DE4424922A1 - Nuclease-resistant oligo:nucleotide conjugates - Google Patents

Abstract

New oligonucleotide conjugates (I) comprise a modified oligonucleotide (Nu) with n substits. of formula B-K, where: Nu has a modification that inhibits degradation by naturally occurring nucleases; n is not defined (but is elsewhere defined as being 1-10); B = a direct bond or a linking gp.; and K = (a) a complexing agent, (b) a complex of a radioactive metal isotope, or (c) a complex of a stable isotope of an element of atomic number 5, 21-29, 31, 39, 42-44, 49, 57-83 or 85 which is converted to a radioactive isotope by external radiation or converts external radiation into radiation of different quality, energy content or wavelength.

Description

The present invention relates to that in the claims marked object, i.e. oligonucleotide Conjugates that are a complexing agent or a complex exhibit. These conjugates can be found in the area of Diagnostics, but also therapy use.

Imaging diagnostics has been used in the past Decades of great strides have been made and developed ongoing. It is possible today, the blood vessel system, most organs and many tissues in the living body without make major interventions visible. Diseases often diagnosed because they are too clear Changes in anatomical shape, size and location  Lead structures in the body. Such anatomical Information from inside the body is through the X-ray technology, ultrasound diagnostics and to obtain magnetic resonance tomography. Each of the Techniques mentioned can be achieved through the use of pharmaceutical Means for enhancing the natural contrasts of the tissues and body fluids in the resulting image in the Performance can be improved. The concerned pharmaceutical agents are introduced into body cavities or injected into blood vessels, aimed at the caves or Change vessels in contrast. You will additionally through the bloodstream in the organism and can Change the visibility of organs and tissues. In In exceptional cases, such substances are sent to certain Structures bound and / or active in the body transported and / or excreted. In this way functions can be made visible in individual cases and used to diagnose diseases.

In contrast, nuclear diagnosis is based on substances that can be made visible themselves. In this case become radioactive isotopes, the far reaching radiation send out, brought into the body. The distribution of this Substances in the organism can be detected using suitable detectors be followed. Advantage of nuclear medicine procedures is the high effectiveness with low dosage of the Radiopharmaceuticals designated signaling radioactive Substances.

Are isotopes used, the α or β radiation or other toxic decay products effective in tissue free radiopharmaceuticals can also be used for therapeutic purposes Purposes, e.g. B. used to destroy tumors. The same goal can also be achieved in that  non-harmful isotopes or substances in the body introduced and only there by z. B. neutron or X-rays, ultrasound or radio waves in one therapeutically effective form are transferred.

A general problem is diagnosis and localization of pathological changes at a time when no significant changes in shape, structure and Blood flow to the organs and tissues in question. Such diagnosis and follow-up is e.g. B. at Tumor diseases including the search for metastases, the assessment of a short supply of tissues with Oxygen and certain infections as well Metabolic diseases are vital.

The therapeutic and imaging available today diagnostic methods are essential to that Availability of pharmaceutical preparations, pathology that is not otherwise recognizable in places enriching changes.

The contrast media currently on the market are complete predominantly so-called non-specific preparations. she are passively distributed in those rooms in which they B. can be introduced by injection.

In the past there are a variety of substances and Substance classes have been identified that have specificity with regard to their distribution in the living organism know or expect. Examples of this are besides that Antibodies lectins, all types of receptor-bound Substances, cells, membranes and membrane components, Nucleic acids, natural metabolites and their derivatives,  as well as countless drugs. With special care and peptides are also being studied.

U.S. Patent No. 4,707,352 deals with one special processes using complex-forming molecules to mark radioactive tsotopes, however, will not be good suitable complexing agent for the binding of metal ions described.

EP-A-0 285 057 describes nucleotide complexing agents Conjugates that u. a. because of the in vivo instability of the nucleotides used for use as in vivo Diagnostics or therapeutics are not suitable and also hardly the other requirements for compatibility and Meet pharmacokinetics.

A variety of U.S. patents, such as that U.S. Patent No. 4,707,440 deals with modified ones Polymers that have a detectable chemical group contain. The polymers can be polynucleotides and Be oligonucleotides, but they are not against one Degradation stabilized by naturally occurring nucleases, still selected by a special process so that they specifically with high binding affinity to target structures tie. Special embodiments of these detectable Molecules are described in U.S. Patent Nos. 4,843,122 and 4,943,523. A modified in this way, single nucleotide is described in U.S. Patent No. 4,952,685 claimed. The use of these funds in imaging The method is disclosed in U.S. Patent No. 4,849,208.

The object of the present invention is to provide specific binding diagnostic agents for detection of target structures, which, for example, makes the display  of organs, tissues and their pathological changes is made possible in vitro and in vivo.

It has now been found that oligonucleotide conjugates which in addition to an oligonucleotide residue, one via a direct Binding or a connecting component bound Have complexing agents and their oligonucleotide residue so modified is that the degradation by naturally occurring Nucleases prevented or at least significantly inhibited will solve this task.

The present invention relates to oligonucleotide Conjugates consisting of an oligonucleotide residue N and n Substituents (B-K), where B is for a direct bond or there is a connecting component to the oligonucleotide residue and K is a complexing agent or complex of radioactive Metal isotopes, or stable isotopes, the

• - by radiation from outside into radioactive isotopes being transformed,
• - radiation from outside in radiation of a different quality, different energy content and / or different wavelength convert,

the elements of atomic numbers 5, 21-29, 31, 39, 42-44, 49, 57-83 or 85 means, characterized in that the Oligonucleotide residue N has a modification that the Degradation prevented by naturally occurring nucleases or at least significantly inhibits.

In a preferred form, the oligonucleotide conjugates have the present invention the general formula

N- (BK) _n (I)

, where n stands for the digits 1 to 10.  

Are the conjugates according to the invention as a diagnostic used contains the Complexing agent is an imaging radioactive isotope Elements of atomic numbers 21, 26-27, 29, 31, 43, or 49, preferably 43 or 49. Should the invention Conjugates used as a therapeutic come - in addition to the aforementioned - also isotopes of Elements of atomic numbers 5, 22-25, 28, 42, 44, 57-83 and 85 in question. About the radioactive isotopes of the above Elements beyond are suitable in the area of therapy in particular also stable isotopes that

• a) by radiation from outside into radioactive isotopes being transformed,
• b) radiation from outside in radiation of a different quality, different energy content and / rather different wavelength convert.

The number of linked to the oligonucleotide residue imaging or therapeutically active substituents B-K is limited by the size of the oligonucleotide, but is never higher than 10. Are preferred according to the invention one or two substituents B-K.

In principle, the size of the oligonucleotide residue N is not limited. Are practical for the present invention 5 to 200 nucleotide oligonucleotides, especially oligonucleotides with 15 to 100 are preferred Nucleotides.

Oligonucleotides which can be used according to the invention are against the Degradation stabilized by nucleases occurring in vivo.

Unmodified oligo- or polynucleotides are generated in vivo cleaved by endo- and exonucleases. The degradation reaction  in the RNA series begins with activation of the 2′- Hydroxy group. Other degrading enzymes are e.g. B. ribozymes, which cleave the phosphodiester bond of RNA (see Science 261: 709 (1993)). The in vivo stability of RNA derivatives can be replaced by partial or complete replacement of the 2'- Increase hydroxyl group against other substituents. Such Substituents are e.g. B. alkoxy groups, especially the Methoxy group (see e.g. Chem. Pharm. Bull. 13, 1273 (1965), Biochemistry 10, 2581, (1971)), a hydrogen atom Fluorine atom, (see e.g. Can. J. Chem. 46, 1131 (1968)) or an amino group (see, e.g., J. Org. Chem. 42, 714 (1977)). Other ways to stabilize the Internucleotide binding is the replacement of one or two Oxygen atoms in the phosphodiester bridge with formation of phosphorothioates (Trends Biochem. Sci. 14, 97 (1989)) or phosphorodithioates (J. Chem. Soc., Chem. Commun. 591 (1983) and Nucleic Acids Res. 12, 9095 (1984)) and the Use of alkyl phosphonates instead of Phosphodiesters (Ann. Rep. N. Y. Acad. Sci. 507, 220 (1988)).

Stabilization can be achieved in that the Hydroxyl groups in the 2'-position of the ribose units be modified. Such a modification can be done by means of replacement of this hydroxyl group by an OR group, a halogen atom, especially a fluorine atom Hydrogen atom or an amine radical, in particular by a primary amine. The rest R of the alkoxy group stands for a straight-chain or branched Alkyl radical with 1 to 20 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl or hexyl or a cyclic unsubstituted or substituted Alkyl radical with 4 to 20 carbon atoms such as cyclopentyl or Cyclohexyl.  

Additionally or alternatively, the Inter Nucleotide binding serving phosphodiester bridge 1 or 2 Oxygen atoms replaced by 1 or 2 sulfur atoms the, whereby phosphorothioates or phosphorodithioates be formed. Another alternative or additional It is possible to modify the oligonucleotides therein, the phosphodi used for internucleotide binding to replace ester bridge with alkyl phosphonates. As an alkyl here come in particular lower alkyl groups with 1 to 10 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl or hexyl or cyclic unsubstituted or substituted alkyl radical with 4 to 14 C atoms such as cyclopentyl or cyclohexyl into consideration.

According to the invention, the oligonucleotide residues used are N not limited to certain oligonucleotide sequences. However, those oligonucleotides which are speci fish with high affinity to target structures Bind exception of nucleic acid.

A method of identifying suitable ones Oligonucleotides that are used as starting materials for the conjugates of the invention are required in U.S. Patent 5,270,163.

The used in the conjugates according to the invention Oligonucleotides are used in a preferred embodiment obtained by the method described below.

Suitable oligonucleotides can be obtained in that a mixture of random sequences containing Matched oligonucleotides to the target structure with certain oligonucleotides increased  Affinity for the target structure in relation to the mixture of the oligonucleotides, these from the rest of the Oligonucleotide mixture are separated, then the Oligonucleotides with increased affinity for the target structure be amplified to a mixture of oligonucleotides to get an increased proportion of oligonucleotides that bind to the target structures.

In the process, a DNA strand is first inserted into preferably produced by chemical synthesis. This DNA strand has a known sequence at the 3′-end, which serves as a promoter for an RNA polymerase and at the same time complementary to a primer sequence for the polymerase Chain reaction (PCR) is. In a particularly preferred Embodiment is the promoter for the T7 RNA polymerase. Subsequent to this promoter synthesized a random sequence on the promoter. The Random sequence can be obtained in that the in Question four bases coming in the same ratio in the Synthesizer can be entered. This creates it completely random DNA sequences. The length of the In a preferred embodiment, the random sequence is approximately 15 to 100 nucleotides. On this piece of DNA with the Random sequence becomes another DNA sequence synthesized, which is complementary to a primer Sequence required for the polymerase chain reaction (PCR) can be used.

After synthesis of this strand of DNA, this is with the help an RNA polymerase into a complementary RNA strand rewritten. In a preferred embodiment used the T7 RNA polymerase. At the transcription the RNA polymerase are offered such nucleotides that are modified. In a particularly preferred embodiment  the ribose is modified at the 2'-position. This can it is a substitution of the hydrogen atom or the Hydroxyl group through an alkoxy group, preferably methoxy, Trade amino or fluorine. That way RNA oligonucleotides produced are then in the Selection process introduced.

The RNA oligonucleotides are included in the selection process brought the target structure together. Under target structure is understood a structure to which the oligonucleotide bind specifically and with high affinity.

Such structures are e.g. B. macromolecules, Tissue structures from higher organisms such as animals or People, organs or parts of organs, cells especially tumor cells or tumors.

The target structure does not necessarily have to be in pure form present, it can also be on a natural occurring organ or on a cell surface.

If this is an isolated protein, can this on a solid phase, for example a Filters are bound. In the selection there is an excess the target structure compared to the RNA mixture. At the incubation bind the specific Oligonucleotide molecules to the target structures, whereas the unbound oligonucleotides from the mixture be separated, for example by washing.

Then the oligonucleotide molecules from the Target molecules separated or by washing with suitable Buffers or solvents removed.  

With the help of the reverse transcriptase, this becomes found RNA oligonucleotide rewritten in the complementary strand of DNA.

Because the DNA strand to be obtained at both ends Has primer sequences (or promoter sequences) can an amplification of the found DNA sequences with the help the polymerase chain reaction can be carried out easily.

The DNA oligonucleotides amplified in this way become then back in with the help of RNA polymerase Rewrote RNA oligonucleotides and the so obtained RNA oligonucleotides can be used in a further selection step (as described above).

