AU2036099A - Conjugates made of metal complexes and oligonucleotides - Google Patents

Description

Our Ref: 722100 P/00/011 Regulation 3:2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): Schering Aktiengesellschaft Mullerstrasse 178 D-13353 Berlin

GERMANY

Nexstar Pharmaceuticals, Inc.

2860 Wilderness Place Boulder Colorado 80301 UNITED STATES OF AMERICA Address for Service: Invention Title: DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 Conjugates made of metal complexes and oligonucleotides The following statement is a full description of this invention, including the best method of performing it known to me:- 5020 WO 96/02274 PCT/EP95102539 CONJUGATES MADE OF METAL COMPLEXES AND OLIGONUCLEOTIDES This invention relates to the object characterized in the claims, i.e., oligonucleotide conjugates, which contain a complexing agent or a complex. These conjugates are used in the fields of diagnosis and treatment.

The imaging diagnosis has achieved great progress in the past decades and is continuously further developing. It is now possible to make visible the vascular system, most organs and many tissues in the living body without major intervention.

Diseases are diagnosed in many cases, because they lead to clear changes of shape, size and position of anatomical structures in the body. Such anatomical data from the inside of the body can be obtained by x-ray technology, ultrasonic diagnosis and magnetic resonance tomography. The efficiency of each of the mentioned technologies can be improved by the use of pharmaceutical agents for enhancement of the natural contrast of the tissues and body fluids in the resulting picture. The pharmaceutical agents in question are introduced in body cavities or injected in blood vessels, with the purpose of changing the contrast of the cavities or vessels. In addition, they are spread by the bloodstream in the organism and can change the visibility of organs and tissues. In exceptional cases, such substances are bound to certain structures in the body and/or actively transported and/or excreted by the latter. In this way, functions can also be made visible in individual cases and used to diagnose diseases.

In contrast to that, the nuclear diagnosis is based on substances that can themselves be made visible. In this case, radioactive isotopes, which emit long-range radiation, are introduced in the body. The spreading of these substances in the organism can be tracked by suitable detectors. The advantage of the nuclear medical process is the high effectiveness at low dosage of the signal-transmitting radioactive substances designated as radiopharmaceutical agents.

If isotopes are used, which release a- or P-radiation or other toxic decomposition products effective in the tissue, radiopharmaceutical agents can also be used for therapeutic purposes, for destruction of tumors. The same end can also be achieved in that nonharmful isotopes or substances are introduced in the body and converted only there by, neutron or x-ray radiation, ultrasound or radio waves, to a therapeutically effective form.

A general problem is the diagnosis and localization of pathological changes at a time at which no clear changes of shape, structure and circulation of the organs and tissues in question are available. Such a diagnosis and follow-up is of decisive QI1 TIT IMrr CUr-r 1r1#I r- WO 96/02274 PCT/IEP95/02539 -2importance, in the case of tumor diseases, including the search for metastases, assessment of a deficient supply of tissues with oxygen and in the case of certain infections as well as metabolic diseases.

The now available therapeutic and imaging diagnostic methods are considerably dependent on the availability of pharmaceutical preparations, which accumulate at sites of otherwise undetectable pathological changes.

The contrast media available commercially at this time are quite predominantly so-called nonspecific preparations. They spread passively in the spaces in which they are introduced, by injection.

In the past, many substances and substance classes have been identified that can detect or can be expected to have a specificity with respect to their spreading in the living organism. Examples in this respect are, in addition to the antibodies, lectins, all types of receptor-bound substances, cells, membranes and membrane components, nucleic acids, natural metabolites and their derivatives, as well as countless 'pharmaceiitical substances. Peptides have been and are also being studied with special I US atcnt No. 4,707,352 deals with a special process to label complexing oleculeswith radioactive isotopes, but no ell-suited complexing agents for the bonding of metal ions are described.

EP-A-0 285 057 describes nucieotide-complexing agent conjugates, which are not suitable; because of the in vivo instability of the nucleotides used, for use as in vivo diagnostic agents or therapeutic agents and also hardly meet the other requirements of compatibility and pharmacokinetics.

