared to non-modified oligonucleotides was strongly enhanced against serum nucleases, while their effect on their hybridization behavior was minimal.

When the half-life of unmodified oligonucleotides in fetal calf serum is about 2 hours, all oligonucleotide analogs of Formula I are sufficiently stable for about 16 hours. In addition, the gonucleotide analogs of formula I are stable against snake venom phosphodiesterase, while the spleen phosphodiesterase is only resistant to those in which R1 is other than hydrogen.

The snake venom phosphodiesterase cleaved exonucleolytically unmodified oligonucleotides at the 3 'end and per-· · · * naphthosphodiesterase at the 5' end.

The oligonucleotide analogs of Formula I form double-stranded hybrids via the Watson-Crick base pair pairing with the * * »complementary single-stranded nucleotide sequences, which in turn form double-stranded nucleic acids with double-stranded nucleic acids. Thus, it is possible to regulate or repress the biological functions of nucleic acids 115214 21 with the oligonucleotide analogs of the invention, for example repression of expression of cellular genes as well as tumor factors or viral genomic functions. Therefore, the oligonucleotide 5 analogs of formula I are useful as therapeutic agents in the therapy or prevention of viral infections or cancerous diseases.

The effect of the oligonucleotides of the invention was demonstrated by inhibition of HSV-1 viral proliferation.

For example, the following oligonucleotides of formula I were found to be effective against HSV-1:

Sequence Point of Action in HSV-1 15 5 'GGG GCG GGG CTC CAT GGG GG IE 110 (start) 5' CCG GAA AAC ATC GCG GTT GT UL 30 (middle) 5 'GGT GCT GGT GCT GGA CGA CA UL 48 (middle) 5 'GGC CCT GCT GTT CCG TGG CG UL 52 (Center) 5' CGT CCA TGT CGG CAA ACA GCT UL 48 (Beginning) 20 5 'GAC GTT CCT CCT GCG GGA AG IE4 / 5 (Looping Point)

The selected sequences are in their natural form, v.j, i.e. without 3'-derivative formation, without effect. ···. 25 HSV-1 in cell culture apparently because of their rapid degradation in serum or their lack of access to cells. In contrast, * * · * oligonucleotides of the formula I having a derivative formed at the 3'-end prevent the proliferation of HSV-1 in varying degrees. 30 rin. As anti-viral inactive control · ... * sequences with corresponding chemical derivative formation, the following were used: • · 5 'CCA GGG TAC AGG TGG CCG GC control; i .: 35 5' GAC TAA TCG GGA ATG TTA AG Control • · 115214 22

The oligonucleotide of Formula I (exemplified by 4S) modified at the 3 'end of the psoralen recognizes the IE4 / 5 region of HSV-2 and prevents HSV-2 replication. The antiviral activity of the psorhalene conjugates can be significantly increased by irradiation with UV light. The HSV-1/2 genome, which has 160,000 bases, naturally provides countless other target sequences with varying potency for inhibition of viral propagation. By varying the nucleotide sequences, the therapeutic principle can also be applied to other viruses, bacteria or other pathogens of interest. The prerequisite for application to other pathogens is only that the genes relevant to the life cycle of this pathogen are known. The sequences of these are predominantly deposited in so-called gene databases. The same applies to tumor factors or other cellular genes whose function is to be repressed. Examples of other cellular genes include those encoding enzymes, receptors, ion channels, immune regulators, growth factors, or other regulatory proteins. Examples of tumor factors include ab, neu, myc, myb, ras, fos, mos, erbB, ets, jun, p53, src and rel. ß ,,, ϊ Non-information or triplex-forming oligonucleotide sequences are known, for example, as inhibitors of the following: cyclo-AMP-dependent protein kinase. ·· *. 25 (L. Sheffield, Exp. Cell Res. 192 (1991) 307), Art. glycine receptors sensitive to yeast (Akagi et al.,

Proc. USA 86 (8103) (1989), chloride channels' (Sorscher et al., Proc.Natl.Acad.Sci. USA 88 (1991) 7759), interleukin-6 (Levy et al., J.Clin.Invest. 88 (1991)). ) - * "d 30 696), basic connective tissue formation cell growth factor (Becker et al., EMBO J. (1989) 3685) and c-myc tumor factor (Postel et al., Proc.Natl.Acad.Sci. USA 8Q). 1991) 8227).

• I

Further sequence examples of other target molecules should make clear the broad utility of the oligonucleotides of the invention.

115214 23 a) Non-knowledge oligonucleotide against HIV-1: 5 'ACA CCC AAT TCT GAA AAT GG 3' (splice site) 5 'AGG TCC CTG TTC GGG CGC CA 3' (primer binding site) 5 b) EGF receptor (epithelial growth factor receptor) 5 'GGG ACT CCG GCG CAG CGC 3' (5 'unread) 10 5' GGC AAA CTT TCT TTT CCT CC 3 '(amino terminus) c) p53 tumor suppressor 5' GGG AAG GAG GAG GAT GAG G 3 '(5' non-code) 15 5 'GGC AGT CAT CCA GCT TCG GAG 3' (start of reading) d) c-fos tumor factor 5 'CCC GAG AAC ATC ATG GTC GAA G 3' (start reading) ) 20 5 'GGG GAA AGC CCG GCA AGG GG 3' (5'-no code) e) ELAM-1 (endothelial leukocyte attachment molecule) * ♦ *; i 5 'ACT GCT GCC TCT TGT CTC AGG 3' (5 '-ei-code) • ·. · *. 25 5 'CAA TCA ATG ACT TCA AGA GTT C 3' (start reading)

* · I

* · £) ICAM-1 (intracellular attachment molecule) * »» 5 'CTC CCC CAC CAC TTC CCC TC 3' (3 'unread) 30 5' GCT GGG AGC CAT AGC GAG G 3 '(start reading) * * * • · »· g) BCR-ABL (Philadelphia chromosomal shift)» · • (5 'GCT GAA GGG CTT CTT CCT TAT TG 3' (BCR-ABL cleavage * ·: · * 35 dots) 115214 24 DNA probes of the oligonucleotide dianalogues of formula I, compared to the oligonucleotide derivatives having the 3'-hydroxyl group known in the literature, have the advantage of having a higher nuclease stability, while allowing the same or different nucleotide residues to be present. Bifunctional derivative formation may also be used to attach one label to one end and an additional function (e.g., Affinity label) at the other end. For this purpose, for example, a biotin recognized by avidin or streptavidin may be included at the 3 'end of the oligonucleotide and inserted at the 5' end via an alkylaminol linker using an acridinium ester chemiluminescent label.

In many cases, the penetration behavior of the oligonucleotide analogs of the invention is more favorable than that of unmodified oligonucleotides, especially when lipophilic residues are incorporated. The increased stability of the oligonucleotides, as well as their heliosponded access to the cells, is expressed as a higher biological activity than unmodified oligonucleotides.

The diagnostic, preventive, and therapeutic uses of the oligonucleotide dianalogues of the invention mentioned above are only one of a selection of representative examples, and therefore their use is not limited to those. In addition, the oligonucleotide analogs of the invention may be used, for example, as tools in biotechnology and molecular biology.

V; In pharmaceutical preparations, an effective amount of one or more compounds of the formula I or a physiologically acceptable salt thereof is formulated in a suitable dosage form, optionally in association with physiologically acceptable auxiliaries. or carriers and / or in combination with other known active ingredients. Preferably, the administration is intravenous, topical or intranasal.