After separating those obtained in the second selection step binding RNA oligonucleotides from the target molecules this back into DNA using reverse transcriptase rewritten, the complementary thus obtained DNA oligonucleotides are made using polymerase Chain reaction amplified and then using the RNA polymerase again to RNA oligonucleotides transcribed that for a further selection step to To be available.

It has been found that the desired high Specificities and high binding affinities are then obtained can be used if the selection steps occur several times be repeated. The one you want is rare Oligonucleotide sequence after one or two Selection steps are obtained. Once the one you want Specificity and binding affinity between target structure and Oligonucleotide is obtained, this or that Oligonucleotide (s) can be sequenced and the  Sequence of the specific binding oligonucleotides determined will.

It is particularly advantageous in the present method that that this method does not only use suitable proteins, but can also be used in vivo. The one above mentioned selection process can also on cleaned Target structures are carried out. Especially for those in In vivo diagnostics it is essential that the specificity the oligonucleotides are given for the target structure in the living environment. Therefore, the selection processes can also on cells or cell cultures, on tissues or Tissue sections, on perfused organs and even on living organisms.

The advantage here is that the modified Oligonucleotides are degraded by the almost ubiquitous Can resist RNs. Doing this yourself Selection processes on living organisms the desired Oligonucleotide sequences enriched because corresponding naturally occurring oligonucleotides from the RNAsen would be broken down.

Linking the substituents B-K with the Oligonucleotide residue N, the nucleotide sequence of which The previously described method is available different positions. So the leftovers preferred to the

• - At the 4'-end around a CH₂-OH group or at the 5'-end around Hydrogen atom-reduced oligonucleotide residue,
• - At the 3'-end reduced by a hydrogen atom Oligonucleotide residue,
• - Phosphorus diester bridged by the OH group (s) ke (n) between two nucleotides and / or  
• - bound to one or more nucleobases.

The connecting component B for the linkages to the 4'- and 3'-end is characterized by the general formula XY-Z¹, where X is a direct bond, an -NH- or -S group,
Y represents a straight, branched chain, saturated or unsaturated C₁-C₂ Alk alkylene chain which optionally contains 1-2 cyclohexylene, 1-5 imino, 1-3 phenylene, 1-3 phenyleneimino, 1-3 phenyleneoxy, 1-3 hydroxyphenylene, 1-5 amido, 1-2 hydrazido, 1-5 carbonyl, 1-5 ethyleneoxy, ureido, thioureido, 1-2 carboxyalkylimino, 1-2 ester groups, 1 -3 groups Ar, in which Ar stands for a saturated or unsaturated 5- or 6-ring which optionally contains 1-2 heteroatoms selected from nitrogen, oxygen and sulfur and / or 1-2 carbonyl groups, 1-10 oxygen, 1- Contains 5 nitrogen and / or 1-5 sulfur atoms and / or optionally by 1-5 hydroxy, 1-2 mercapto, 1-5 oxo, 1-5 thioxo, 1-3 carboxy, 1-5 carboxy -C₁-C₄-alkyl, 1-5 esters, 1-3 amino, 1-3 hydroxy-C₁-C₄-alkyl, 1-3 C₁-C₇-alkoxy groups, and Z¹ for a group -CONH -CH₂-4 ′, -NH-CO-4 ′, -OP (O) R¹-NH-CH₂-4 ′, -OP (O) R¹-O-CH₂-4 ′ or -OP (O) R¹-3 ′ St eht, where 4 'and 3' indicates the link to the terminal sugar unit (s) and R¹ is O⁻, S⁻, a C₁-C₄-alkyl or NR²R³ group, with R² and R³ in the Significance of hydrogen or C₁-C₄-alkyl radicals, the connecting component B being connected on the X side with the complexing agent or complex and on the Z side with the oligonucleotide.

Ar are particularly suitable as cyclic structures cyclic saturated or unsaturated alkylenes with 3 to 6, in particular 5 or 6 carbon atoms, optionally  Heteroatoms such as N, S, or O can contain. For example, cyclopentylene, pyrrolylene, Furanylene, thiophenylene, imidazolylene, oxazolylidene, Thiazolylene, pyrazolylene, pyrrolidylene, pyridylene, Pyrimidylene, maleimidylene and phthalimidylene groups in Consideration.

In case of connection to the phosphorus diester bridge (s) is the connecting component B by the formula X-Y-Z² characterized with Z² in the two neighboring Bridge linking sugar units

represents the group -NR² -, - O- or -S- and X, Y and R² the have previously mentioned meaning.

Examples of the radicals Y of the connecting component Z¹-YX or Z²-YX are - (CH₂) ₆-NH-CS-NH-C₆H₄- CH (CH₂CO₂H) -CH₂-CO-NH-CH₂-CH (OH) -CH₂- ,
- (CH₂) ₆-NH-CS-NH-C₆H₄-CH₂-, - (CH₂) ₆-NH-CO-CH₂-,
- (CH₂) ₆-NH-CO-CH₂-CH₂-, - (CH₂) ₂-, - (CH₂) ₆-,
- (CH₂) ₆-S- (CH₂) ₂-, - (CH₂) ₆-S- (CH₂) ₆-, - (CH₂) ₂-NH-CO-,
- (CH₂) ₆-NH-CO-, - (CH₂) ₆-S- (CH₂) ₂-NH-CO-,
- (CH₂) ₆-S- (CH₂) ₆-NH-CO-,

called.

If the link is made via the nucleobase (s), then the Compound B is characterized by the formula X-Y-Z³, where Z³ for an NH group on or a direct bond to Nucleobase stands and X and Y the meaning given to have.

As residues Y of the connecting component Z³-Y-X are for example the radicals -CH₂-CO-NH-CH₂-CH (OH) -CH₂-, -NH-CO-CH₂-CO-NH-CH₂-CH (OH) -CH₂-, -CO-NH-CH₂-CH₂-NH-, -CH₂-S-CH₂-CH₂-NH-, -CH₂-S-CH₂-CH₂-, - (CH₂) ₄-S-CH₂-CH₂-NH-, -CO-CH₂-S-CH₂-CH₂-NH-, -CO-CH₂-S- (CH₂) ₆-NH-, -CH = CH-CO-NH-CH₂-CH₂-NH-, -CH = CH-CH₂-NH-, -C≡C-CH₂-NH- or -CO-CH₂-CH₂-NH-CH₂-CH₂-NH- called.

In particular, the binding sites in the purine bases are position 8 and position 5 for the pyrimidine bases suitable. A hydrogen atom becomes purely formal each base substituted by the radical B-K. A Linking can also be carried out in the Positions 2, 4 or 6 contained amino groups, so z. B. via the 2-amino group in guanine, via the 6- Amino group in adenine or via the 4-amino group in Cytosine. In this case there is one hydrogen atom the respective amino group substituted by the radical B-K.

The oligonucleotide residue N can be one or more Connection components B, or substituents B-K, the can be selected independently of one another. Oligonucleotide conjugates containing 1 to 10 are claimed  same or 2 to 10 different Connection components B included. Particularly preferred are oligonucleotide conjugates with one or two Connection components B.

The connecting component B connects the oligonucleotide Radical N with a complexing agent or complex K.

Multi-toothed, open-chain or cyclic complex-forming ligands with O, S and N. Use donor atoms.

Examples include the complexing agent residues K a hydrogen atom, a hydroxy and / or one Acaminic group decreased polyaminopolycarboxylic acids Diamine tetraacetic acid, diethylenetriaminepentaacetic acid, trans-1,2-cyclohexanediaminetetraacetic acid, 1,4,7,10-tetra azacyclododecanetetraacetic acid, 1,4,7-triazacyclononane triacetic acid, 1,4,8,11-tetraazatetradecanetetraacetic acid, 1,5,9-triazacyclododecanetriacetic acid, 1,4,7,10-tetraaza cyclododecane triacetic acid and 3,6,9,15-tetraaza-bicyclo- [9,3,1] -pentadeca-1 (15), called 11,13-trientrieslacetic acid.

Suitable complexing agents are e.g. B. in EP 0 485 045, EP 0 071 564 and EP 0 588 229, in DE 43 10 999 and DE 43 11 023 and US 4,965,392.

In order to clarify the various possibilities for the complexing agents K according to the present invention, reference is made to FIGS. 1 and 2, in which some advantageous structures are compiled. These figures are to be understood as a selection and in no way limit the present invention to the complexing agents shown.

The complexing agent K can do everything in nuclear medicine in use for diagnostic and therapeutic purposes used radioactive isotopes in the form of their metal ions contain. Stable isotopes can also be generated by external Radiation to deliver diagnostic or therapeutic Radiation excited, or isotopes caused by radiation from be converted into radioactive isotopes on the outside will.

Isotopes suitable according to the invention are formed from the elements of atomic numbers 5, 21-29, 31, 39, 42-44, 49, 57-83 or 85 selected.

To use the compound according to the invention as a radiopharmaceutical, the complexing agent contains a radioactive element. All radioactive elements that are able to achieve a therapeutic or diagnostic effect in vivo or in vitro are suitable for this. Radioactive isotopes of the elements copper, bismuth, technetium, rhenium or indium are preferred. ^99m Tc complexes are particularly preferred.

Contain the compounds of the invention general formula I positron emitting isotopes such as e.g. B. Sc-43, Sc-44, Fe-52, Co-55, Ga-68 or Cu-61, see above these can be done with positron emission tomography (PET) be used.

Contain the compounds of the invention general formula I gamma radiation emitting isotopes such as B. Tc-99m or In-111, these can be used in the Single photon emission tomography (SPECT) used will.  

The compounds of the invention can be in the form of their Complexes with radioisotopes, e.g. B. Ir-192, also in the Radio therapy can be used.

The compounds of the invention can also in the Radioimmunotherapy or radiation therapy can be used. These only differs from the corresponding diagnostics by the amount and type of isotope used. the goal is the destruction of tumor cells by high-energy short-wave radiation with the lowest possible Range. Suitable β-emitting ions are for Example Sc-46, Sc-47, Sc-48, Ga-72, Ga-73, Y-90, Re-186 or Re-188. Appropriate short half-lives α-emitting ions are, for example, At-209, At-211, Bi- 211, Bi-212, Bi-213 and Bi-214, with Bi-212 being preferred. A suitable photon and electron emitting ion is ¹⁵⁸Gd obtained from ¹⁵⁷Gd by neutron capture can be.

Is the agent according to the invention for use in the R.L. Mills et al. (Nature 336, 787 (1988)) Variant of radiation therapy determined, so it must Central ion from a Mössbauer isotope such as Derive ⁵⁷Fe or ¹⁵¹Eu.

Those carboxylic acid groups that are not used for complexation the metal ions of the elements of atomic numbers 21 to 29, 31, 39, 42 to 44, 49, 57 to 83 or 85 are required can, if desired, as salts of an inorganic or organic base such as alkali or alkaline earth metal hydroxides and carbonates, especially sodium and potassium hydroxide, or ammonia and alkylamines, or amino acid or as Ester or amide are present.  

Furthermore, compounds that use neutrons to Emission of α-particles are used will. Gadolinium is particularly effective. Advantage Such connections can also be used contain the isotope boron-10. In such cases, K can structure

have x for a whole Number from 1 to 10 stands.

The invention further relates to methods of manufacture of the conjugates according to the invention.

For example, conjugates in which the substituent on the 5′- End of the oligonucleotide is bound by reaction of the Obtained oligonucleotide with a phosphoramidite derivative (Tetrahedron 49, 1925-1963 (1993)). For this, the 5'-hydroxy group or 3'-hydroxy group of the oligonucleotide with a phosphoramidite of the general formula PR '(NR₂' ') OR' '' implemented. R 'stands for one optionally containing N, NO₂, Si or SO₂ alkyl, Alkoxy or arylalkoxy group with 1 to 20 carbon atoms such as Methyl, ethyl, propyl, butyl, pentyl, hexyl, methoxy, Ethoxy, propyloxy, butyloxy, benzyloxy or phenylethoxy, which may be substituted. As In particular, substituents become cyano and nitro groups used. For example, methoxy, β-cyanoethoxy or nitrophenylethoxy groups used will. Β-Cyanoethoxy groups are particularly preferred. R '' is a C₁-C₄ alkyl radical, with ethyl and propyl radicals are particularly suitable. Isopropyl radicals are preferred. R '' 'is an optionally S, O, N, CN, NO₂ or halogen containing alkyl or arylalkyl group with 1 to 20 C- Atoms. Protected amino and  Thioalkyl residues and protected amino and Thiooxaalkylreste used. 6- are particularly preferred Aminohexyl-, 6-thiohexyl-, 3,6,9-trioxa-11-amino-undecyl- and 3,6-dioxa-8-amino-octanyl groups. As protective groups can use commonly used N or S protecting groups will. For example, trifluoroacetyl, Phthalimido and monomethoxytrityl groups.