Many US patents, such as, for example, US Patent No. 4,707,440, deal with modified polymers, which contain a detectable chemical group. The polymers can be polynucleotides and oligonucleotides, but they are neither stabilized against a degradation by naturally occurring nucleases nor selected by a special process, so that they bond specifically with high bonding affinity to target structures. Special embodiments Sof these detectable molecules are mentioned in US Patents No. 4,843,122 and 4,943,523. An individual nucleotide, modified in this way, is claimed in US Patent No. 4,952,685. The use of these agents in imaging processes is disclosed in US Patent No. 4,849,208.

The object of this invention is the preparation of specifically bonding diagnostic agents for the detection of target structures, by which, for example, the visualization of organs, tissues and their pathological changes in vitro and in vivo is made possible.

It has now been found that this object is achieved by oligonucleotide conjugates, which in addition to an oligonucleotide radical exhibit a complexing agent, P:\WWDOSCRN\SPBC 2 6 M2sE 29/1/99 -3bound by a direct bond or a connecting component, and whose oligoneucleotide radical is modified so that the degradation by naturally occurring nucleases is prevented or at least significantly inhibited.

Object of the invention are: S1. Oligoneuclotide conjugates consisting of an oligonucleotide radical N and n substituents in which n is a number between 1 and 10, B stands for a direct bond or a connecting component to the oligoneucleotide radical, and K means a complexing agent or complex of radioactive metal isotopes, or stable isotopes, which are converted by radiation from outside to radioactive isotopes, convert radiation from outside to radiation of different quality, different energy content and/or different wavelength, of elements of atomic numbers 5, 21-29, 31, 39, 42-44, 49, 57-83 or 85, characterized in that oligonucleoide radical Nexhibits a modification, which prevents or at least significantly inhibits the degradation by naturally occurring nucleases and in which oligonucleotide bonds specifically with high bonding affinity to a target structure with the proviso that the oligoneucleotide radical N is obtained by a process of identifying a nucleic acid ligand of a target from a candidate mixture of nucleic acids, said process comprising:ap contacting the candidate mixture with said target volume; partitioning the nucleic acids having an increased affinity to the target molecule relative to the candidate mixture; and amplifying the increased affinity mixture of nucleic acids, whereby nucleic acid ligands of the target molecule are identified.

2. In a preferred embodiment, the oligoneucleotide conjugates of this invention exhibit general formula

(I)

in ich N is an oligocleotde, w h i ch in which N is an oligonuleotide which bonds specifically with high bonding affinity to a target structure and exhibits modifications that significantly reduce the degradation by naturally B is a chemical bond or a connecting component, which produces the connection between N and K, and K ve is a complexing ligand, which can contain a signal-transmitting or therapeutically active element, and n is a number between 1 and with the proviso thatthe oligoneucleotide radical N is btained by a poces f identifying a nucleic acid ligand of a target from a candidate mixture of nucleic acids, said process comprising: a) contacting the candidate mixture with said target molecule, b) artitioning the nucleic acids having an increased affinity to the target molecule relative P:\WPDOCS\CRN\SPECA620W6SPE 2911/99 -3ato the candidate mixture; and c) amplifying the increased affinity mixture of nucleic acids, whereby nucleic acid ligands of the target molecule are identified.

3. Compound according to 1 or 2, in which N is an oligonucleotide with to 200 nucleotides, wherein a) the position of the sugar unit, independently of one another, is occupied by the following groups: a group OR, in which R means an alkyl radical with I to 20 carbon atoms, which optionally contains up to 2 hydroxyl groups and which optionally is interrupted by 1-5 oxygen atoms, a hydrogen atom, a hydroxyl group, S ,a .fluorine atom, anr aminogroup, WO 96/02274 PCT/EP95/02S39 -4and hydroxyl groups present in terminal positions 3' and independently of one another, optionally are etherified with radical R and/or b) the phosphodiesters, optionally being used as the internucleotide bond, independently of one another, are replaced by phosphorothioates, phosphorodithioates or alkylphosphoates, preferably methyl phosphonate, and/or c) the terminal radicals in and 5'-positions are linked in an intramolecular manner with one another by an internucleotide bond as described in b) and/or d) it contains an internucleotide bond as described in which links or and/or e) it contains a phosphodiester bond as described in which connects, esterlike, two thymidines by a C 2 -C2 hydroxyalkyl radical respectively in 3-position or connects an analogously substituted thymidine radical, esterlike, with a hydroxyl group of another sugar in or or 5'-position and/or 0 the terminal radicals in and 5'-positions contain internucleotide bonds optionally modified as described in b).