Example 1: Preparation of a support of formula IV a) Preparation of a support of formula IVa by reacting aminopropyl CPG with bishydroxyethylsulfonide dimethoxytrityl ether succinate 4.56 g of bis- (2-hydroxyethyl) sulfone simple-dimethoxytrityl (DMTr) -eth 10 mmol) is dried by slurrying the abs. pyridine and by evaporation twice, it is dissolved in 25 ml of abs. pyridine and 1.78 g (14 mmol) of DMAP (dimethylaminopyridine) and 1.4 g of succinic anhydride 15 (14 mmol) are added to the solution, followed by stirring for 3 hours at room temperature. After completion of the reaction, the mixture is evaporated to dryness and the residue is slurried in toluene and evaporated to dryness 3 times to remove pyridine, followed by slurry in 220 ml of methylene chloride. The organic phase is washed with 10% citric acid (110 ml) and 3 times with 110 ml of water, dried over sodium sulfate and evaporated to dryness. The resulting solid residue is dried under vacuum (5.64 g). From this succinate, 1.67 g (3 mmol) is slurried in abs. pyridine and • ·. ·· *. After evaporation to dryness 2 times, it is dissolved in a mixture of 0.65 ml abs. pyridine and 6 ml

• I

abs. tetrahydrofuran (THF). A solution of 420 mg (3 mmol) of p-nitrophenol and 687 mg of DCC (dicyclohexylcarbodiimide, 3.3 mmol) in 2.1 ml “··· 30 abs. THF, and stirred for 2 hours at room temperature.

• · I

mode. After the reaction, the completely precipitated dicyclohexylurea is centrifuged off. The precipitate is slurried in 1 ml abs. ether and centrifuged again for separation. 1.5 g of aminopropyl CPG carrier from 35 Fluka (500 Å, 100 pmol / g amino function) is slurried in a mixture of 115214 with 1.8 ml of abs. DMF and 350 μΐ triethylamine, add the combined solutions decanted from the precipitate of succinate nitrophenyl ester and shake for 16 hours at room temperature. The solid carrier 5 is separated and shaken with 3 ml of blocking reagent (acetic anhydride / 2,6-lutidine / DMAP, each in 0.25 M in THF) for 1 hour at room temperature to block reactive groups. The derivatized CPG carrier is then filtered off with suction, washed with methanol, THF, methylene chloride and ether and finally dried under vacuum at 40 ° C. The carrier of formula IVa comprises a dimethoxytrityl containing component of 38 pmol / g.

b) Preparation of Tenta6el R (R = Registered Trade Mark of Fa. Rapp, Tuebingen) of a carrier of formula IVb with bishydroxyethylsulfonimethoxytrityl ether succinate

The amino form of TentaGel resin, a PS / POE copolymer having 250 pmol / g of amino function (100 to 20 mg) is slurried in a mixture of 360 μΐ DMF and 70 μΐ triethylamine, 400 μιηοΐ succinate p-nitro is added. - · *,, * phenyl ester (preparation see e.g. la) and shake for 16 hours at room temperature. It is then further processed as described in Example 1a). The TentaGel resin of formula IVb comprises a dimethoxytrityl-containing component of 98 pmol / g.

C) Preparation of the carrier ZVc by reacting TentaGel tl · (hydroxyl form) with a phosphitylation reagent of formula IX (Z "= DMTr-O-CH 2 CH 2 -S-CH 2 CH 2 -O-; · ;;; R 9 = N (i-). C 3 H 7) 2; R 10 = O-CH 2 CH 2 CN)

The hydroxyl form of the TentaGel resin comprising; ·, · 500 pmol / g of the hydroxyl function (50 mg) is reacted with 10 equivalents of the phosphisylation reagent of formula IX (Z “= DMTr-O-CH 2 CH 2 -S-CH 2 CH 2). -0-, R 9 = '··· 35 N (i-C 3 H 7) 2; R 10 = O-CH 2 CH 2 CN) in the presence of 25 equivalents .115214 of 27 tetrazole in acetonitrile at 22 ° C. , 3 g of iodine in THF / water / pyridine; 70: 20: 5, v / v / v), followed by further workup as in Example 1a The carrier of formula IVc comprises a dimeric toxin-containing component of 247 μπιοΐ / g.

Example 2: Preparation of Protected Nucleoside 3'-Phosphoramidites of Formula VIII a) Preparation of Villa (B '= Cyti_Bu, Z = O-CH 2 CH 3, R 5 = R 6 = X-C 3 H 7) 10 2 mmol of nucleoside 3' of formula VI phosphorbis-amidite (B '= Cyti_Bu, R5 = R6 = R7 = R8 = 1-C3H7) is slurried in 20 ml of abs. acetonitrile and evaporated to dryness 2 times and dissolved in 20 ml abs. acetonitrile. A solution of 2.4 mmol ethanol and 1.2 mmol sublimated tetrazole in 5 mL abs is then added dropwise to the solution. acetonitrile, over 15 minutes. The mixture is then stirred for a further 2.5 hours, after which it is diluted by adding 75 ml of methylene chloride and the organic phase is extracted with 50 ml of 5% sodium bicarbonate solution. The aqueous solution is washed twice with 50 ml of methylene chloride, the combined organic phases are dried over sodium sulfate and evaporated in vacuo to dryness. To purify the residue, it is subjected to chromatography on a silica gel column using methylene chloride / 25 h / n-heptane / triethylamine (45:45:10, v / v / v). 0.7 g of the desired diastereomeric material is obtained in the form of a thin-layer chromatographically homogeneous compound (31 P nmr δ = 146.7, · 147.5 ppm). As a by-product, small amounts of the corresponding bisethyl phosphite (31 P-nmr δ = 139.3 30 ppm) can be isolated.

B) Preparation of VUIbrn (B '= Thy, Z = O-X-C 3 H 7, R 5 = R 6 = X-C 3 H 7)

Preparation is carried out by phosphorylating 5'-O-dimethoxytritylthymidine of formula X (B '= Thy (β-position); 5 R = DMTr, V = 0, a-0, Y "= 0; 2 mmol) with bisamidite of formula IX (Z "= O-1-C 3 H 7, R 9 = R 10 = N (i-C 3 H 7) 2; 4 mmol) in the presence of tetrazole (0.5 mmol) in 10 mL abs. of methylene chloride. The mixture is further worked up as in Example 2a (31 P nmr δ = 145, 04, 145, 66 ppm).

C) Preparation of Ville (B> = Cyti_Bu, Z = -O-n-C 6 H 13, R 5 = R 6 = 1-C 3 H 7) This preparation is carried out analogously to Example 2a by carrying out the bisamidite of formula VIa (B '= Cyt 1 -Bu, R5 = R6 = R7 = R8 = 1-C3H7) with 1 eq-15 equivalent of n-hexanol with tetrazole catalyzing (31 P nmr 148.1, 148.5 ppm).

d) Preparation of VIIId (B '= CytiBu, Z = -O-n-C 18 H 37, R 5 = R 6 = X-C 3 H 7) This preparation is carried out analogously to Example 20a by introducing bisamidite of formula VIa (B' = Cyt 1 -Bu, R 5 = R 6 = R 7 = R 8 = 1-C 3 H 7) with 1 equiv-Valent to catalyze n-octadecanol with tetrazole (31 P nmr 147.2, 147, 9 ppm).