In a particularly preferred embodiment of this Er invention is used as a phosphoramidite derivative β-cyanoethyl-N, N- diisopropylamino-6- (trifluoroacetamido) -1-hexyl-phosphorus amidite used.

In a further preferred embodiment of this Invention is used as a phosphoramidite derivative β-cyanoethyl-N, N- diisopropylamino- (3,6,9-trioxa-11-phthalimido-1-undecyl) - phosporamidit used.

In a further embodiment of the invention, the Compound component B at the 3'-end of the oligonucleotide N in a manner analogous to that described above bound a phosphorus-containing group.

The reaction between oligonucleotide and Phosphoramidite can take place as a solid phase reaction, whereby the oligonucleotide is still on the column of a synthesis machine is located. After an oligonucleotide the desired sequence is obtained and exposure has taken place the 5'-hydroxy group of the oligonucleotide, e.g. B. with Trichloroacetic acid is reacted with the phosphoramidite and the reaction product is oxidized and released. Then the oligonucleotide derivative thus obtained at the terminal amino or thiol group with the Complexing agent or complex K optionally via a  further linker group coupled. The first step over the phosphorus-containing group bound to the oligonucleotide The rest then forms together with the if necessary existing linker group the connection component B.

Linking of the oligonucleotide derivative via the linker with the complexing agent or complex K can also be used as a solid phase reaction on the column of an automatic synthesizer respectively. The compound of the invention can then be isolated from the solid support by detachment.

Linking of the polynucleotide to the linker can not just about the 5′-OH group of the terminal sugar Nucleotides are done, but also via other functional ones Groups that can be generated from the 5′-OH group such as B. an amino or carboxy group. Such amino or nucleotides carrying carboxy groups are known and easy to manufacture. The synthesis of a 5'-aminouridine is in J. Med. Chem. 22, 1273 (1979) and in Chem. Lett. 6, 601 (1976). 4'-carboxyuridine is as in J. Med. Chem. 21, 1141 (1978), or Nucleic Acids Symp. Ser. 9, 95 (1981).

The link to the complexing agent is then via a linker carrying a carboxylic acid or amino group in the person skilled in the art. The linker then forms together with the -NH-CH₂-4 'or the -CO-4' group Connection component B.

It should be noted that the division of the conjugates according to the invention in a nucleotide residue, a Compound component and a complexing agent or complex purely formal and therefore independent of the actual one  synthetic structure. So z. B. in the above mentioned case the group -NH-CH₂-4 'or -CO-4' as for Connection component B viewed properly, whereas that decreased in the 4'-position by a CH₂-OH group Oligonucleotide is designated as oligonucleotide residue N.

A process for the preparation of conjugates in which the Connection component to those reduced by the OH groups Phosphodiester bridges are carried out, that is, initially two sugar units are linked to form a dinucleotide (see e.g. Chem. Lett. 1305 (1993)). This creates initially a trieste of formula

where U is a corresponding alkylene radical and V is a protected amino or sulfur group. To Cleavage of the amino protecting group can be done in the skilled person known manner of the complexing agent, if appropriate a linker with the amino group - e.g. B. in the form of a Amide bond - to be linked. The linker then forms together with the group O-U-V ′ (where V ′ for a group - NH-) the connection component B.

An alternative method is that the intermediate phosphotriester (e.g. through Reaction with 1,5-diaminopentane) subjected to aminolysis (see Biochemistry 27, 7237 (1988) or J. Am. Chem. Soc. 110, 4470 (1988)).  

The compounds of the formula thus obtained

can as described above with the complexing agent possibly linked via a linker.

Dinucleoside phosphate are also suitable for coupling purposes monothiotriester (see J. Am. Chem. Soc. 111, 9117 (1983) and Nucl. Acids Res. 20, 5205 (1992)).

A particularly large variety to link the The nucleobases offer complexing agents with the nucleotides. A link in positions 2 for the purines and 4 with the pyrimidines can be done directly. However, it is Often, the purines or pyrimidines are initially cheaper modify and these derivatized bases with the Complexing agents (possibly via further linkers) link. Suitable derivatized nucleobases e.g. B. in Biochemie 71, 319 (1989), Nucl. Acids Res. 16, 4937 (1988) or Nucleosides Nucleotides 10, 633 (1991) described.

An alternative method of linking through the Nucleobases consist of the palladium-catalyzed coupling of bromine or iodine nucleobases with functionalized residues (Biogenic and Medical Chemistry Letter V, 361 (1994)). about these functionalized residues can then be known Methods of complexing agents, if necessary, via a additional linkers can be linked to the nucleobase. As Functionalized residues are an example of a 5-acrylic ester or called a 5-allylamine (see Nucl. Acids Res. 14, 6115 (1986) and Nucl. Acids Res. 16, 4077 (1988)). As  Halogen derivatives used as precursors are such. B. in Biophys. J. 44, 201 (1983), J. Am. Chem. Soc. 86, 1242 (1964) or Chem. Commun. 17 (1967), available.

The production of the metal complexes according to the invention The metal-free oligonucleotide conjugates are carried out as in DE 34 01 052 discloses by using the metal oxide or a metal salt (e.g. the nitrate, acetate, Carbonate, chloride or sulfate) of the desired Metal isotopes in water and / or a lower alcohol (such as methanol, ethanol or isopropanol) dissolves or suspended and with the solution or suspension of equivalent amount of the complexing agent Reacts oligonucleotide conjugate and then if desired, existing acidic hydrogen atoms through cations of inorganic and / or organic bases or Amino acids substituted or free carboxylic acid groups in Converts amino acid amides.

The neutralization of any remaining free Acid groups occur with the help of inorganic bases (for Example hydroxides, carbonates or bicarbonates) from to Example sodium, potassium, lithium, magnesium or calcium and / or organic bases such as primary, secondary and tertiary amines, such as Ethanolamine, morpholine, glucamine, N-methyl and N, N- Dimethyl-glucamine, as well as basic amino acids, such as Example lysine, arginine and ornithine, or of amides originally neutral or acidic amino acids.

The preparation of the pharmaceutical according to the invention Means are also carried out in a manner known per se, by the oligonucleotide conjugates according to the invention  - if necessary with the addition of the usual in galenics Additives - suspended or dissolved in aqueous medium and then the suspension or solution if necessary sterilized or sterile filtered. Suitable additives are for example physiologically safe buffers (such as Example tromethamine), addition of complexing agents (such as Example diethylenetriaminepentaacetic acid) or - if required - electrolytes such as sodium chloride or - if necessary - antioxidants such as Ascorbic acid or, especially for oral administration forms, mannitol or other osmotically active substances.

If suspensions or solutions of the agents according to the invention in water or physiological saline solution are desired for enteral administration or other purposes, they can be mixed with one or more adjuvant (s) (for example methyl cellulose, lactose, mannitol) and / or surfactant (s) ) (for example Lecithine, Tween ^TR , Myrj ^TR ) can be mixed.

The pharmaceutical compositions according to the invention contain preferably 0.1 µmol / l to 3 mmol / l of the invention Oligonucleotide conjugates are usually used in quantities dosed from 0.01 nmol / kg - 60 µmol / kg. You are for enteral and parenteral application.

In the case of nuclear medical in vivo use, the labeled compounds are generally dosed in amounts of less than 10 ^-10 mol / kg body weight, the exact dose depending on the region of the body examined, but in particular also the particular examination method chosen, can vary widely. Starting from an average body weight of 70 kg, the amount of radioactivity for diagnostic applications is between 40 and 1100 MBq, preferably 200-800 MBq, for therapeutic applications 1-500 MBq, preferably 10-100 MBq per application. The application is usually intravenous, intraarterial, interstitial, peritoneal or intra-tumoral, with intravenous application being preferred. Generally 0.1 to 20 ml of the agent in question is administered per test.

The present invention further relates to a method for the detection of target structures. Doing one or several of the compounds described above with the to un probing sample in vivo or in vitro. The oligonucleotide residue N binds specifically and with high binding affinity to the target structure to be demonstrated.

If the target structure is present in the sample, it can can be detected there using the signal. Especially the procedure is suitable for a non-invasive Diagnosis of diseases. One or more of them of the compounds described above administered in vivo and the signal can be used to demonstrate whether the Target structure to which the oligonucleotide residue N is specific and binds with high affinity in the subject to be examined Organism is present.

In addition to the mere detection of target structures in too Examining samples can also be targeted be destroyed. The connections of the present invention particularly in radiotherapy, e.g. B. in cancer therapy.

Another embodiment of this invention includes a Diagnostic kit for the detection of target structures in vivo, the contains one or more of the above compounds.  

The conjugates and agents according to the invention fulfill the diverse requirements for a pharmaceutical for radiotherapy and diagnostics are to be provided. she are characterized in particular by high specificity or Affinity for the target structure concerned. Compared to known oligonucleotide conjugates, the conjugates according to the invention a particularly high in vivo Stability on. This was achieved by substituting the 2'-hydroxyl group reached. Surprisingly, the Specificity of the oligonucleotide neither by this Modification, still by coupling with the Complexing agents significantly impaired. Additional advantages are the controllable pharmacokinetics and the necessary Compatibility.

Fig. 1 shows a selection of cyclic complexing agents K, which can be used advantageously for the present invention.

Fig. 2 shows a selection of open-chain complexing agents K, which can be used advantageously for the present invention.

The following examples are intended to illustrate the present Illustrate inventions in more detail.  

example 1 a) 5 ′ - (6-amino-1-hexyl-phosphoric acid ester) of the 30mer Oligonucleotides 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUA-3 ' (Ligand for Nerve Growth Factor NGF, Seq. No. 21) (im U.S. Patent No. 5,270,163)

The 30-mer identified by the SELEX method Oligonucleotide 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUA-3 'is found in customarily in an automatic synthesizer from Pharmacia prepared (see Oligonucleotides and Analogues, A Practical Approach, Ed. F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1991), where the oligonucleotide is still located on the column of the solid support. By reaction with trichloroacetic acid solution in dichloromethane, the 5′- Hydroxy group exposed. The column is loaded about 10 mg of the 30mer oligonucleotide. To tie up the The column becomes linker with a solution of 50 µmol β-cyanoethyl-N, N-diisopropylamino-6- (trifluoroacetamido) -1- hexyl phosphoramidite (manufactured according to Nucl. Acids. Res. 16, 2659-2669 (1988)) in the presence of tetrazole. The oxidation of the phosphite formed to the fully protected Phosphotriester is done with iodine in tetrahydrofuran. The column is then washed in succession with methanol and Washed water. To replace the modified Solid support oligonucleotides, transfer the contents the column into a multivial, mixed with 5 ml of 30% Ammonia solution, closes the vessel and shakes over Night at 55 ° C. It is then cooled to 0 ° C., centrifuged, washes the carrier with 5 ml of water and subjects the combined aqueous phases of freeze drying.

For cleaning, take the solid material in 2 ml of water , mixed with 2 ml of 0.5 M ammonium acetate solution and  10 ml of ethanol are added and the mixture is left at -20 ° C. overnight standing, centrifuged, washes the residue with 1 ml Ethanol (-20 ° C) and finally dries in a vacuum Room temperature.