4. Compound according to 3, wherein oligonucleotide N comprises 15 to 100 nucleotides.

Compound according to anyone of points 1 to 4, wherein N is an oligonucleotide, which bonds specifically with high bonding affinity to a target structure and which can be obtained in that a mixture of oligoneucleotides containing random sequences is brought together with the target structure, wherein oligonucleotides which exhibit an increased affinity to the target structure relative to the mixture of the oligonucleotides are separated from the remainder of the oligonucleotide mixture, then the oligoneucleotides with increased affinity to the target structure are amplified to obtain a mixture of oligoneucleotides that exhibits an increased portion of oligonucleotides that bond on the target structures.

6. Compounds as described in anyone of points 1 to 5, wherein N is an oligonucleotide, which specifically bonds with high bonding affinity to a target structure, and which can be obtained in that a) first, a DNA strand is produced by chemical synthesis, so that on the 3'end, this DNA strand exhibits a defined sequence, 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 so that this DNA strand exhibits a defined sequence on the 5'-end, which is complementiry to a primer sequence for the polymerase chain reaction, and the sequence between thedefined sequences contains a random sequence, and in that WO 96/02274 PCTAEP9/02539 b) this DNA strand is transcribed in a complementary RNA strand with the help of an RNA-polymerase, and nucleotides are offered to the polymerase, which are modified in the 2'-position of the ribose unit, and in that c) the RNA oligonucleotides, produced in this way, are brought together with the target structure on which the oligonucleotide specifically is to bond, and in that d) those oligonucleotides that have bound on the target structure are separated first together with the target structure from the nonbinding oligonucleotides and then the bound oligonucleotides are separated again from the target structure, and in that e) these target-structure-specific RNA oligonucleotides are transcribed with the to help of reverse transcriptase in a complementary DNA strand, anti in that f) these DNA strands are amplified with the polymerase chain reaction with use of the defined primer sequences, and in that g) the DNA oligonucleotides amplified in this manner are then transcribed again with the help of the RNA-polymerase and with modified nucleotides in RNAoligonucleotides, and in that h) above-mentioned selection steps c) to g) optionally are repeated often until the oligonucleotides, which are characterized by a high bonding affinity to the target structure, are sufficiently selected, and then the sequences of the thus obtained oligonucleotide optionally are able to be determined.

7. Compound according to 6, wherein the target structure is selected among macromolecules, tissue structures of higher organisms, such as animals or humans, organs or parts of organs of an animal or human, cells, tumor cells or tumors.

8. Compound according to anyone of points 1 to 7, wherein connecting component(s) B is (are) bound a) to the 4'-end of oligonucleotide radical N reduced in 4'-position by the

CH

2 -OH group and/or b) to the 3'-end of oligonucleotide radical N reduced in 3'-position by a hydrogen atom and/or c) to the phosphodiester bridge(s), reduced by the OH group(s), between two nucleotides each and/or d) to 1 to 10 nucleobase(s), which is (are) reduced by a hydrogen atom respectively in 8-position(s) and/or the amino group(s) in 4- and 6-position(s).