; ·, · E) Preparation of Ville (B '* CytBz, Z = 3-Pyrxdyylpropyl-3-oxy, R 5 = R 6 = R 7 = R 8 = X-C 3 H 7) This preparation is carried out analogously to Example • · *. ! 2a, by reacting the bisamidite of formula Via · (B '= CytBz, R 5 = R 6 = R 7 = R 8 = i-C 3 H 7, R 2' = H) with 1 equivalent of 3-pyridine (propan-3-ol) tetrazole · * ! 30 purple catalysts. In this case, both diastereoisomers could be separated by column chromatography (31 P nmr diastereomer 1: 147.7 ppm, diastereomer 2:> 148.2 ppm).

F) Preparation of VTIIf (B '= CytBz, Z = p-nitrophenylethyl-2-oxy, R5 = R6 = X-C3H7) This preparation is carried out analogously to Example 2a by carrying out the formula VIa bisamidite 5 (B '= CytBz, R 5 = R 6 = R 7 = R 8 = 1-C 3 H 7) with 1 equivalent of p-nitrophenylethan-2-ol by tetrazole catalyzing (31 P-nmr 148.1, 148, 6 ppm).

g) Preparation of VIIIg (B '= CytBz, Z * - (OCH 2 CH 2) 3 OCH 3, R 5 = R 6 = X-C 3 H 7) This preparation is carried out analogously to Example 2a by reacting the bisamidite of formula VIa (B' = CytBz, R 5 = R 6). = R 7 = R 8 = 1-C 3 H 7) with 1 equivalent of triethylene glycol monomethyl ether by tetrazole catalyzing (31 P nmr 148.5, 148.9 ppm).

h) Preparation of VXIIhr (B '= CytBz, Z = - (OCH 2 CH 2) 4 O (CH 2) 9 CH 3, R 5 = R 6 = X-C 3 H 7) This preparation is carried out analogously to Example 2a by introducing bisamidite 20 of formula VIa (B' = CytBz, R 5). = R 6 = R 7 = R 8 = 1-C 3 H 7) with 1 equivalent of tetraethylene glycol monodecyl ether with tetrazole to catalyze (31 P-nmr 148.4, 148.8 ppm).

i) Preparation of wild-type (B * = CytBz, Z = - (OCH 2 CH 2) 5 O (CH 2) 4 CH 3,; R 5 = R 6 = X-C 3 H 7) This preparation is carried out analogously to Example «2a by bisamidite of formula VIa * = CytBz, R5 = R6 = R7 = R8 = 1-C3H7) with 1 equivalent of pentaethylene glycol monopentyl ether · ;; 30 tetrazole catalyzed (31 P nmr 148.4, 148.9 ppm).

: k) Preparation of VIIIk (B · = CytBz, Z = - (OCH 2 CH 2) 80 (CH 2) 13 CH 3, R 5 = R 6 = X-C 3 H 7) • # This preparation is carried out analogously to Example '·! * 35 2a by bisamidite 115214 (B '= CytBz, R5 = R6 = R7 = R8 = i-C3H7) is reacted with 1 equivalent of octaethylene glycol monotetradecyl ether with tetrazole to catalyze (31 P nmr 148.4, 148.8 ppm).

5 1) Preparation of VIIIp (B '* Thy, Z = CH 3, R 5 = R 6 = i-C 3 H 7) This preparation is carried out analogously to Example 2b from 5'-O-dimethoxytrityl thymidine by phosphorylating a reagent of formula IX (Z " = CH 3, R 9 = Cl, 10 R 10 = N (i-C 3 H 7) 2), whereby catalysis is carried out with 2 equivalents of diisopropylethylamine (31 P nmr 120.6, 121.0 ppm) instead of tetrazole.

m) Preparation of VIIIm (B '= CytBz, Z = acridine-9- (butyl-4-oxy) -, R5 = R6 = i-C3H7) This preparation is carried out analogously to Example 2a by carrying out the bisamidite of formula VIa (B 1 = CytBz, R 5 = R 6 = R 7 = R 8 = 1-C 3 H 7) with 1 equivalent of 9- (4-hydroxybutyl) acridine by tetrazole catalyzing (31 P nmr 146.7, 147.4 ppm).

Example 3: Preparation of a Nucleotide Binded to a Carrier of Formula VII: a) Method A: Preparation of a Carrier of Formula VIIa-1 by Coupling Nucleoside 3 'to VHIb. Phosphoramidite 7.5 mg of the support of Example 1a, containing 0.2 pmol of bishydroxyethylsulfone dimethoxyethyl ether bound, is treated with 3% trichloroacetic acid, whereupon the DMTr protecting group is cleaved, washed * * · 30 acetonitrile and then reacting with 2 pmol of t »nucleoside 3'-phosphoramidite of formula VHIb (B '= Thy, Z = O-i-C 3 H 7, R 5 = R 6 = i-C 3 H 7) tetrazole (10 pmol) acetonitrile. The reaction time is • (2.5 minutes. Then oxidized with iodine (when W = 0, 1.3 g '·' ·· ') 35 iodines in THF / water / pyridine; 70:20: 5, v / v / v, 5 minutes ).

31

1152 U

b) Method B: Preparation of a Supporting Formula VIIa-2 Using a Reaction with a Phosphorylation Reagent of Formula IX

The phosphithylation reagent of formula IX (Z "= 5 n-octyl, R 9 = R 10 = Cl; 1 equivalent) is reacted in the presence of 1.2 equivalents of diisopropylethylamine (DIPEA) in abs. Acetonitrile or methylene chloride (1 equivalent). 5'-O-dimethoxytrityl-thymidine, B '= at the β-position, Y "' = O) 10 at -78 ° C to give the corresponding nucleoside-3'-On-octylphosphonmonochloride. The support of Formula IVa is treated to cleave the protecting group D = DMTr as described in Method A, washed with acetonitrile and reacted with an excess of 15 situ nucleoside 3'-O-n-octylphosphonmonochloride in the presence of DIPEA. Oxidation with iodine water provides a carrier-bound nucleotide of formula VIIa-2, which is available for subsequent oligonucleotide synthesis.

Example 4: Preparation of Oligonucleotides of Formula I (monomer is always BD deoxyribonucleoside) a) Oligonucleotide of Formula Ia (R 1 = R 2 = H, Z = O-i-C 3 H 7, a = U = V = W = X = Y = Y '= O, B = Thy,: ns 9) Preparation • · «* * · · * *» • t 25 TpTpTpTpTpTpTpTpTpTp- (0-i-C3H7) «* · • * 0.2 pmol of carrier VIIa-1 ( B * = Thy, W = 0, Z = 0-1 (→ C 3 H 7) from Example 3a is treated sequentially with the following reagents: 1. Abs. acetonitrile · ... * 2. 3% trichloroacetic acid in dichloromethane 3. Abs. acetonitrile 4. 4 pmol 5'-O-dimethoxytritylthymidine-3'-fo- (foroic acid B-cyanoethyl ester isopropylamidite and '··· · 35 25 pmol tetrazole in 0.15 ml abs. acetonitrile 115214 32 5. Acetonitrile 6. 20% Acetic anhydride in THF with 40% lutidine and 10% dimethylaminopyridine 7. Acetonitrile 5. 8. Iodine (1.3 g in THF / water / pyridine; 70: 20: 5, v / v / v)