8 mg of the title compound are obtained as a colorless powder.

b) 10- [5- (2-carboxyphenyl) -2-hydroxy-5-oxo-4-aza-pentyl] - 1,4,7-tris (carboxymethyl) -1,4,7,10-tetraazacyclo dodecan

50 g (144.3 mmol) 1,4,7-tris (carboxymethyl) -1,4,7,10-tetra azacyclododecane (DO3A) are dissolved in 250 ml water and the pH value is adjusted to 13 with 5N sodium hydroxide solution set. A solution of 38.12 g (187.6 mmol) of N- (2,3-epoxypropyl) phthalimide in 100 ml of dioxane is then added dropwise within one hour, the mixture is stirred at 50 ° C. for 24 hours and the pH is added by adding 5 N sodium hydroxide solution kept at pH 13. The solution is adjusted to pH 2 with 10% hydrochloric acid and evaporated to dryness in vacuo. The residue is dissolved in a little water and purified on an ion exchange column (Reillex ^TR = poly (4-vinyl) pyridine, eluted with water). The main fractions are evaporated in vacuo and the residue is finally purified by chromatography on RP-18 (LichroPrep ^TR / mobile solvent: gradient of tetrahydrofuran / methanol / water). After evaporation of the main fractions, 63.57 g (71% of theory) of an amorphous solid are obtained.
Water content: 8.5%
Elemental analysis (based on anhydrous substance):
calc .:
C 52.90; H 6.57; N 12.34;
found:
C 52.65; H 6.68; N 12.15.

c) 10- (3-Amino-2-hydroxypropyl) -1,4,7-tris (carboxy methyl) -1,4,7,10-tetraazacyclododecane

50 g (88.1 mmol) of the title compound from Example 1b are concentrated in 300 ml. Hydrochloric acid heated under reflux for 24 hours. It is evaporated to dryness, the residue is dissolved in a little water and cleaned on an ion exchange column (Reillex ^TR = poly- (4-vinyl) pyridine (eluted with water)). The main fractions are evaporated to dryness.
Yield: 39.0 g (95% of theory) of a glassy solid.
Water content: 10.3%
Elemental analysis (based on anhydrous substance):
calc .:
C 48.68; H 7.93; N 16.70;
found:
C 48.47; H 8.09; N 16.55.

d) 10- [7- (4-nitrophenyl) -2-hydroxy-5-oxo-7- (carboxy methyl) -4-aza-heptyl] -1,4,7-tris (carboxymethyl) - 1,4,7,10-tetraazacyclododecane

To 14.62 g (34.86 mmol) of the title compound from Example 1c) in 200 ml of dimethylformamide / 20 ml of triethylamine, 9.84 g (41.8 mmol) of 3- (4-nitrophenyl) glutaric anhydride (J. Org Chem. 26, 3856 (1961)) and stirred overnight at room temperature. It is evaporated to the dry state in a vacuum. The residue is recrystallized from isopropanol / acetic acid 95: 5.
Yield: 21.68 g (95% of theory) of a yellowish solid
Water content: 0.9%
Elemental analysis (calculated on anhydrous substance):
calc .:
C 51.37; H 6.47; N 12.84;
found:
C 51.18; H 6.58; N 12.67.

e) 10- [7- (4-aminophenyl) -2-hydroxy-5-oxo-7- (carboxy methyl) -4-aza-heptyl] -1,4,7-tris (carboxymethyl) - 1,4,7,10-tetraazacyclododecane

21.0 g (32.07 mmol) of the title compound from Example 1d) are dissolved in 250 ml of methanol and 5 g of palladium catalyst (10% Pd on C) are added. It is hydrogenated overnight at room temperature. The catalyst is filtered off and the filtrate is evaporated to dryness in vacuo.
Yield: 19.63 g (98% of theory) of an off-white solid
Water content: 0.8%
Elemental analysis (calculated on anhydrous substance):
calc .:
C 53.84; H 6.35; N 12.60;
found:
C 53.73; H 6.45; N 12.51.

f) 10- [7- (4-isothiocyanatophenyl) -2-hydroxy-5-oxo-7- (carboxymethyl) -4-aza-heptyl] -1,4,7-tris (carboxy methyl) -1,4,7,10-tetraazacyclododecane

12.4 g (19.27 mmol) of the title compound from Example 1e) are dissolved in 200 ml of water and 6.64 g (57.8 mmol) of thiophosgene in 50 ml of chloroform are added. The mixture is stirred at 50 ° C. for 1 hour. The mixture is cooled to room temperature, the organic phase is separated off and the aqueous phase is shaken out twice with 100 ml of chloroform. The aqueous phase is evaporated to dryness and the residue is stirred in 100 ml of isopropanol at room temperature. The solid is filtered off and washed with ether. After drying overnight in vacuo (40 ° C.), 12.74 g (97% of theory) of a cream-colored solid are obtained.
Water content: 3.1%
Elemental analysis (calculated on anhydrous substance):
calc .:
C 52.24; H 6.35; N 12.60; S 4.81;
found:
C 52.37; H 6.44; N 12.48; S 4.83.

g) conjugation of the oligonucleotide 1a) with the Thioisocyanate ligands 1f)

8 mg of the oligonucleotide obtained in Example 1a) are in 2.5 ml of a mixture of 1 M NaHCO₃ / Na₂CO₃ (pH 9.0) dissolved and with 1 mg 10- [7- (4-isothiocyanatophenyl) -2- hydroxy-5-oxo-7- (carboxymethyl) -4-aza-heptyl] -1,4,7-tris- (carboxymethyl) -1,4,7,10-tetraazacyclododecane (title compound from Example 1f) added. Stir for 5 hours at room temperature, adjust the pH by adding 0.01M Hydrochloric acid to 7.2 and subject the solution to one Ultrafiltration through a membrane with the exclusion limit 3000 (Amicon YM3) and then freeze drying. 7 mg of the desired conjugate are obtained.

h) ¹¹¹Indium complex of the thiourea conjugate 1 g)

15 µl of a 111 indium (III) acetate solution (350 µCi), (Made from 111 indium (III) chloride in 2 M sodium ace tat solution and adjust the pH with 0.1 M hydrochloric acid to pH 4.0) in 135 µl of a solution of 1 mg Title compound from Example 1 g) in MES buffer, pH 6.2  given (MES = 2- (N-morpholino) ethylsulfonic acid). The pH The value is adjusted to pH 4.2 by adding 0.01 M hydrochloric acid brought. The mixture is stirred at 37 ° C. at pH 4.2 for 1 hour. It will brought to pH 6 with 2 M sodium acetate solution and 10 ul a 0.1 M Na₂EDTA solution (Na₂EDTA = ethylenediamine tetraacetic acid disodium salt) added to excess ¹¹¹Indium to complex. The final cleaning of the sun Labeled conjugate obtained (1h) is carried out by HPLC (Exclusion chromatography: TSK-400 / MES buffer). The the fractions containing labeled conjugate are marked with physiological saline solution diluted with 0.01 M Sodium hydroxide solution adjusted to pH 7.2 and filtered. Such a thing prepared solution then provides a suitable preparation for represents radio diagnostics.

Example 2 a) Conjugation of the oligonucleotide (1a) with N- [2-amino-3- (p-isothiocyanatophenyl) propyl] -trans-cyclohexane-1,2- diamine-N, N ′, N ′, N ′ ′, N ′ ′ - pentaacetic acid

8 mg of the oligonucleotide obtained in Example 1a) are in 2.5 ml of a mixture of 1 M NaHCO₃ / Na₂CO₃ (pH 9.0) dissolved and 1 mg of N- [2-amino-3- (p-isothiocyanatophenyl) propyl] -trans-cyclohexane-1,2-diamine-N, N ′, N ′, N ′ ′, N ′ ′ - penta acetic acid added (manufactured according to Bioconjugate Chem. 1, 59 (1990)). The mixture is stirred for 5 hours at room temperature, then adjust to pH 7.2 with 0.1 M hydrochloric acid and subject the solution of an ultrafiltration through a membrane with the Exclusion limit 3000 (Amicon YM3). After freeze drying 6 mg of the thiourea conjugate 2a) are obtained.  

b) ²¹² bismuth complex of the thiourea conjugate Example 2a)

A 122 bismuth tetraiodide solution in 0.1 M hydrogen iodide acid is brought to pH 4 with 2 M acetic acid. On Aliquot of this solution becomes about 3 mCi in activity 1 mg of the title compound from Example 2a) dissolved in 0.5 ml Added 0.02 M MES buffer and 0.5 ml 0.15 M sodium chloride Solution added. The mixture is stirred for 20 minutes at room temperature. It is brought to pH 6 with 2 M sodium acetate solution and 20 ul of a 0.01 M Na₂EDTA solution added. You stir 20 Minutes. The complex is purified by HPLC (Exclusion chromatography: TSK-400 / MES buffer). The radio active conjugate fractions are pooled with physiological saline diluted, and with 0.01 M Sodium hydroxide solution adjusted to pH 7.2. Obtained after filtration a preparation suitable for radiotherapy.

Example 3 a) 111 indium complex of the thiourea conjugate 2a)

15 ml of a 111 indium (III) acetate solution (350 μCi), (made from 111 indium (III) chloride in 2 M sodium acetate) Solution and adjust the pH with 0.1 M hydrochloric acid pH 4.0) are in 0.5 ml of a solution of 1 mg Title compound from Example 2a) in MES buffer, pH 6.2 given (MES = 2- (N-morpholino) ethylsulfonic acid). The pH The value is adjusted to pH 5.0 by adding 0.01 M hydrochloric acid brought. The mixture is stirred at pH 5.0 for 30 minutes. It comes with 2 sts Sodium acetate solution brought to pH 6 and 10 ul one 0.1 M Na₂EDTA solution (Na₂EDTA = ethylenediamine tetra acetic acid disodium salt) added to excess  ¹¹¹Indium to complex. The final cleaning of the sun Labeled conjugate (3a) obtained is obtained by HPLC (Exclusion chromatography: TSK-400 / MES buffer). The the fractions containing labeled conjugate are marked with physiological saline solution diluted with 0.01 M Sodium hydroxide solution adjusted to pH 7.2 and filtered. Such a thing prepared solution then provides a suitable preparation for represents radio diagnostics.

Example 4 a) Conjugation of the title compound 1a) with 2- (p-isothiocyanato-benzyl) -diethylenetriamine- N, N, N ′, N ′ ′, N ′ ′ - pentaacetic acid

8 mg of the oligonucleotide obtained in Example 1a) are in 2.5 ml of a mixture of 1 M NaHCO₃ / Na₂CO₃ (pH 9.0) dissolved and 1 mg 2- (p-isothiocyanato-benzyl) - diethylenetriamine-N, N, N ′, N ′ ′, N ′ ′ - pentaacetic acid added (produced by: Bioconjugate Chem. 2, 187 (1991)). Man stir for 5 hours at room temperature, then adjust with 0.01 M. Hydrochloric acid to pH 7.2 and subject the solution to a Ultrafiltration through a membrane with the exclusion limit 3000 (Amicon YM 3). After freeze-drying, 6 mg are obtained of the thiourea conjugate.

b) ⁹⁰Yttrium complex of the thiourea conjugate Example 4a)

To 1 mg of the thiourea derivative from Example 4a) in 0.5 ml of 0.05 M ammonium acetate solution of pH 6 is added Solution of ⁹⁰Yttrium, dissolved in 0.05 M ammonium acetate solution  (approx. 380 mCi), adjust to pH 5.2 and with 3 M acetic acid stir for 1 hour at room temperature. It is set with 0.01 M. Sodium hydroxide solution to pH 7.0 and purifies the conjugate by HPLC to (TSK-400 / MES buffer). The main fractions will be combined, diluted with physiological saline and brought to pH 7.2 with 0.01 M sodium hydroxide solution. After filtration a preparation suitable for radiotherapy is obtained.

Example 5 a) 5 ′ - (6-mercapto-1-hexyl-phosphoric acid ester) of the 30mer Oligonucleotides 5′-AGGAGGAGGAGGGAGAGCGCAAAUGAGAUU-3 ′ (Ligand for serine protease)

The 30-mer identified by the SELEX method Oligonucleotide 5′-AGGAGGAGGAGGGAGAGCGCAAAUGAGAUU-3 ′ (Seq. No. 13 from U.S. Patent No. 5,270,163) is more common Way in an automatic synthesizer from Pharmacia prepared (see Oligonucleotides and Analogues, A Practical Approach, Ed. F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1991), where the oligonucleotide is still located on the column of the solid support. By reaction with trichloroacetic acid solution in dichloromethane, the 5′- Hydroxy group exposed. The column is loaded about 10 mg of the 30mer oligonucleotide. To tie up the The column becomes linker with a solution of 50 µmol β-cyanoethyl-N, N-diisopropylamino-S-trityl-6-mercapto) - phosphoramidite in acetonitrile in the presence of tetrazole implemented. The oxidation of the phosphite to full Protected phosphotriester is made with iodine Tetrahydrofuran. Then the column is successively washed with methanol and water. To replace the modifi decorated oligonucleotide from the solid support, one transfers the  Contents of the column in a multivial, mixed with 5 ml of 30% ammonia solution, seals the vessel and shakes overnight at 55 ° C. You then cool to 0 ° C, centrifuged, washed the carrier with 5 ml of water and subjects the combined aqueous phases to one Freeze drying.

For cleaning, take the solid material in 2 ml of water , mixed with 2 ml of 0.5 M ammonium acetate solution and 10 ml of ethanol are added and the mixture is left at -20 ° C. overnight standing, centrifuged, washes the residue with 1 ml Ethanol (-20 ° C) and finally dries in a vacuum Room temperature.