9. Compound according to point 8a) or 8b), wherein B has general formula X-

Y-Z

1 which is connected on the X side with the complexing agent or complex and on the Z side with the oligonucleotide, in which X-stands for a direct bond, an -NH or -S group, Y stands for a straight-chain, branched-chain, saturated or unsaturated CI-C 20 alkylene chain; which optionally contains WO %102274 PCrM"M2539 -6- 1-2 cyclohexylene, 1-5 irnino, 1-3 phenylene, 1-3 phenylenimino, 1-3 pheim1enoxy, 1-3 hydroxyphenylene, 1-5 amido, 1-2 hydrazido, 1-5 carbonyl, 1-5 ethylenoxy, a ureido, a thioureido, 1-2 carboxyalkylimino, 1-2 ester groups, 1-3 groups of Ar, in whiichbAr stands for saturated or unsaturated 5- or 6- memnbered ring, which optionally contains 1-2 heterotaonis selected from nitrogen, oxygen and sulfur and/or 1-2 carbonyl groups; 1 -10 oxygen 1-5 nitrogen and/or 1-5 sulur atoms, and/or optionally is substituted by 1-5 hydroxy, 1-2 merc~to, 1-5 oxo, dhioxo, 1-3 carboxy, 1-5 carboxy-C 1

-C

4 alkyl, 1-5 ester, 1-3 amino, 1-3 hydroxy-C 1

-C

4 alkYl, 1-3 C,-C 7 alkoxy groups, and

Z

1 stands for -CONH-CH 2 -NH-CO-41, -0-P(O)R'-NH-CH 2 -O-P(0)R 1

-O-CH

2 -0-P(S)R 1 or in which 4' or 3' indicates the linkage to the terminal sugar unit(s) and R 1 stands for 0% a C 1

-C

4 alkyl or WO 2 R group, with e 2 and R 3 meaning hydrogen and C 1

-C

4 allkyl radicals.

As cyclic stnicture especially cyclic saturated or unsaturated alkylenes with 3 to 6, especialy or 6 C atoms, which optionally contain heteromxns, such -as N, S or 0, are suitable. As examples, there can be mentioned: cyclopetylene, pyrrolylene, furanylene, thiop henylene, imidaolylene, oxAzolylidene, thiazolylene,, pyrazolylene, pyrrolidylene, pyridylene, pyrimidylene, maleinimidylce and phtbalimidylene groups.

compound according to 8c), wherein B has general formula X-Y-Z 2 which is connecte on the X side with the complexing agent or complex and on the Z side with the oligonucleotide, in which zin the bridge linking two adjacent sugar units, 0 S ZP-0- S' and/or Z P-0 3' 3' stands for the group or and X, Y and R 2 have the meaning indicated in point 9.

As radicals Y of connecting component Z 1 -Y-X (according to point 9) or. 2Y X (according to point 10), there can be mentioned as examples the radicals -(CH2) 6

-NI{-CS-NH-C

6 Rr-CH(CH 2

CO

2

H)-CH

2

-CO-NH-CH

2

-CH(OH)-CH

2 .0

-(CH

2 6

-NH-CS-NH-C

6

H

4 -C1 2

-(CH

2 6 -NH-C 0-CH 2

-(CH

2 6

-NH-CO-CH

2

-CH

2

-(CH

2 2

-(CH

2 6

-(CH

2 6

-S-(CH

2 2 -(CI{2) 6

-S-

(CH

2 6 -(CH2)2-NH-CO-, -(CH 2 6

-NH-CO-,

WO 96/02274 PCTIEP95102539 PCTAEP9=/2539 -7-

-(CH

2 )6-S-(CH 2 )-NH-CO, -(CH2)6-S-(CH 2 )6-NH-CO-,

(CH

2 6

-S-CH-CH

2 -CO-N- (CH 2 5-NH-NH-CO-CH 2 -0-C 6

H

4

C

2

-(C

2 6 -S-CH-CH 2 -CO-N- (CH 2 2-0- (CH 2 2 -NH-CO-CH2 (CH2) 6

-S-CH-CH

2 -CO-N- (CHM2 2)-Co-NH-

(CH

2 3

(CH

2 6

-S-CH-CH

2 -CO-N-

(CH

2 2 -CO-NH-

(CH

2 4 (cmH)

(CH

2 2 -c=-CC=CH -o (cH 2 or 2 -CH s CH)-C=CH-CH C- CH -O-CH2-CO-NH-CH2 6CH3(OH)- -c- 2 6 -_S7 (CH 2 2 N~ 11. C mpound according to 8d), wherein B has general formula X-Y-Z3, in which Z 3 stands for an -NH group or a direct bond to the nucleobase and X and Y have the meaning indicated in claim 9.