Steps 1-8, hereinafter referred to as the reaction cycle, are repeated 8 times for the construction of the decathymidylate derivative. Once the synthesis is complete, cleavage of the dimethoxytrityl group is carried out as described in steps 1-3. After treatment with ammonia for 1.5 hours, the oligonucleotide is cleaved from the carrier thereby removing the β-cyanoethyl groups. Since the oligonucleotide does not contain amino protecting groups, no further treatment with ammonia is required. The resulting decatymidylate 3'-phosphate isopropyl ester crude product is purified by polyacrylamide gel electrophoresis or HPLC.

b) Preparation of Oligonucleotide of Formula Ib (R 1 = R 2 = H, Z = OX-C 3 H 7, a = U = V = W = X = Y = Y '= 0) 20 d (CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCpTp-O-1 · C) »· · • ·» • ·

However, the synthesis is carried out analogously to Example 4a * 25 using different nucleobases in the monomer. In the synthesis steps 1 to 8, monomers are generally used for the 5 '-O-dimethoxytrityl nucleoside-3'

t t I

β-cyanoethyl ester alkylalkamide, wherein the amino functions of adenine (Ade), cytosine (Cyt) or guanine (Gua) are provided with suitable protecting groups. In this example, N6-benzoyl-Ade (AdeBz), N4-benzoyl-Cyt; N (CytBz) and N2-isobutyryl-Gua (Gua1-Bu) are used. The chain is constructed starting from the carrier of formula VIIa-1 (B '= · _ Thy, W = O, Z = O-i-C 3 H 7) as described in Example 4a,' ·! ** 35 wherein monomers 115214 correspond to the above sequence. is condensed. However, ammonia treatment is still carried out (16 hours at 50 ° C) to cleave the amino protecting groups.

c) Preparation of Oligonucleotide of Formula Ie (R 1 = R 2 = H, 5 Z = O-CH 2 CH 3, a = U = V = W = X = Y = Y '= 0) d (CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-O-CH 2 CH 3) -3 (B '=

Cyt 1 -Bu, W = O, Z = O-C 2 H 5), which is prepared using the monomer of formula VIIIa according to Method A (Example 3a), is synthesized analogously to Example 4b. However, the most labile amino protecting groups of N6-phenoxyacetyl-Ade (AdePΔC), N4-isobutyryl-Cyt (Cyt1-Bu), N2-phenoxyacetyl-Gua (preferably with the most labile amino protecting groups) are used for the preparation of base labile substitutions (such as here Z = O-C2H5). , which can be more easily cleaved off at the end of the synthesis. Removal of the protecting groups with 20 ammonia then takes only 2 hours at 50 ° C. If the product is further treated with ammonia at 50 ° C for 6 hours, about 5-10% of oligonucleotide 3'-phosphate is obtained as a by-product by cleavage of the phosphate ethyl ester: ', 1.

(D) the oligonucleotide of formula Id (R 1 = R 2 = • · 25 H, Z = O- (CH 2) 17 CH 3, a = U = V = X = Y = Y '= O, W = O, except for the last 5 'end phosphorothioate tinucleotide intermediate bond, where W = S (designated ps)); 30 d (CpsGpsTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-O- (CH2) 17CH3) »· I months -.j Starting from the carrier of formula VIIa-4 (B '= ·, C is prepared using the monomer of formula VHId according to Method A 34115214 (Example 3a), the synthesis is carried out analogously to that described in Example 4c using the most labile protecting groups. Second, after the last (G) and last nucleotide (C) coupling, a solution of TETD-5 (0.4 M tetraethylthiuram sulfide in acetonitrile) is used in place of iodine water in sulfur oxidation. Treatment with ammonia for 2 hours removes the protecting groups. An oligonucleotide of formula Id having two 5'-main phosphorothioate nucleotide spacers and one 3'-O-n-octadecyl-10 phosphate ester residue is obtained.

e) with the exception of each of the 5'-end methylphosphonate nucleotide spacings where U = CH3, the oligonucleotide of formula Ie (R1 = R2 = H, Z = CH3, a = V = W = X = Y = Y '= O; U = O); (labeled Pms)) Preparation 15 d (CpMeGpMeTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCpTpMe) Starting from the carrier of formula VIIa-5 (B '=

Thy, W = O, Z = CH 3), prepared using the monomer of Formula VIIlp according to Method A (Example 3a), the synthesis is carried out analogously to Example 4c. Instead of the normal cyanoethyl protected monomers (Formula VIII, Z = OCH 2 CH 2 CN), the corresponding methyl phosphonamidites (Formula VIII, Z = CH 3) are used to link the last two nucleotide units (G and C):. Cleavage from the support (1.5 hours at room temperature) by conc.

Ammonia is followed by treatment for 6 hours with ethylene diamine / ethanol / water (5: 4: 1, v / v / v) to release the amino functions of the bases. An oligonucleotide 3'-methylphosphonate of formula Ie having two '·· * 5' parent methylphosphonate nucleotide intermediate bonds is obtained.

f) Preparation of Oligonucleotide of Formula If (R 1 = R 2 = H, Z = CH 3, X = S, α = U = V = W = Y = Y '= O) 35 d (CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp (s) Me) From the support of Example IVa from Example 1a, the first reaction cycle is coupled with methyl phosphonamidite of formula VIII (Z = CH 3, B '= Cyt 1 -Bu, R 5 = R 6 = 1 -C 3 H 7) as described in Example 3a. Oxidation 5 is performed on TETD. The synthesis is then continued as in Example 3c. When the protecting groups are cleaved analogously to Example 3e, an oligonucleotide 3'-methylphosphonothioate of the formula If is obtained.

g) Preparation of oligonucleotide of formula Ig (R1 = R2 = 10H, Z = X = S, a = U = V = W = Y = Y '= O) d (CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp <s> s)

The preparation is carried out analogously to the synthesis described in Example 4b, starting from the support of formula IVa from Example 1a but with the difference that in the first condensation step the nucleoside 3'-phosphoramidite of formula VIII (Z = 2,4-dichlorothiobenzyl, R 5 = R 6 = ethyl) is used. instead of methylphosphonamidite 20. The second S atom is coupled again by oxidation with TETD (0.4 M in acetonitrile). The dichloro-1-benzylthio group is cleaved in a known manner with thiophenol / triethylamine. By cleaving the protecting groups in conc. ammonia provides an oligonucleotide 3'- * 25 phosphorodithioate of formula Ig.

h) Preparation of Oligonucleotide of Formula Ih (R 1 = R 2 = * H, Z = p-Nitrophenylethyl-2-oxy, a = U = V = W = X = Y = Y '= O); 30 d (CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-O-CH2CH2- ** .. (phenyl) -NO2) •% Starting from the carrier of formula VIIa-β (B '= ·, CytBz, W = 0, Z = p-nitrof) which is prepared using the monomer of formula VHIf according to Method I (1152144) 36 (Example 3a), the synthesis is carried out analogously to Example 4b. Cleavage of the protecting groups by treatment with ammonia at 55 ° C for 10 hours gives the oligonucleotide 3'-O- (p-nitrophen-5-ylethyl) phosphate of formula Ih.

i) Preparation of Oligonucleotide of Formula I (R1 = R2 = H, Z = 3-Pyridylpropan-3-oxo, a = U = V = W = X = Y = Y '= O) for 10 d (CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-O) ) 3-Pyridyl) Starting from the support of formula VIIa-7 (B '= Cyt 1 -Bu, W = O, Z = -O- (CH 2) 3-pyridyl) prepared as described in Example 3a using the amidite of formula Ville, oligonucleotide synthesis is carried out analogously to Example 4c.

k) Preparation of Oligonucleotide of Formula Ik (R 1 = R 2 = H, Z = -O- (CH 2 CH 2 O) 3 CH 3, a = U = V = W = X = Y = Y '= O) 20 d (GpApGpGpApCpGpTpTpCpCpTpCpCpTpGpCpGpGpGpAp). o- (ch2ch2O) 3CH3) ## STR3 ## Starting from the carrier of formula VIIa-8 (B * =: v. CytBz, W = 0, Z = -O- (CH2CH2O) 3CH3) as described in Example 3a using the amidite of formula VUIg, oligonucleotide synthesis according to the preceding sequence is carried out analogously to Example 4b.