9 mg of the S-tritylated title compound are obtained. For Cleavage of the trityl protective group dissolves the product in 0.5 ml of water, mixed with 0.1 ml of 1 M silver nitrate solution and stirred for 1 hour at room temperature. Subsequently 0.1 ml of 1 M dithiothreitol solution is added. After 15 Centrifuged for minutes and the supernatant extracted Solution several times with ethyl acetate. From the aqueous solution 8 mg of the desired are obtained after freeze-drying Title link.

b) 4-Benzyloxy-N-methanesulfonylphenylalanine methyl ester

19.58 g of methanesulfonic acid chloride are added dropwise to 50 g of 4-benzyloxyphenylalanine methyl ester hydrochloride in 300 ml of pyridine at 0 ° C. and the mixture is stirred at 0 ° C. for 3 hours. It is evaporated to dryness in vacuo and the residue is dissolved in 500 ml of dichloromethane. It is shaken twice with 300 ml of 5N hydrochloric acid, dried over magnesium sulfate and evaporated in vacuo. The residue is recrystallized from 150 ml of methanol.
Yield: 53.64 g of a colorless crystalline powder.

c) 2- (4-benzyloxybenzyl) -1-methanesulfonyl-1,4,7- triazaheptan-3-one

37.2 g of 4-benzyloxy-N-methanesulfonyl-phenylalanine-methyl ester and 1.2 l of 1,2-diaminoethane are 3 hours at 80 ° C. touched. The residue is evaporated to dryness and stirred with 200 ml of water, sucks off the precipitate, washes neutral with water and dries overnight at 60 ° C. Yield: 37.68 g of a cream-colored, amorphous powder.

d) 2- (4-benzyloxybenzyl) -1-methanesulfonyl-7- (tert.- butyloxycarbonyl) -1,4,7-triazaheptan-3-one

A solution of 16.23 g of 2- (4-benzyloxybenzyl) -1-methanesulfonyl-1,4, 7-triazaheptan-3-one and 4.76 g of triethylamine in 200 ml of chloroform are dissolved at 0 ° C. with a solution of 10 , 27 g of di-tert-butyl dicarbonate in 50 ml of chloroform. The mixture is stirred for 5 hours at room temperature, shaken with 5% sodium carbonate solution and water, dried over magnesium sulfate and evaporated in vacuo. The residue is recrystallized from 100 ml of methanol.
Yield: 20.19 g of a foamy solid.

e) 2- (4-hydroxybenzyl) -1-methanesulfonyl-7- (tert.- butyloxycarbonyl) -1,4,7-triazaheptan-3-one

20 g of 2- (4-benzyloxybenzyl) -1-methanesulfonyl-7- (tert-butyloxycarbonyl) -1,4,7-triazaheptan-3-one dissolved in 300 ml of dichloromethane are mixed with 4 g of palladium-carbon (10% ) stirred overnight under a hydrogen atmosphere. It is filtered and the solution is evaporated in vacuo.
Yield: 16.17 g of a glassy foam which solidifies after a few minutes.

f) 2- [4- (3-oxapropionic acid benzyl ester) benzyl] -1-methane sulfonyl-7- (tert-butyloxycarbonyl) -1,4,7- triazaheptan-3-one

15 g of 2- (4-hydroxybenzyl) -1-methanesulfonyl-7- (tert-butyl oxycarbonyl) -1,4,7-triazaheptan-3-one, 8.56 g of benzyl bromoacetate and 13.18 g of potassium carbonate are heated under reflux in 300 ml of acetonitrile for 24 hours. It is filtered and evaporated to dryness in vacuo. The residue is dissolved in 200 ml of dichloromethane and extracted twice with 50 ml of water. The organic phase is dried over magnesium sulfate and evaporated in vacuo. The residue is chromatographed with dichloromethane-hexane-acetone (20/10/1) as the eluent.
Yield: 10.06 g of foamy solid

g) 2- [4- (3-oxapropionic acid benzyl ester) benzyl] -1- methanesulfonyl-1,4,7-triazaheptan-3-one

10 g of 2- [4- (3-oxapropionic acid benzyl ester) benzyl] -1-methane sulfonyl-7- (tert-butyloxycarbonyl) -1,4,7-triazaheptan-3-one are mixed with 100 at room temperature for 1 hour ml trifluoroacetic acid stirred. It is evaporated to the dry state in a vacuum.
Yield: 9.2 g of glassy foam that solidifies on standing.

h) 9-Chloro-1-methanesulfonyl-2- [4- (3-oxapropionic acid benzyl ester) benzyl] -1,4,7-triaza-3,8-dione

9 g of 2- [4- (3-oxapropionic acid benzyl ester) benzyl] -1-methane sulfonyl-1,4,7-triazaheptan-3-one and 1.78 g of triethylamine are dissolved in 200 ml of chloroform. At 0 ° C., 1.98 g of chloroacetyl chloride dissolved in 20 ml of chloroform are added dropwise in the course of 30 minutes and then stirred at 0 ° C. for 2 hours. It is washed with 100 ml of 5% hydrochloric acid, twice with 50 ml of water each time, dried over magnesium sulfate and evaporated to dryness in vacuo. The residue is chromatographed on silica gel using dichloromethane-ethyl acetate (20/1) as the eluent.
Yield: 6.97 g waxy solid

i) 9-chloro-1-methanesulfonyl-2- [4- (3-oxapropionic acid) - benzyl] -1,4,7-triaza-3,8-dione

6.5 g of 9-chloro-1-methanesulfonyl-2- [4- (3-oxapropionic acid benzyl ester) benzyl] -1,4,7-triaza-3,8-dione are dissolved in 150 ml of dichloromethane with 2 g of palladium -Carbon (10%) stirred overnight under a hydrogen atmosphere. It is filtered and the solution is evaporated in vacuo.
Yield: 5.33 g of glassy solid

j) 10-acetyl-2- [4- (3-oxapropionic acid) benzyl] -1- (methanesulfonyl) -10-thia-1,4,7-triazadecane-3,8-dione

5 g 9-chloro-1-methanesulfonyl-2- [4- (3-oxapropionic acid) - benzyl] -1,4,7-triaza-3,8-dione dissolved in 80 ml chloroform are mixed with 1.98 g of triethylamine and 0.74 g of thioacetic acid 10 Heated under reflux for minutes. You pour the solution in 200 ml ice-cold 5% hydrochloric acid, separates the organic Phase, dries over magnesium sulfate and evaporates in Vacuum on. After chromatography on silica gel with hexane Ethyl acetate (3/1) gives 4.64 g of the desired Connection as a glassy solid.

k) 10-Acetyl-2- [4- (3-oxapropionic acid- (2,5-dioxo pyrrolidin-1-yl) ester) benzyl] -1- (methanesulfonyl) -10- thia-1,4,7-triazadecane-3,8-dione

4 g of 10-acetyl-2- [4- (3-oxapropionic acid) benzyl] -1- (methanesulfonyl) -10-thia-1,4,7-triazadecane-3,8-dione, 1.91 g of dicyclohexylcarbodiimide , 4 g of N-hydroxysuccinimide and 30 mg of 4-dimethylaminopyridine are stirred in 20 ml of chloroform at room temperature for 24 hours. 20 ml of diethyl ether are then added, the mixture is filtered and the residue is evaporated in vacuo. The residue is chromatographed on silica gel with dichloromethane-dioxane (10/1) as the eluent.
Yield: 3.47 g of an off-white solid
Elemental analysis:
calc .:
C 46.15; H 4.93; N 9.78; S 11.20;
found:
C 46.03; H 4.83; N 9.64; S 11.05.

l) 10-Acetyl-2- {4- [3-oxapropionic acid (6-maleimido hexanoyl) hydrazide] benzyl} -1-methanesulfonyl-10-thia- 1,4,7-triazadecane-3,8-dione

3 g of 10-acetyl-2- [4- (3-oxapropionic acid (2,5-dioxopyrrol din-1-yl) ester) benzyl] -1- (methanesulfonyl) -10-thia-1,4 , 7-triazadecane-3,8-dione and 1.17 g of 6-maleimidocaproic acid hydrazide (Science 261, 212 (1993)) are stirred in 40 ml of dimethylformamide at 60 ° C. for 5 hours. With vigorous stirring, 100 ml of water are added dropwise and the precipitate which has separated out is filtered off. It is dried in vacuo and the residue is purified by FLASH chromatography on a silica gel column using dichloromethane / dioxane (10/1) as the eluent. 2.8 g of the title compound are obtained as a white solid.
Elemental analysis:
calc .:
C 49.25; H 5.61; N 12.30; S 9.39;
found:
C 49.33; H 5.95; N 12.43; S 9.11.

m) conjugation of the oligonucleotide according to Example 5a) with the title compound from Example 5l)

5 mg of the thiol-containing prepared according to Example 5a) Oligonucleotides are mixed in 2 ml of phosphate buffer (pH 7.4) 1 mg of the maleimide derivative prepared according to Example 5l) dissolved in 0.2 ml of dimethylformamide. You leave 2 Stand for hours at room temperature and subject the solution an ultrafiltration through a membrane with the off final limit 3000 (Amicon YM 3) and then one Freeze drying. 5 mg of the desired are obtained Conjugate.  

n) Tc-99m complex of the conjugate from Example 5m)

1 ml of a solution of potassium D-glucarate (12 mg / ml) and Tin (II) chloride (100 µg / ml) in 0.2 M NaHCO₃ are in one Vial freeze-dried and then with [Tc-99m] - Sodium pertechnetate solution (1 ml, 1 mCi) from a Mo-99 / Tc-99m generator offset. After standing at room temperature take an aliquot for 15 minutes and add the same volume of the solution in 0.2 M NaHCO₃ solution Conjugate from example 5m) (1 mg / ml). After 15 minutes showed both thin layer chromatography and HPLC, that <98% of the radioactivity was absorbed by the conjugate were.

Example 6 a) Conjugation of the title compound of Example 1a) with S-Benzoyl-MAG₃-2,3,5,6-tetrafluorophenyl ester to the amide

To 8 mg of the title compound from Example 1a) dissolved in 0.5 ml of 0.1 M phosphate buffer (pH 7) is added to 3 mg of S-benzoyl MAG₃-2,3,5,6-tetrafluorophenyl ester (manufactured by No. 4,965,392) dissolved in 0.2 ml of dimethylformamide and stirred 3 Hours at room temperature. It is diluted with water and subjecting the solution to ultrafiltration (Amicon YM 3, Exclusion limit 3000). After freeze-drying, 5 are obtained mg of conjugate 6a).  

b) ^99m technetium complex from the amide conjugate 6a)

1 mg of the title compound from Example 6a) is dissolved in 200 μl of water and 1 ml of 0.1 M phosphate buffer pH 8.5 is added. 200 μl of ^99m technetium (V) gluconate solution (about 15 mCi) are added to this mixture and the mixture is left to stand at room temperature for 15 minutes. The labeling yield (determined by HPLC) is approx. 95%

Example 7 a) Conjugation of the title compound from Example 1a) with the ^99m technetium complex of 2,3,5,6-tetrafluorophenyl-4,5-bis (mercaptoacetamido) pentanoic acid ester

To the ^99m technetium complex of 2,3,5,6-tetrafluorophenyl-4,5- bis (mercaptoacetamido) pentasate (manufactured according to: J. Nucl. Med. 32, 1445 (1991)) approx. 100 mCi, dissolved in 2 ml of phosphate buffer, pH 7.2, are given 3 mg of the title compound from Example 1a), dissolved in 0.6 ml of phosphate buffer. The pH is adjusted to 10 with 1.0 M potassium carbonate buffer and the mixture is stirred for 20 min. at room temperature. For final cleaning, the solution is placed on a Sephadex column (Pharmacia) and eluted with 75 mM sodium chloride solution. The main fractions are combined, diluted with physiological saline and filtered. The solution thus obtained can be used for radiodiagnostic examinations.