<I

•; t; 30 den.

• »» 1 t · »1>» • 1 »I * '* 1 1 2» »* * I t 37 115214 1 2 l) Oligonucleotide of formula II (R = R = H, Z = -O- CH2CH2O) 5 (CH2) 4CH3, a = U = V = W = X = Y = Y '= O) Preparation of 5d (CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-O- (CH2CH2O) 5 (CH2) 4CH3) B '= CytBz, W = 0, Z = -O- (CH 2 CH 2 O) 5 (CH 2) 4 CH 3 prepared as described in Example 3a using the amidite of formula Wild, the oligonucleotide is synthesized analogously to Example 4b.

m) Preparation of Oligonucleotide of Formula Im (R 1 = R 2 = H, Z = -O- (CH 2 CH 2 O) s (CH 2) 13 CH 3, a = U = V = W = X = Y = 15 Y 1 = O) d (CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpAp) - (CH 2 CH 2 O) 8 (CH 2) 13 CH 3) 20 Starting from the carrier of formula VIIa-10 (B 1 =

CytBz, W = 0, Z = -O- (CH 2 CH 2 O) s (CH 2) 13 CH 3 prepared as described in Example 3a using the amidite of formula VHIk I, the oligonucleotide is synthesized analogously to Example 4b.

: ''; N) Preparation of Oligonucleotide of Formula In (R 1 = R 2 = H, Z = - (CH 3) N (CH 2) 2 N (CH 3) 2, a = U = V = W = X = Y = Y '= O) • 1 * 1 d (CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-N (CH 3) - 30 (ch 2) 2n (ch 3) 2) • 1 1 The 35 condensation reactions employ methoxide of formula VIII.

<> * I

115214 38 ziphosphoramidite (B '= Cyt 1 -Bu, Z = OCH 3, R 5 = R 6 = N (i-C 3 H 7) 2 and oxidative amidation is carried out using 0.1 M iodine in THF / N, N', N'-trimethylethylenediamine (2 : 1, v / v) twice for 15 min., After constructing the oligonucleotide sequence, the base stable sulpho-5 dicander is oxidized using NaJO4 in a manner known per se to the base labile sulfone carrier, both for the carrier and for protecting groups (PAC for Ader and Gua; i-Bu Cyt). is performed with t-butylamine / methanol (1: 1, 10 v / v) at 50 ° C for 16 hours to give the oligonucleotide 3'-trimethylethylenediamine phosphoramidate of formula In.

o) Preparation of Oligonucleotide of Formula Io (R 1 = R 2 = H, Z = -HN (CH 2) 2 O -CH 3, a = U = V = W = X = Y = Y '= O) 15 d (CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp- ) 20-CH3) 20 Preparation is carried out analogously to Example 4n, wherein oxidative amidation is carried out using; 0.1 M iodine solution in THF / 2-methoxyethylamine (2: 1, v / v) • twice for 15 minutes. Protecting groups by clipping. : oligonucleotide 3'- (2-methoxyethyl-25 .mu.l) phosphoramidate of formula Io.

p) Preparation of Oligonucleotide of Formula Ip (R 1 = Formula II, R 2 = H, Z = S, a = U = V = W = X = Y = Y '= Z' s O) '·' ' d (psCpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCps) i <1> »t ·, \: The synthesis is carried out starting from the carrier of formula IVa as described in Example 4b. However, first

«I

. after coupling of the parent compound (Formula VIII; B '= CytBz, Z = O-CH 2 CH 2 CN,;? 35 R 5 = R 6 = N (i-C 3 H 7) 2) by oxidation with? 115214 39 TETD. After removal of the DMTr protecting group from the last addition base, the free 5'-hydroxyl group is phosphorylated with bissyanoethyloxy phosphoramidite of formula IX (R 9 = N (1-C 3 H 7) 2, Z "= R 10 = OCH 2 CH 2 CN), then oxidized with TETD. The cyanoethyl protecting groups are removed by treatment with ammonia to give the oligonucleotide 3'5'-bistophosphate of formula Ip.

q) Preparation of the oligonucleotide of formula Iq (R1 = formula II, R2 = H, Z = O-i-C3H7, a = U = V = W = X = Y = Y '= 10 Z' = O) d (i- C3H7-0-pCpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCpTp-O-1-C3H7) The synthesis is carried out as described in Example 4b.

After removal of the DMTr protecting group from the last base added, the free 5'-hydroxyl group is phosphorylated with cyanoethyloxy-i-propyloxy phosphoramidite of formula IX (R 9 = N (i-C 3 H 7) 2, R 10 = OCH 2 CH 2 CN, Z C3H7) followed by oxidation with iodine water. An oligonucleotide 3'5'-bisopropyl phosphate ester of formula Iq is obtained.

r) Preparation of Oligonucleotide of Formula Ir (R 1 = Formula II, R 2 = H, Z = n-C 8 H 17, a = U = V = W = X = Y = Y '=; Z' = O) 25, d (CH 3 (CH2) 7-pCpGpTpCpCpApTpGpTpCpGpGpCpApApApCpAp-

GpCpTp- (CH2) 7CH3) Starting from the carrier of formula VIIa-2 (B '= 30 Thy, W = 0, Z = (O ^) 7 (¾), the preparation of which is described in Example 3b. , the synthesis is carried out analogously to Example 4c. After removal of the DMTr protecting group from the last addition of base, the free 5'-hydroxyl group is phosphorylated using n-octyldichlorophosphane of formula IX (Z "= (CH 2) 7 CH 3, R 9). = R10 = Cl) and DIPEA 11521440 (diisopropylethylamine) After oxidation and hydrolysis, the oligonucleotide is cleaved from the support as in Example 4e to give an oligonucleotide 3 * 5 * -bis- (n-octylphosphonate of formula Ir).