Example 8 a) Conjugation of the oligonucleotide (Example 5a) with 5- (N-Maleimido) -3-oxapentyl {2 - [(3-carboxybenzoyl) thio] acetyl} glycylglycylglycinate

5 mg of the thio-containing prepared according to Example 5a) Oligonucleotides are under N₂ in 2 ml of phosphate buffer (pH 7.4) with 1 mg of 5- (N-maleimido) -3-oxapentyl} 2 - [(3-carboxy benzoyl) thio] acetyl} glycylglycylglycinate (manufactured by Bioconj. Chem. 1, 431 (1990)) dissolved in 0.2 ml Dimethylformamide added. Allow 2 hours Stir room temperature and subject the solution to one Ultrafiltration through a membrane (Amicon YM 3) and then freeze drying. 5.5 mg of the are obtained desired conjugate.

b) Tc-99m complex of that prepared according to Example 8a) Conjugate

1 ml of a solution of potassium D-glucarate (12 mg / ml) and Tin (II) chloride (100 µg / ml) in 0.2 M NaHCO₃ are in one Vial freeze-dried and then with [Tc-99m] - Sodium pertechnetate solution (1 ml, 1 mCi) from a Mo-99 / Tc-99m generator offset. After standing at room temperature take an aliquot for 15 minutes and add the same volume of the solution in 0.2 M NaHCO₃ solution Conjugate from Example 8a) (1 mg / ml). The substance can be isolated by freeze drying. The by HPLC certain marking yield is <96%.  

Example 9 a) Conjugation of the oligonucleotide from Example 5a) with 1- [6- (2-vinyl-6-hexyloxymethyl) pyridine] -1,4,8,11- tetraazacyclotetradecane

A solution of 4 mg of the solution according to Example is added under N₂ 5a) prepared thio-containing oligonucleotide in 2 ml Phosphate buffer (pH 7.4) with 1 mg 1- [6- (2-vinyl-6-hexyl oxymethyl) pyridine] -1,4,8,11-tetraazacyclotetradecane (produced according to EP 0 588 229) dissolved in 0.5 ml Dimethylformamide. The mixture is stirred at 35 ° C. for 4 hours, mixed with 10 ml of ethanol and isolated the product Centrifuge. The cleaning is done by reversed phase Chromatography on a 1 × 25 cm column with a 50 mM Triethylammonium acetate (pH 7) acetronitrile gradient. The pooled fractions are freeze-dried, in 1 ml of water dissolved and desalted on a Sephadex G-10 column. The title compound (approx. 4 mg) is freeze-dried isolated.

b) Tc-99m complex of that prepared according to Example 9a) Conjugate

The procedure is as described in Example 8b). By HPLC-determined labeling yield is 92%.  

Example 10 a) Conjugation of the oligonucleotide from Example 5a) with N- [4-Hydroxy-3- (1,4,8,11-tetraaza-cyclotetradec-5-yl) - benzyl] -2- (6-vinyl-pyridin-2-ylmethoxy) acetamide

A solution of 6.5 mg of that of Example 5a) is added. prepared thiol-containing oligonucleotide in 2 ml Phosphate buffer (pH 8.0) with 1.2 mg N- [4-hydroxy-3- (1,4,8,11-tetraaza-cyclotetradec-5-yl) benzyl] -2- (6-vinyl pyridin-2-ylmethoxy) acetamide (manufactured according to J. Chem. Soc., Chem. Commun. 156 (1988)) dissolved in 0.1 ml Dimethylformamide. The mixture is stirred for 4 hours under N₂ at 35 ° C. mixed with 10 ml of ethanol and isolated the product Centrifuge. The cleaning is done by reversed phase Chromatography on a 1 × 25 cm column with a 50 mM Triethylammonium acetate (pH 7) / acetonitrile gradient. The Pooled fractions are on a Sephadex G-10 column desalinated. Freeze drying gives 5 mg of Title compound as a white powder.

b) Cu-64 complex of that prepared according to Example 10a) Conjugate

1 mg of the conjugate obtained according to 10a) are in 1 ml Phosphate buffer (pH 8) incubated with ⁶⁴CuCl₂ (0.2 mCi). The Labeling yield determined after 1 hour by HPLC, is <98%. The product is freeze-dried isolated.  

Example 11 a) Conjugation of the oligonucleotide from Example 5a) with 1,4,7,10-tetraazacyclododecan-2 - [(5-aza-8-maleimido-6- oxo) octane] -1,4,7,10-tetraacetic acid

To a solution of 5 mg of that according to Example 5a) prepared thiol-containing oligonucleotide in 2 ml Phosphate buffer (pH 8) is added under N₂ 1 mg 1,4,7,10- Tetraazacyclododecan-2 - [(5-aza-8-maleimido-6-oxo) octane] - 1,4,7,10-tetraacetic acid (manufactured according to J. Chem. Soc., Chem. Commun. 796, (1989)). The mixture is stirred at 35 ° C. for 3 hours, mixed with 10 ml of isopropyl alcohol and isolated Product by centrifugation. The cleaning is done by Reversed phase chromatography on a 1 × 25 cm column with a 25 mM triethylammonium acetate (pH 7) / acetonitrile Gradients. The combined fractions are in a vacuum gently evaporated, dissolved in a little water and with the help desalinated on a Sephadex G-10 column. By freeze drying 4 mg of the title compound are obtained as a white powder.

b) Y-90 complex of that prepared according to Example 11a) Conjugate

⁹⁰Y acetate (1 mCi), dissolved in 1 ml 0.05 M ammonium acetate Solution is made with 1 mg of that prepared according to Example 11a) Conjugate added and heated to 85 ° C for 1 hour. By HPLC-determined labeling yield is <95%. The Product is isolated by freeze drying.  

Example 12 a) Conjugation of the oligonucleotide from Example 5a with 1,4,7-triazacyclononan-2 - [(5-aza-8-maleimido-6-oxo) - octane] -1,4,7-triacetic acid

To a solution of 5 mg of that according to Example 5a) prepared thiol-containing oligonucleotide in 2 ml Phosphate buffer (pH 8) is added under N₂ 1 mg 1,4,7- Triazacyclononan-2 - [(5-aza-8-maleimido-6-oxo) octane] -1,4,7- triacetic acid (manufactured according to J. Chem. Soc., Chem. Commun. 794, (1989)). The mixture is stirred at 35 ° C. for 6 hours, mixed with 10 ml of isopropanol and isolates the product by centrifugation. The cleaning is done by reversed Phase chromatography on a 1 × 25 cm column with a 25th mM triethylammonium acetate (pH 7) / acetonitrile gradient. The combined fractions are gently in a vacuum evaporated, dissolved in a little water and with the help of a Desalinated Sephadex G-10 column. Obtained by freeze drying 3 mg of the title compound as a white powder.

b) Ga-67 complex of that prepared according to Example 12a) Conjugate

20 mg of the conjugate prepared according to Example 12a) dissolved in 0.5 ml 0.1 M citrate buffer pH 4.5 and with 0.1 ml a gallium 67 citrate solution (0.2 mCi) was added. You leave Stand for 2 hours at room temperature and desalinate it Product on a Sephadex G-10 column. After freeze drying 17 mg of the title compound are obtained as a white powder.  

Example 13 a) Phosphitylation of 5′-O- (4,4′-dimethoxytrityl) -5- (prop-2-en-1-one) -2'-deoxyuridine

To a stirred solution of 2.1 g (3.59 mmol) of 5'-O- (4,4'-dimethoxytrityl) -5- (prop-2-en-1-one) -2'-deoxyuridine (made according to Nucleosides & Nucleotides 13, 939-944, (1994)) in 50 ml of tetrahydrofuran are successively added 50 mg of 4-dimethylaminopyridine, 3 ml of diisopropylethylamine and 962 μl (4.31 mmol) of 2-cyanoethyl-N, N-diisopropylchloro phosphoramidite. A white precipitate forms after about 30 minutes. It is filtered, the solution is concentrated in vacuo and the residue is partitioned between dichloromethane and 5% sodium hydrogen carbonate solution. The dichloromethane phase is dried over magnesium sulfate and evaporated in vacuo. The residue is purified by rapid chromatography on silica gel, eluting with dichloromethane / hexane / diisopropylethylamine (80: 18: 2). 1.8 g of the desired compound are obtained as a white foam.
Elemental analysis:
calc .:
C 64.28; H 6.29; N 7.14; P 3.95;
found:
C 64.02; H 6.60; N 7.21; P 4.09.

b) implementation of the phosphoramidite from Example 13a) with of oligonucleotide sequence No. 21 from example 1a)

The 30-mer identified by the SELEX method Oligonucleotide is used in a conventional manner Synthesizers manufactured by Pharmacia (see Oligonucleotides and Analogues, A Practical Approach, Ed. F. Eckstein, Oxford University Press, Oxford, New York,  Tokyo, 1991), with the oligonucleotide still on the Column of the solid support is located. By reacting with Trichloroacetic acid solution in dichloromethane is the 5'- Hydroxy group exposed. The column is loaded about 10 mg of the 30mer oligonucleotide. The 5'-hydroxy group is in the presence of tetrazole with that according to Example 13a) obtained phosphoramidite implemented. Then will by treatment with iodine solution the phosphite in the Phosphotriester transferred and by reaction with Trichloroacetic acid solution in dichloromethane cleaved terminal DMT remainder. To add one Thiol group at the terminal 2'-deoxyuridine existing α, β-unsaturated carbonyl system is used a solution of 10- (4-aza-2-hydroxy-5-imino-8-mercapto octane) -1,4,7-tris (carboxymethyl) -1,4,7,10-tetraaza cyclododecane * in tetrahydrofuran and washes one after the other with methanol and water. To replace the modified Solid oligonucleotides, the contents are transferred the column into a multivial, mixed with 5 ml of 30% Ammonia solution, closes the vessel and shakes over Night at 55 ° C. It is then cooled to 0 ° C., centrifuged, washes the carrier with 5 ml of water and subjects the combined aqueous phases of freeze drying.

For cleaning, take the solid material in 2 ml of water , mixed with 2 ml of 0.5 M ammonium acetate solution and 10 ml of ethanol are added and the mixture is left at -20 ° C. overnight standing, centrifuged, washes the residue with 1 ml Ethanol (-20 ° C) and finally dries in a vacuum Room temperature.

6 mg of the title compound are obtained as a colorless powder.  

* 10- (4-Aza-2-hydroxy-5-imino-8-mercapto-octane) 1,4,7-tris (carboxymethyl) -1,4,7,10-tetraazacyclododecane is obtained as described below:
To a solution of 1.46 g (3.49 mmol) of 10- (3-amino-2-hydroxypropyl) -1,4,7-tris (carboxymethyl) -1,4,7,10-tetra azacyclododecane (see Example 1c) in a mixture of 50 ml of water and 50 ml of methanol, 15.7 ml of 1N sodium hydroxide solution and 480 mg (3.49 mmol) of 2-iminotetrahydrothiophene hydrochloride are added and the mixture is stirred for 3 hours at room temperature, concentrated in vacuo to approx. 1/4 of the initial volume and stirring with an anion exchanger (IRA 410) until a pH of 11 is reached. The solution is filtered and, with stirring, the solution is added in small portions with as much cation exchanger IRC 50 until a pH of 3.5 is reached. After filtering, the solution is freeze-dried. 1.39 g of the desired substance are obtained as a white powder with a water content of 4.9%.
Elemental analysis (based on anhydrous substance):
calc .:
C 48.45; H 7.74; N 16.14; S 6.16;
found:
C 48.30; H 7.98; N 16.05; S 6.44.

c) Y-90 complex of that prepared according to Example 13b) Conjugate

To 1 mg of the thio derivative from Example 13b) in 0.5 ml 0.05 M solution of ammonium acetate of pH 6 is given a solution of ⁹⁰Yttrium, dissolved in 0.05 M ammonium acetate solution (approx. 380 mCi), adjust to pH 5.2 with 3 M acetic acid and stir 1 Hour at room temperature. It is set with 0.01 M. Sodium hydroxide solution to pH 7.0 and purifies the conjugate by HPLC to (TSK-400 / MES buffer). The main fractions will be  combined, diluted with physiological saline and brought to pH 7.2 with 0.01 M sodium hydroxide solution. After filtration a preparation suitable for radiotherapy is obtained.

Example 14 a) S- (triphenylmethyl-mercaptoacetyl) glycyl glycine methyl ester

3.34 g (10 mmol) of S-triphenylmethylmercaptoacetic acid and 1.83 g (10 mmol) of glycylglycine methyl ester hydrochloride are suspended in 250 ml of absolute dichloromethane. After adding 1.01 g (10 mmol) of triethylamine, 2.06 g (10 mmol) of dicyclohexylcarbodiimide, dissolved in 50 ml of absolute dichloromethane, are added dropwise with ice cooling. The mixture is stirred at 0 ° C. for 1 h and at room temperature for 18 h. It is filtered, concentrated and chromatographed on silica gel (eluent: CH₂Cl₂ / MeOH: 10% - 30%).
Yield: 3.56 g (77.0% of theory), white powder
Elemental analysis:
calc .:
C 67.51; H 5.67; N 6.06; O 13.84; S 6.93;
found:
C 67.37; H 6.02; N 5.91; S 6.73.

b) [S- (triphenylmethyl-mercaptoacetyl) glycyl-glycine amidyl] -6-hexanol

4.63 g (10 mmol) of the S- (triphenyl-methyl-mercaptoacetyl) -glycyl-glycine methyl ester prepared in Example 14a) are dissolved in 11.72 g (100 mmol) of 6-aminohexanol / 50 ml of 1,4-dioxane under an argon atmosphere Heated to 100 ° C for 2 h. The reaction mixture is then poured onto a mixture of 100 ml of dichloromethane and 100 ml of water. With stirring and ice cooling, a pH of 6 is set using 10 molar hydrochloric acid, the organic phase is separated off and dried over sodium sulfate. After evaporation of the solvent, the crude product is purified on silica gel (eluent: CH₂Cl₂ / MeOH: 10% -50%).
Yield: 2.97 g (54.2% of theory), white powder
Elemental analysis:
calc .:
C 67.98; H 6.81; N 7.67; O 11.68; S 5.85;
found:
C 67.72; H 6.93; N 7.39; S 5.64.

c) O - {[S- (triphenylmethyl mercaptoacetyl) glycyl glycine amidyl] -6-hex-1-yl} -diisopropylamide-O'-methyl phosphoric acid diester

5.48 g (10 mmol) of the [S- (triphenyl-methyl-mercaptoacetyl) -glycyl-glycine-amidyl] -6-hexanol prepared in Example 14b) are dissolved in 100 ml of absolute dichloromethane. 5.17 g (40 mmol) of diisopropylethylamine are added under an argon atmosphere and 3.95 g (20 mmol) of monomethyl phosphorous acid diisopropylamide chloride, dissolved in 50 ml of absolute dichloromethane, are added dropwise at 0.degree. The mixture is stirred at 0 ° C. for 0.5 h and finally at room temperature for 2 h. For working up, 320 mg (10 mmol) of absolute methanol are added while cooling with ice and, after concentration, chromatographed on silica gel (eluent: CH₂Cl₂ / MeOH: 95: 5/5% triethylamine).
Yield: 1.98 g (27.9 d. Th.), Colorless oil

d) conjugation of the oligonucleotide 5′-CUCAUGGAGCGCAAGACGAAUAGCUACAUA with the connection after Example 14c)

A 30-mer identified by the Selex method Ologonucleotide is generated using an automatic synthesizer Manufactured by Pharmacia (see Oligonucleotides and Analogues, A Practical Approach, Editor F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1991) the oligonucleotide in protected form still on the column of the solid support. The loading of the column is approx. 15 mg of 30mer oligonucleotide. After splitting off the 5′-DMT Protective group (trichloroacetic acid / dichloromethane) is according to the Standard methods with that described in Example 14c Coupled phosphoramidite. After oxidation with iodine in The conjugate is split off from the carrier by tetrahydrofuran. For this, the material is mixed with 10 ml of 30% Ammonia solution, closes the jar and shakes overnight at 55 ° C. It is cooled to 0 ° C., centrifuged, the carrier is washed with 10 ml of water and subject the combined aqueous Freeze drying phases.

For cleaning, take up the material in 5 ml of water, mixed with 4 ml of 0.5 molar ammonium acetate solution and mixed with 20 ml of ethanol. To complete the precipitation put it cold overnight (-20 ° C), centrifuge, wash it Residue with 1 ml of ethanol (-20 ° C) and dried in vacuo. Man receives 9 mg of a white powder.

To split off the S-trityl protecting group, the material in 5 ml 50 mmolar triethylammonium acetate solution (pH = 7.0) taken up and with 500 ul 0.1 molar silver nitrate solution 30 min incubated. Then 500 µl 0.14 molar is placed Add dithiothreitol solution and incubate for a further 30 min. To The clear supernatant is centrifuged on Sephadex G 10 desalinated. The fractions contained in the product will be  lyophilized. 4 mg of the conjugate are obtained as white Powder.

e) Tc-99m complex of the conjugate according to Example 14d)

1 mg of conjugate 14d) in 1 ml of 0.1 molar disodium hydrogen phosphate buffer (pH = 9.5) dissolved. After adding 10 mg Disodium tartrate is mixed with sodium pertechnetate solution (1 mCi) and then with 10 ml of stannous chloride solution (5 mg SnCl₂ / 1 ml 0.01 molar HCl). The marking yield (approx. 93%) is determined by HPLC.

Example 15 a) O - {[S- (triphenylmethyl mercaptoacetyl) glycyl glycine amidyl] -6-hex-1-yl} toluenesulfonic acid ester

5.48 g (10 mmol) of the [S- (triphenylmethyl-mercaptoacetyl) -glycyl-glycine-amidyl] -6-hexanol prepared in Example 14b) are dissolved in 100 ml of absolute dichloromethane. 1.01 g (10 mmol) of triethylamine and 1.91 g (10 mmol) of p-toluenesulfonic acid chloride are added and the mixture is stirred at room temperature for 24 h. It is then concentrated and chromatographed on silica gel (eluent: CH₂Cl₂ / MeOH: 95: 5).
Yield: 4.32 g (61.5% of theory), colorless oil
Elemental analysis:
calc .:
C 65.03; H 6.18; N 5.99; O 13.68; S 9.14;
found:
C 64.93; H 6.32; N 5.78; S 8.87.

b) Conjugation of the oligonucleotide 5′-AGGAGGAGGAGGGAGAGCGCAAAUGAGAUU with the connection after Example 15a)

A 30-mer identified by the Selex method Ologonucleotide is generated using an automatic synthesizer Manufactured by Pharmacia (see Oligonucleotides and Analogues, A Practical Approach, Editor F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1991) the oligonucleotide in protected form still on the column of the solid support. The loading of the column is approx. 15 mg of 30mer oligonucleotide. After splitting off the 5′-DMT Protective group (trichloroacetic acid / dichloromethane) is according to the Standard methods with S-trityl-6-mercapto-hexyl-phosphoramidite coupled. After oxidation with iodine in tetrahydrofuran, the Trityl-protected compound split from the carrier, isolated and cleaned (see example 14d).

The separation of the S-trityl protective group, the isolation of the SH- group-bearing oligonucleotide and the purification happens as described under Example 14d) (6 mg).

For coupling, the SH group-bearing 30mer oligonucleotide (6 mg) in 500 µl 0.1 molar sodium carbonate solution Argon atmosphere with 100 mg toluenesulfonic acid ester 15a), dissolved in 500 ul dimethylformamide. After 30 min neutralized and with water to a volume of 5 ml diluted. After centrifugation, the clear supernatant becomes lyophilized.

To split off the S-trityl groups, as in 14d) proceed as described. 4 mg are obtained after purification Conjugate.  

c) Tc-99m complex of the conjugate according to Example 15b)

1 mg of the conjugate described in Example 15b) is described in 1 ml 0.1 molar disodium hydrogen phosphate buffer (pH = 9.5) solved. After the addition of 10 mg disodium tartrate with sodium pertechnetate solution (1 mCi) and then with 10 µl tin-II-chloride solution (5 mg SnCl₂ / 1 ml 0.01 molar HCl). The labeling yield (approx. 95%) is determined by HPLC.

Example 16 a) N- [3-Thia-5- (triphenylmethylmercapto) -1-oxopent-1-yl] -S- triphenylmethyl cysteine methyl ester

2.69 g (10 mmol) of N- [3-thia-5-mercapto-1-oxopent-1-yl] - cysteine methyl ester (preparation according to DE 43 10 999) together with 5.58 g (20 mmol) Triphenylmethyl chloride dissolved in 100 ml of absolute dichloromethane. After addition of 2.02 g (20 mmol) of triethylamine, the mixture is stirred overnight under an argon atmosphere at room temperature. For working up, the organic phase is washed three times with 1% aqueous citric acid solution, saturated sodium bicarbonate solution and with water. After drying over sodium sulfate, the mixture is concentrated and purified on silica gel (eluent: CH₂Cl₂ / MeOH: 95: 5).
Yield: 4.53 g (60.1% of theory) of a colorless oil
Elemental analysis:
calc .:
C 73.27; H 5.75; N 1.86; O 6.37; S 12.76;
found:
C 73.31; H 5.48; N 1.63; S 12.49.

b) N- [3-Thia-5- (triphenylmethylmercapto) -1-oxopent-1-yl] -S- triphenylmethyl-N ′ - (6-hydroxy-hex-1-yl) cysteinamide

7.54 g (10 mmol) of the N- [3-thia-5- (triphenylmethylmercapto) -1-oxopent-1-yl] -S-tri phenylmethyl-cysteine methyl ester described in Example 16a are dissolved in 11.72 g ( 100 mmol) 6-aminohexanol / 50 ml 1,4-dioxane heated to 100 ° C. under an argon atmosphere for 2 h. The reaction mixture is then poured onto a mixture of 100 ml of dichloromethane and 100 ml of water. With stirring and ice cooling, a pH of 6 is set using 10 molar hydrochloric acid, the organic phase is separated off and dried over sodium sulfate 08063 00070 552 001000280000000200012000285910795200040 0002004424922 00004 07944. After evaporation of the solvent, the crude product is purified on silica gel (eluent: CH₂Cl₂ / MeOH: 5% -50%).
Yield: 3.73 g (44% of theory) of a yellowish oil
Elemental analysis:
calc .:
C 72.99; H 6.49; N 3.34; O 5.72; S 11.46;
found:
C 72.73; H 6.62; N 3.11; S 11.17.

c) O - {{[N- [3-thia-5- (triphenylmethylmercapto) -1-oxopent-1- yl] -S-triphenylmethyl-cysteinyl} -2-amino-eth-1-yl} - diisopropylamide-O'-methylphosphoric diester

8.39 g (10 mmol) of the N- [3-thia-5- (triphenylmethylmercapto) -1-oxopent-1-yl] -S-tri phenylmethyl-N ′ - (6-hydroxy) prepared under Example 16b) -hex-1-yl) cysteinamids are dissolved in 200 ml of absolute dichloromethane. 5.17 g (40 mmol) of diisopropylethylamine are added under an argon atmosphere and 3.95 g (20 mmol) of phosphorous acid monomethyl ester diisopropylamide chloride, dissolved in 50 ml of absolute dichloromethane, are added dropwise at 0.degree. The mixture is stirred at 0 ° C. for 0.5 h and then at room temperature for 1.5 h. For working up, 320 mg (10 mmol) of absolute methanol are added while cooling with ice and, after concentration, chromatographed on silica gel (eluent: CH₂Cl₂ / MeOH: 95: 5/5% triethylamine).
Yield: 5.37 g (53.7% of theory) of a yellowish oil

d) conjugation of the oligonucleotide 5′-CUCAUGGAGCGCAAGACGAAUAGCUACAUA with the connection according to example 16c)

A 30-mer identified by the Selex method The oligonucleotide is generated using an automatic synthesizer Manufactured by Pharmacia (see Oligonucleotides and Analogues, A Practical Approach, Editor F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1991) the oligonucleotide in protected form still on the column of the solid support. The loading of the column is approx. 15 mg of 30mer oligonucleotide. After splitting off the 5′-DMT Protective group (trichloroacetic acid / dichloromethane) is according to the Standard methods coupled with phosphoramidite (16c) and then oxidized.

The separation from the carrier, the base protecting groups and the Purification of the bis-S-trityl-protected conjugate happens as described under 14d (approx. 12 mg).

To split off the S-trityl protective groups, the material is in 5 ml 50 mmolar triethylamonium acetate solution (pH = 7.0) recorded and with 500 ul 0.1 molar silver nitrate solution Incubated for 30 min. Then 500 µl 0.14 molar is placed Add dithiothreitol solution and incubate for a further 30 min. To The clear supernatant is centrifuged on Sephadex G 10 desalinated. The fractions contained in the product will be lyophilized. 5 mg of white powder are obtained.  

e) Tc-99m complex of the conjugate according to Example 16d)

1 mg of the conjugate described in Example 16d is described in 1 ml 0.1 molar disodium hydrogen phosphate buffer (pH = 9.5) solved. After the addition of 10 mg disodium tartrate with sodium pertechnetate solution (1 mCi) and then with 10 ml of tin-II-chloride solution (5 mg SnCl₂ / 1 ml 0.01 molar HCl). The labeling yield (approx. 96%) is determined by HPLC certainly.