5 s) Preparation of oligonucleotide of formula Is (R1 = R2 = H, Z = psoralen, a = U = V = W = X = Y = Y '= O) d (GpGpCpGpCpCpCpGpGpCpCpTpGpCpGpApGpApApApGpCpGpCiGp)

Synthesis is carried out analogously to Example 4c starting from the support of formula VIIa-11 (B '= Gua, Z = psoralen, W = O) prepared analogously to Example 3a from the mono-15 monomer of formula VIII (B' = GuaPAC, Z = psoralen , R5 = R6 = 1-C3H7) previously obtained from bisamidite Via (B '= GuaPAC, R5 -R = 1-C3H7) which was reacted with psoralen-H (U. Pieles and U. Englisch, Nucl. Acids Res., 17, 285 (1989)) in analogy to Example 2a. Cleavage of the protecting groups with ammonia yields an oligonucleotide of the formula Is having a psoralenic → phosphate ester bound at the 3 'end.

t) Preparation of Oligonucleotide of Formula It (R 1 = R 2 = H, Z = Biotin, a = U = V = W = X = Y = Y '= 0). "·! 25 • 1: ·. ·, d (GpGpCpGpCpCpCpGpGpCpCpTpGpCpGpApGpApApApGpApApApG - t »biotiini) t

The synthesis is carried out analogously to Example 4c 30 starting from the support of formula VIIa-12 (B '=

GuaPÄC, Z = biotin, W = O) prepared analogously to Example 3a from the monomer of formula VIII (B '= GuaPÄC, Z = biotin, R 5 = R 6 = 1-C 3 H 7) previously obtained from bisamidite Via ( B '= GuaPAC, R 5 - R 8 = ** ·' * '35 1-C 3 H 7) which was reacted with biotin-H 11521441 (R. Pon, Tetrahedron Lett. 32 (1715) 1715) analogously to Example 2a. . Cleavage of the protecting groups with ammonia yields an oligonucleotide of formula I containing a biotin phosphate ester bound at the 3 'end, 5 µ) of an oligonucleotide of formula Iu (R 1 = R 2 = H, Z = fluorescein, a = U = V = W = X = Y = Y' = 0) Preparation d (GpGpCpGpCpCpCpGpGpCpCpTpGpCpGpApGpApApApGpCpGpCpGp-10 Fluorescein)

The synthesis is carried out analogously to Example 4c starting from the support of formula VIIa-13 (Bf = GuaPÄC, Z = f fluorescein, W = 0) prepared analogously to Example 3a from the monomer of formula VIII (B '= GuaPÄC, Z = f luorescein, R5 = R6 = 1-C3H7) previously obtained from bisamidite Via (B '=

GuaPAC, R5-R8 = 1-C3H7), which was reacted with fluorescein-H (Schubert et al., Nucl.Acids Res. 18 20 (1991) 3427) in analogy to Example 2a. Cleavage of the protecting groups with ammonia yields an oligonucleotide of formula Iu containing the fluorescein phosphate ester bound at the 3 'end.

: v) Preparation of oligonucleotide of formula lv (R = R = R ··· 25 H, Z = acridin-9-yl-but-4-oxy, a = U = V = W = X = Y = Y '= 0) • • 1 1 · • 1 »d (CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp- (Acridin-9-yl-but-4-oxy);:) Starting from the carrier of formula VIIa-14 (B '= CytBz,? -yl-but-4-oxy) prepared using the monomer of formula VIIIllm analogously to Example 3a, the oligonucleotide synthesized as described in Example 4b. thereby obtaining an oligonucleotide of formula lv containing an acridin-9-yl-but-4-yl phosphate ester at the 3'-end.

w) Preparation of oligonucleotide of formula Iw (R 1 = R 2 = 5 H, Z = H N (CH 2) 3 NH (CH 2) 4 NH (CH 2) 3 NH 2, a = U = V = W = X = Y = Y '= O) d. CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-HN (CH 2) 3 NH (CH 2) 4 NH (CH 2) 3 NH 2) 10

The synthesis is carried out analogously to Example 4n, wherein the oxidizing amidation is carried out using spermine. The blocking reaction is then performed using trifluoroacetic anhydride instead of acetic anhydride.

Cleavage of the protecting groups yields an oligonucleotide of formula Iw containing a spermine phosphoramidate residue at the 3 'end.

x) Preparation of oligonucleotide of formula Ix (R1 = R2 = H, Z = aziridyl-N-ethyl-2-oxy, a = U = V = W = X = Y = 20 Y '= O) ch2

d (CpGpTpCpCpApTpGpTpCpGpGpCpApApApCpApGpCp-0 (CH 2) 2 N

:! '· Ί ch2

The synthesis is carried out analogously to Example 4c

• · I

'·' * 25 starting from the carrier of formula VIIa-15 (B '=

CytBz, Z = aziridyl-N-ethyl-2-oxy, W = O) prepared analogously to Example 3a from the monomer of formula VIII 't "' (B '= CytBz, Z = aziridyl-N-, ·, ethyl 2-oxy, R 5 = R 6 = 1-C 3 H 7) previously obtained from the bisamidite of formula VIa (B '= CytBz, R 5 -R 8 = .1-C 3 H 7) which was reacted with N- (2- hydroxyethyl) aziridine analogous to Example 2a.

1152 U

Cleavage of the protecting groups with ammonia yields an oligonucleotide of formula Ix containing the aziridine N-eth-2-yl phosphate ester bound at the 3 'end.

y-1) Preparation of oligonucleotide n of formula y-1 (R 1 = 5 R 2 = H, Z = -O-farnesyl, for ps W) S) 5 '

The synthesis is analogous to Example 4d starting from the support of formula VIIa-16 (B * =

Thy, Z = O-farnesyl) prepared analogously to Example 3a from the monomer of formula VIII (B '= Thy, Z = O-farnesyl, R 5 = R 6 = 1-C 3 H 7) previously obtained from bisamidite Via (B' = Thy). , R5-R8 = 1-C3H7) which was reacted with farnesol analogously to Example 2b. However, the oxidation is then carried out in each case using a TETD solution as described in Example 4d. Cleavage of the protecting groups with ammonia yields an allphosphorothioate oligonucleotide of formula Iy-1, 20 di, containing the farnesyl thiophosphate ester bound at the 3 'end.

• · · * y-2) Preparation of Oligonucleotide of Formula Iy-2 (R1 = • · * '·' 'R2 = H, Z = -O-Phytyl, for ps (s) W = U = S). : CpS (S) CpS (S) GpGpApApApApCpApT p CpGpCpGGpTpTpS (S) GpS (S) T p-0- '· *' Phytyl 3 '25 ·· * The synthesis is carried out analogously to Example 4y-1 starting from formula VIIa-17 carrier (B '= *: Thy, Z = O-phytyl) prepared analogously to Example 3a from the monomer of formula VIII., (B * = Thy, Z = O-phytyl, R 5 = R 6 = X -C3H7) previously obtained from 44115214 previously obtained from bisamidite Via (B '= Thy, R5-R8 = 1-C3H7) which was reacted with phytol analogously to Example 2b. Nucleotides 2, 3, 19 and 20 (for the nucleotide corresponds to the synthesis direction). 3 '5') was incorporated as the moieties of formula VIII (Z = 2,4-dichlorothiobenzyl, R 5, R 6 = C 2 H 5) .The oxidation of these nucleotides is performed using TETD. In other reaction cycles, oxidation with iodine water. the ammonium protecting groups provide an oligonucleo-10 tide of formula Iy-2 containing both phosphorodithioate nucleoside intermediates at both the 3 'and 5' ends and a farnesyl phosphate ester bonded at the 3 'end.

y-3) Preparation of Oligonucleotide of Formula Iy-3 (R1 = R2 = H, Z = -O-Cholesterol, for pMe U = CH3) 15 'CpMeCpMeGpGpApApApApCpApTpCpGpCpGpGpTpTpMeGpMeTp-O

The synthesis is analogous to Example 4y-1 starting from the support of formula VIIa-18 (B '= t -. T 20 Thy, Z = O-cholesterol) prepared analogously to Example 3a from monomer of formula VIII. · ·; ; (B '- Thy, Z = O-cholesterol, R 5 = R 6 = i-C 3 H 7) previously obtained from bisamidite Via (Bf = Thy, R 5 - R 8 = • · 1-C 3 H 7), which was reacted with cholesterol in an analogous manner to Example 2b. Nucleotides 2, 3 19 and • · · 20 were included as described in Example 4e as methylthosphonamidites of formula VIII (Z = CH 3). The oxidation ·· was carried out using iodine water in each case. Cleavage of the protecting groups (cf. Example 4d) yields the formula Iy-3 * (30 oligonucleotides containing at both the 3 'and 5' ends 'of each of the two methylphosphonate nucleoside intermediates * and the 3' end bound cholesterol phosphate ester).