Example 17 a) O- {N- [3-Thia-5- (triphenylmethylmercapto) -1-oxopent-1- yl] -S-triphenylmethyl-N ′ - (6-hydroxy-hex-1-yl) cysteine imidyl} -p-toluenesulfonic acid ester

8.39 g (10 mmol) of the N- [3-thia-5- (triphenylmethylmercapto) -1-oxopent-1-yl] -S-tri phenylmethyl-N ′ - (6-hydroxy- hex-1-yl) cysteinamids are dissolved in 200 ml of absolute dichloromethane. 1.01 g (10 mmol) of triethylamine, 1.91 g (10 mmol) of p-toluenesulfonic acid chloride are added and the mixture is stirred at room temperature for 20 h. It is then concentrated and chromatographed on silica gel. (Eluent: CH₂Cl₂ / MeOH: 97: 3).
Yield: 6.44 g (67.0% of theory) of a yellowish oil
Elemental analysis:
calc .:
C 72.47; H 6.29; N 2.91; O 8.32; S 10.01;
found:
C 72.19; H 6.47; N 2.68; S 9.83.

b) Conjugation of the oligonucleotide 5′-CUCAUGGAGCGCAAGACGAAUAGCUACAUA with the connection according to example 17a)

A 30-mer identified by the Selex method The oligonucleotide is generated using an automatic synthesizer Manufactured by Pharmacia (see Oligonucleotides and Analogues, A Practical Approach, Editor F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1991) the oligonucleotide in protected form still on the column of the solid support. The loading of the column is approx. 15 mg of 30mer oligonucleotide. After splitting off the 5′-DMT Protective group (trichloroacetic acid / dichloromethane) is according to the Standard methods with S-trityl-6-mercaptohexyl-phosphoramidite coupled.

After oxidation with iodine in tetrahydrofuran, the Trityl-protected compound split from the carrier, isolated and cleaned (see example 14d).

The splitting off of the S-trityl protecting group, the isolation of the SH group-bearing oligonucleotide and purification happens as described under Example 14d) (7.5 mg). For coupling, the SH group-bearing 30mer oligonucleotide (7 mg) in 550 µl 0.1 molar sodium carbonate solution Argon atmosphere with 180 mg of toluenesulfonic acid ester 17a) dissolved in 500 ul dimethylformamide. After 30 min neutralized and with water to a volume of 5 ml diluted. After centrifugation, the clear supernatant becomes lyophilized. How to split off the S-trityl groups proceed as described under 14d). You get after cleaning 4.3 mg conjugate.  

c) Tc-99m complex of the conjugate according to Example 17b)

1 mg of the conjugate described under Example 17b) is in 1 ml 0.1 molar disodium hydrogen phosphate buffer (pH = 9.5) solved. After the addition of 10 mg disodium tartrate with sodium pertechnetate solution (1 mCi) and then with 10 µl tin-II-chloride solution (5 mg SnCl₂ / 1 ml 0.01 molar HCl). The labeling yield (approx. 93%) is determined by HPLC certainly.

Example 18 a) Conjugation of the oligonucleotide according to Example 1a) with N- (tetrahydro-2-oxo-thiophene-3-yl) thiodiglycolic acid monoamide

A 30-mer identified by the Selex method The oligonucleotide is generated using an automatic synthesizer Manufactured by Pharmacia (see Oligonucleotides and Analogues, A Practical Approach, Editor F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1991) the oligonucleotide in protected form still on the column of the solid support. The loading of the column is approx. 15 mg of 30mer oligonucleotide. After splitting off the 5′-DMT Protective group (trichloroacetic acid / dichloromethane) is according to the Standard methods with N-trifluoroacetylaminohexyl phosphoramidite coupled.

After oxidation with iodine in tetrahydrofuran, this becomes cleaved amino group carrying oligonucleotide from the support and isolated and purified as described under 1b (9.3 mg). For coupling with the N- (tetrahydro-2-oxo-thiophene-3-yl) - thiodiglycolic acid monoamide (DE 43 11 023) is dissolved in 5 mg amino group-bearing oligonucleotide in 1 ml 2 molar  Sodium carbonate solution. After adding 100 mg of Thiolactone derivatives are incubated for 4 h at room temperature. It is then neutralized and desalination is carried out Ultrafiltration through a membrane with an exclusion limit reached by 3000 (Amicon YM3). After lyophilizing subject to freeze drying. 6.3 mg are obtained desired conjugate.

b) Tc-99m complex of the conjugate according to Example 18a)

1 mg of the SH group-bearing described under Example 18a) Conjugate is made in 1 ml of 0.1 molar disodium hydrogen phosphate buffer (pH = 9.5) dissolved. After adding 10 mg Disodium tartrate is mixed with sodium pertechnetate solution (1 mCi) and then with 10 µl tin-II-chloride solution (5 mg SnCl₂ / 1 ml 0.01 molar HCl). The marker yield (94%) is determined by HPLC.

Claims (18)

1. Oligonucleotide conjugates consisting of an oligonucleotide residue N and n substituents (BK), wherein B stands for a direct bond or a connecting component to the oligonucleotide residue and K is a complexing agent or complex of radioactive metal isotopes, or stable isotopes, the

• - are converted into radioactive isotopes by external radiation,
• - convert radiation from outside into radiation of different quality, different energy content and / or different wavelength,

of the elements of atomic numbers 5, 21-29, 31, 39, 42-44, 49, 57-83 or 85, characterized in that the oligonucleotide residue N has a modification which prevents degradation by naturally occurring nucleases or at least significantly inhibits. 2. A compound according to claim 1, characterized in that the compound has the general formula N- (BK) _n (I), wherein N is an oligonucleotide which binds specifically with high binding affinity to target structures with the exception of nucleic acids and has modifications which significantly reduce the breakdown by naturally occurring nucleases,
B is a chemical bond or a connecting component that creates the connection between N and K and
K is a complex-forming ligand, which can have a signaling or therapeutically active element and
n is a number between 1 and 10. 3. A compound according to claim 1 or 2, wherein N is an oligonucleotide having 5 to 200 nucleotides, which is stabilized against degradation in that

• a) the hydroxyl group at the 2'-position of the ribose unit is replaced by a group -OR, in which R is an alkyl radical having 1 to 20 carbon atoms, or by a halogen radical, a hydrogen atom or an amine radical and / or
• b) in the phospho diester bridge serving for internucleotide binding, 1 or 2 oxygen atoms are replaced by 1 or 2 sulfur atoms, whereby phosphorothioates or phosphorodithioates are formed and / or
• c) the phosphodiester bridge serving the internucleotide bond is replaced by alkylphosphonates.

4. Connection according to claim 3, characterized in that the oligonucleotide N comprises 15 to 100 nucleotides. 5. Connection according to one of claims 1 to 4, characterized characterized in that N is an oligonucleotide that specifically with high affinity to others Binds target structures and is obtainable by the fact that a mixture of random sequences containing Matched oligonucleotides to the target structure with certain oligonucleotides increased Affinity for the target structure in relation to the mixture of the oligonucleotides, these from the rest of the Oligonucleotide mixture are separated, then the Oligonucleotides with increased affinity for the target structure be amplified to a mixture of oligonucleotides  to get an increased proportion of oligonucleotides that bind to the target structures. 6. A compound according to any one of claims 1 to 5, characterized in that N is an oligonucleotide which binds specifically with high binding affinity to other target structures with the exception of nucleic acids and which is obtainable in that

• a) a DNA strand is first prepared by chemical synthesis in such a way that this DNA strand has a defined sequence at the 3'-end which is complementary to a promoter for an RNA polymerase and at the same time complementary to a primer of the polymerase chain reaction (PCR) and that this DNA strand at the 5'-end has a defined DNA sequence which is complementary to a primer sequence for the polymerase chain reaction, the sequence between the defined sequences containing a random sequence and that
• b) this DNA strand is rewritten with the aid of an RNA polymerase into a complementary RNA strand, the polymerase being offered nucleotides which are modified at the 2'-position of the ribose unit and that
• c) the RNA oligonucleotides produced in this way are brought together with the target structure to which the oligonucleotide is to bind specifically, and that
• d) those oligonucleotides which have bound to the target structure are first separated from the non-binding oligonucleotides together with the target structure and then the bound oligonucleotides are separated again from the target structure and that
• e) these target structure-specific RNA oligonucleotides are transcribed into a complementary DNA strand with the aid of the reverse transcriptase and that
• f) these DNA strands are amplified using the defined primer sequences with the polymerase chain reaction and that
• g) the DNA oligonucleotides amplified in this way are then rewritten with the aid of RNA polymerase and with modified nucleotides in RNA oligonucleotides and that
• h) the above-mentioned selection steps c) to g) are repeated as often as necessary until the oligonucleotides, which are characterized by a high affinity for binding to the target structure, have been sufficiently selected and the sequences of the oligonucleotides thus obtained can then optionally be determined.

7. Connection according to claim 6, characterized in that the target structure is selected from macromolecules, Tissue structures from higher organisms such as animals or People, organs or parts of organs of an animal or of humans, cells, tumor cells or tumors. 8) Connection according to one of claims 1 to 7, characterized in that the connection component B

• a) at the 4'-end of the oligonucleotide residue N reduced in the 4'-position by the CH₂-OH group,
• b) at the 3'-end of the oligonucleotide residue N reduced by one hydrogen atom in the 3'-position,
• c) to the phospho diester bridge (s) reduced by the OH group (s) between two nucleotides and / or
• d) at 1 to 10 nucleobase (s), which are each in the position (s) 5, 8 and / or the amino group (s) in position (s) 2, 4 and 6 reduced by one hydrogen atom (are) ),

is (are) bound. 9. A compound according to claim 8a) or 8b), characterized in that B has the general formula XY-Z¹, which is connected on the X side with the complexing agent or complex and on the Z side with the nucleotide, wherein
X represents a direct bond, an -NH or -S group,
Y represents a straight-chain, branched-chain, saturated or unsaturated C₁-C₂₀ alkylene chain, which optionally 1-2 cyclohexylene, 1-5 imino, 1-3 phenylene, 1-3 phenyleneimino, 1-3 phenyleneoxy , 1-3 hydroxyphenylene, 1-5 amido, 1-2 hydrazido, 1-5 carbonyl, 1-5 ethyleneoxy, a ureido, a thioureido, 1-2 carboxyalkylimino, 1-2 ester groups 1-3 groups Ar, in which Ar stands for a saturated or unsaturated 5- or 6-ring, which optionally contains 1-2 heteroatoms selected from nitrogen, oxygen and sulfur and / or 1-2 carbonyl groups, 1-10 oxygen Contains 1-5 nitrogen and / or 1-5 sulfur atoms and / or optionally by 1-5 hydroxy, 1-2 mercapto, 1-5 oxo, 1-5 thioxo, 1-3 carboxy, 1 -5 carboxy-C₁-C₄-alkyl, 1-5 ester, 1-3 amino, 1-3 hydroxy-C₁-C₄-alkyl, 1-3 C₁-C₇-alkoxy groups is substituted and
Z¹ for -CONH-CH₂-4 ′, -NH-CO-4 ′, -OP (O) R¹-NH-CH₂-4 ′, -OP (O) R¹-O-CH₂-4 ′ or -OP (O ) R¹-3 ′ where 4 ′ or 3 ′ indicates the link to the terminal sugar unit (s) and R¹ is O⁻, S⁻, a C₁-C₄-alkyl or NR²R³ group with R² and R³ in the meaning of hydrogen and C₁-C₄ alkyl radicals. 10. A compound according to claim 8c), characterized in that B has the general formula XY-Z², wherein
Z² in the bridge linking two neighboring sugar units represents the group -NR²-, -O- or -S- and X, Y and R² have the meaning given in claim 9. 11. Connection according to claim 8d), characterized in that B has the general formula X-Y-Z³, where Z³ for a -NH group on or a direct link to the nucleobase and X and Y have the meaning given in claim 9 to have. 12. Connection according to one of the preceding claims characterized in that the metal complex as imaging element a radioactive isotope, selected from the elements copper, bismuth, technetium, rhenium or Contains indium. 13. Procedure for the detection of a target structure thereby characterized in that one or more of the compounds  according to one of the preceding claims with the to brings together the investigating sample in vivo or in vitro and uses the signal to prove whether the target structure is on which the oligonucleotide N specific and with high Binding affinity binds in the sample. 14. Procedure for the non-invasive diagnosis of Diseases, characterized in that one or several of the compounds according to one of claims 1 to 12 with the target structure to be investigated in vivo matches and uses the signal to prove whether the Target structure to which the oligonucleotide N is specific binds in the organism to be examined. 15. Use of a compound according to one of claims 1 to 12 in radio diagnostics and / or in Radiotherapy. 16. Diagnostic kit for the detection of target structures in vivo and / or in vitro, characterized in that the Diagnostic kit at least one connection according to one of the Claims 1 to 12.