45 11521 &.

y-4) Preparation of Oligonucleotide of Formula Iy-4 (R 1 = R 2 = H, Z = -O-Testosterone, Silica U = CH 3) 5 '

The synthesis is analogous to Example 4y-3 starting from the support of formula VIIa-19 (B '=

Thy, Z = O-testosterone) prepared analogously to Example 3a from monomer 10 of formula VIII (B '= Thy, Z = O-testosterone, R 5 = R 6 = 1-C 3 H 7) previously obtained from bisamidite Via (B 1 = Thy , R5-R8 = 1-C3H7) which was reacted with testosterone analogously to Example 2b. Nucleotides 2-7 as well as 15-20 were incorporated as described in Example 4e 15 as methylphosphonamidites of formula VIII (Z = CH 3). The oxidation was performed using iodine water at each time. Cleavage of the protecting groups yields an oligonucleotide of formula Iy-4 which contains at both the 3 'and 5' ends of each of the 6 methylphosphonate nucleoside intermediates and a testosterone phosphate ester bound at the 2 3 'end.

• · y-5) for the oligonucleotide of formula Iy-5 (R1 =: R2 = H, Z = -O-vitamin A, pMe (S) O = CH3 and W = S, • · · pS U = S and W = O) Preparation •>: 5 'CpMe (S) CpM * (S) GpMe (S) GpMe (S) ApMe (S) ApMe (S) ApSApSCpSApSTpSCpSGpSCpM © (S)

GpMe (S) GpMe (S) T pMe (S) T pMe (S) GpMe (S) Tp * 0 '"Vitamin A" 25 - "h The synthesis is analogous to Example 4y-4, starting from Formula VIIa-20. a carrier (B '=' Thy, Z = Vitamin A) prepared analogously to fit 3 * in Example 3a of the monomer of formula VIII; (B '= Thy, Z = Vitamin A, R 5 = R 6 = 1-C 3 H 7) previously obtained from bisamidite Via (B' = Thy, R 5 - R 8 = 1-C 3 H 7) which was reacted with vitamin -A alcohol analogously to Example 2b. Nucleotides 2 -7 as well as 15-20 are incorporated as described in Example 54e using methylphosphonamidites of formula VIII (Z = CH 3). The oxidation is in each case performed by TETD as described in Example 4d. Cleavage of the protecting groups yields an oligonucleotide of formula Iy-5 containing 3 'and 5' ends at each of 6 methylphosphonothioate and internally 7 phosphorothioate nucleoside linkages. This oligo nucleotide has a vitamin A phosphate ester at the 3 'end.

y-6) Preparation of Oligonucleotide of Formula ly-6 (R 1 = H, R 2 = O-CH 3, R 2 = H, Z = -O Vitamin E) 5 '2'-O-CH 3 (CpCpGpGpApApApApCpApUpCpGpCpGpGpUpUpGp) -0- 15 "Vitamin E"

The synthesis is carried out analogously to Example 4y-4 starting from the support of formula VIIa-21 (B '=

Thy, Z = Vitamin E) prepared analogously. Monomer of formula VIII with respect to Example 3a. ; (B '= Thy, Z = Vitamin E, R 5 = R 6 = 1-C 3 H 7) which • 1 l t. * t J was previously obtained from bisamidite Via (B' = Thy, R 5 - R 8 = • · » 1-C3H7) reacted with tocopherol to give

• I

·· ** logically relative to Example 2b. Nucleotides 2-20 ligands were added as 2'-O-methyl ribonucleo-V 'cidophosphoramidites of formula V (R = DMTr, R 2 = O-CH 3 H 7). The oxidation is carried out using iodine water as described in Example 4a. Cleavage of labile phenoxyacetyl protecting groups: 'yields a 2'-O-methyloligorobonucleotide of formula Iy-6 containing at the 3'-end the vitamin E-phosphate thiol; ter.

115214 47

Example 5: Nuclease stability assay 10 nmol of the oligonucleotide to be examined is dissolved in 450 µl of 20% fetal calf serum in RPMI medium and 50 ml of double-distilled water and incubated at 37 ° C. Immediately then, at 1, 2, 4, 7 and 24 hours, take a 10 μ1: η sample for gel electrophoresis or a 20 μ1: η sample for HPLC, add 5 μΐ or 10 μΐ of formamide to stop the reaction and heat for 5 minutes 95 ° C. For gel electrophoresis-10, samples are placed in a 15% polyacrylamide digest (2% BIS) developed at approximately 3,000 volts. The strips are exposed by silver staining. For HPLC analysis, samples are injected onto a Gen-Pak Fax HPLC column (Fa. Waters / Millipore) and chromatographed at a flow rate of 1 mL / min using 5 to 50% buffer in AB (buffer A: 10 mM sodium dihydrogen phosphate, 0.1 M NaCl in acetonitrile / water 1: 4 (v / v), pH 6.8; buffer B: as in A but 1.5 M in NaCl).

Example 6: Antiviral Activity The antiviral activity of the compounds of the invention is demonstrated by in vitro experiments. To this end, various diluents are added to the compounds of the invention. j as wells to cell cultures of HeLa and Vero cells with microtiter • · »1 '| plates. After 3 hours, cultures are infected with various viruses of human pathogenicity (e.g., herpesviruses HSV-1, HSV-2, orthomyxoviruses influenza A2, Pi * * »: Corna viruses, rhinovirus-2). 48-72 hours after the required V ', the therapeutic result is determined microscopically for the cytopathogenic effect and after photometric (Finter, NB, "Interferones", NB Finter et al., North Holland / Publishing Co., Amsterdam) for neutral red staining (Finter color test). The minimum concentration at which approximately one-half of the infected cells show no cyto- • * * pathogenic effect is considered to be the minimum inhibitory concentration: 35 (MHK).

* · * S »» · 115214 48

Example 7: Preparation of DMTr-O-CH 2 CH 2 -S-CH 2 CH 2 O-P- {OCH 2 CH 2 CN} Phosphorylation Reagent (Claim 8; X '= O, x' = O, R 9 = OCH 2 CH 2 CN, R 10 = N ( i-C3H7) 2)

Bishydroxyethylsulfide (3.05 g) is dissolved in 75 5 (ml) of absolute pyridine and this solution is cooled to 0 ° C. With stirring, a solution of dimethoxytrityl chloride (8.04 g) in 60 ml abs is then added dropwise over 1 hour. pyridine. The reaction mixture is warmed to room temperature and then stirred for a further 1.5 hours. Water (5 ml) is added to the solution, followed by evaporation in vacuo. The residue is dissolved in 250 ml of methylene chloride, the resulting solution is extracted three times with 125 ml of 0.1 M phosphate buffer, pH 7 each time, the organic phase is dried over sodium sulfate and evaporated in vacuo. The crude product is subjected to chromatography on a silica gel column using ethyl acetate / n-heptane / triethylamine (gradient 6: 14: 1 to 2: 2: 1, v / v / v). 5.3 g of bishydroxyethylsulphide mono (dimethoxytrityl) et-20 ter are obtained in DMTr-O-CH 2 CH 2 -S-CH 2 CH 2 OH (52%).

A solution of this dimethoxytrityl compound: tϊ (1.06 g) and tetrazole (88 mg) in 12.5 mL of absolute acetonitrile is added slowly (20 min) to a solution of cyanoethoxydisopropylaminophosphate → 25 nia ( 0.75 g) abs. in acetonitrile (20 mL). After an additional 3 hours reaction time, the reaction solution is diluted by the addition of 95 ml of methylene chloride and washed with 5% sodium carbonate solution (65 ml). The organic phase is dried over sodium sulfate and evaporated in vacuo to dryness. The residue is purified by chromatography on a silica gel column using ethyl acetate / n-hexane / triethylamine (11: 8: 1, v / v / v) to give 1.25 g (80%) of the desired product. phosphorylation reagent (31P nmr δ 148 ppm [d], 99% of total phosphorus content).

115214 49

The residues present in the compounds y-1 to y-6 described in Example 4 are defined as: farnesyl-phytyl-V / sX ySS. yNS 2 <χ / \, '^ O "Vitamin A" 5 I I I I "Vitamin E": - "° V ^ H 0 115214 50 rSrj5 Testosterone

Claims (10)

115214 51 A process for the preparation of an oligonucleotide analogue of formula I: fί f · (I) L '^ r * R * i PX II w 5 and physiologically acceptable salts thereof, wherein R 1 is hydrogen, C 1-8 alkyl, C 2 -is-alkenyl, C2-18 alkynyl, C2-18 alkylcarbonyl, C3-19 alkenylcarbonyl, C3-19 alkynylcarbonyl, C6-20 aryl, C6-14 arylC1-8 alkyl, or a residue of the formula II: z-p-z ': (II); ··; 11: v. W 1 I R 2 is hydrogen, hydroxyl, C 1-6 alkoxy, halogen, azido or NH 2; 15. is a base conventional in nucleotide chemistry; a is oxy or methylene; 1 · *; * n is an integer from 1 to 100; • 1 '/; W is oxo, thioxo or selenoxo; •: * *: V is oxy, sulfandiyl or imino; 20. is oxy, sulfandiyl, imino or methylene; 115214 52 Y 'is oxy, sulfandiyl, imino, (CH 2) m or V (CH 2) m wherein m is an integer from 1 to 18; X is hydroxyl or mercapto; 5. is hydroxyl, mercapto, SeH, C 1-8 alkoxy, C 1-8 alkyl, C 6-20 aryl, C 6-14 arylC 1-8 alkyl, NHR 3, NR 3 R 4 or a residue of formula III: (OCH 2 CH 2) PO (CH 2) q CH 2 R 11 (III) wherein: R 3 is C 1-8 alkyl, C 6-20 aryl, C 6-14 arylC 1-8 alkyl, 2- (CH 2) c - [NH (CH 2) c] d -NR 12 R 12 wherein c is an integer from 2 to 6 and d is an integer from 0 to 6, and R 12 are each independently hydrogen or C 1-6 alkyl or C 1-4 alkoxy C 1-6 alkyl; R 4 is C 1-8 alkyl, C 6-20 aryl or C 6-10 arylC 1-8 alkyl, or, in the case of NR 3 R 4, together with R 3 and the nitrogen atom to which they are attached form a 5-6 membered hetero- a cyclic ring which may additionally contain another heteroatom from the group O, S, N, p is an integer from 1 to 100, 20. is an integer from 0 to 22, R 11 is hydrogen or a functional group; Z = Z 'mercapto, C 1 -C 22 -alkoxy, C 1 -C 8 -alkyl, C 6 -C 14 -arylC 1 -C 8 -alkoxy, wherein aryl is also heteroaryl and aryl is optionally substituted with one, two or three identical or different residues carboxyl, amino, nitro, C 1-4 alkylamino, C 1-6 alkoxy, hydroxyl, halogen and cyano, or a residue of formula III wherein the parentheses indicate that R 2 and the adjacent substituents; The 30 phosphoryl residue may be in the 2 'and 3' position or vice versa in the 3 'and 2' position, with each nucleotide in its D or L configuration and base B in the α or β position, provided that when Z is mercapto, C 1 -C 8 alkyl or ethoxy, at least one of X, Y, Y ', V, W is not hydroxyl, thio, oxy or oxo, or R x is not hydrogen, known a) reacting a nucleotide unit having a 3 '(2') - main phosphorus (V) group and a free 5'-hydroxyl or mercapto group with another phosphorus (III) at the 3 'end; or a phosphorus (V) group, or with activated derivatives thereof, or b) the oligonucleotide analog is constructed in the same way, the oligonucleotides obtained according to (10) (a) or (b) are cleaved to protect other functions and thus obtained the oligonucleotide analogues are optionally physiologically modified into their salt. 2. A method according to claim 1, characterized in that the base B is in the β position, the nucleotides are in the D configuration, R2 is in the 2 'position and a is oxy. Method according to claim 1 or 2, characterized in that R 1 is hydrogen, C 1-6 alkyl, especially methyl, or a residue of formula II; R 2 is hydrogen or hydroxyl, especially hydrogen; n is an integer from 10 to 40, in particular 12 to 30; m is an integer from 1 to 6, especially 1; 25 is hydroxyl, mercapto, C1-6 alkoxy, C1-6 alkyl, NR3R4 or NHR3, especially hydroxyl or C1-6 alkyl, wherein R3 is C1-8 alkyl; alkyl, preferably C 1-4 alkyl, or methoxyethyl, and? B, W, V, Y, Y ', X and Z are defined as above. Process according to one or more of Claims 1, 2 or 3, characterized in that V, Y and ·.: Y 'are each an oxy. · _ Process according to one or more of claims 1 to 3 · 35 or 4, characterized in that W is oxo. 115214 54 Process according to one or more of Claims 1 to 4 or 5, characterized in that U is hydroxyl. Process according to one or more of Claims 1 to 4 5 or 5, characterized in that R 1 is hydrogen. 8. A phosphorylation reagent of the formula: / R 9 DMTr-X'-CH 2 CH 2 -S (O) y. * CH 2 CH 2 -X, -PN 4 - wherein R 9 and R 10 are independently Cl, NR 5 R 6, NR 7 R 8 or Z "where Z" = Z, provided that the mercapto must be a protected derivative, R 5, R 6, R 7 and R 8 are each independently C 1 -C 12 alkyl, or R 5 and R 6 or R 7 and R 8 together form a 5-6 membered ring, X 'is oxy or sulfandiyl, x' is an integer from 0 to 15 or 1 and DMTr is dimethoxytrityl, characterized in that it is carried out in the process according to one or more of claims 1 to 7. Use in vitro of an oligonucleotide analogue according to one or more of claims 1 to 6, 10 or 7, characterized in that it occurs as an inhibitor of gene expression:. • · Use in vitro of an oligonucleotide analogue according to one or more of claims 1 to 6 or 7, characterized in that it is carried out as a probe for detection of nucleic acids or as an aid in molecular biology. <* * 115214 55