There can be mentioned as examples the radicals -CH2-CO-NH-CH 2

-CH(OH)-CH

2

NH-CO-CH

2

-CO-NH-CH-CH(OH)-CH

2 to -CO-NH-CH 2

-CH

2 -CH2-S-CH 2

-CH

2

-CH

2

-S-CH

2

-CH

2 -(CH2) 4

-S-CH

2

-CH

2

-CO-CH

2

-S-CH

2

-CH

2

-CO-CH

2

-S-(CH

2 6

CH=CH-CO-NH-CH

2

-CH

2

-CH=CH-CH

2

-NH-,

-C2C-CH 2 -NH- or -CO-CH2-CH 2

-NH-CH

2

-CH

2

-NH-.

As bonding sites in the case of the purine bases, especially 8-position is suitable, and in the case of the pyrimidine bases, 5-position is suitable. Purely formally, in this case, a hydrogen atom of the respective base is substituted by radical B- K. But a linkage can also take place by amino groups optionally contained in 4- or 6 -position, thus, by the 2-amino group in guanine, by the 6-amino group in adenine or by the 4-amino group in cytosine. In this case, a hydrogen atom of the respective amino group is respectively substituted by radical B-K.

12. Compounds according to any one of the preceding points, wherein the metal complex, which acts as an imaging element, contains a radioactive isotope, selected from the elements copper, bismuth, technetium, rhenium or indium.

13. The invention also comprises a process for detecting a target structure, wherein one or more of the compounds according to any one of the preceding points are Wo 002274 brought together with the sample to be studied in vivo or in vitro and based on the signal, it is detted whether the target structue, on which oligonecled N bonds specificaly with high bondin~g affinity, is present in the sample, being studied, as weil as a 14. Process for noninvasive diagnosis of diseases, wherein one or more of the omipoundjs according to anyone of points I to 12 is brought together with the target structure to b~e studied in vivo and based on the signal, it is detected whether the target structure, on which 01i9onueclotd N specificaly bonds, is present In the organism to be studied.

The object of the invention is also the use of a compojud according to anyone of points I to12 nroiansior nradrthey, swellas 16. Diagnosis kit for in vivo and/or in vitro dettion of target stuctures, wherein the di agnosis kit contains at least one Compond according to anyone of points I to 12.

Mf the copgae according to the invention arc to be used asa diagnostic agnt, the complexig agents) contain (COMMai) an Imgig rdociIsotope Of the OlCeMtS Of atomic numbers 21, 26-27, 29, 31, 43 or 49, Preferably 43 or 49. If the c-Onjugates according to the invention are to be Used as a. therapuic agent, besides the IbOVe-mentioed, in addition isotopes of the ele'Ments of atomic nunjbers5, 22-25, 28, 42, 44, 57-83 and 85 are also suitable. Beyond .the radioactive Isotopes of t .he above- Mentionedl elements, especially also stable isotopes, which a) are convertedi by radiation from outside to radioactive isotopeS. b) convert radiation from Outside to radiation of different quality, different Mrgy content and/or different wavelength, *re suitable in the range of the treatment.

The numbier of imaging or therapetiticallY effective substituents B-K linked With the oligonucleotidje radical is, on the one hand, limited by the 'Value of the oligo.

oucleotide, but is never greater than 10. According to the invention, one or two mubstituents; B-K are preferred.

The value of oligonucleotide radical N in principle is not limited. For this Ovention, oligonucleotides with 5 to 200 nucleotides are Practicable, especially pecferred are oligonucleotjdes with 15 to 100 nucleotides.

WO 96/02274 PCT/EP95/02539 -9- Oligonucleotides usable according to the invention are stabilized against degradation by nucleases occurring in vivo.

Unmodified oligonucleotides or polynucleotides are cleaved in vivo by endonucleases and exonucleases. The degradation reaction in the RNA series begins with an activation of the 2'-hydroxy group. Other catabolic enzymes are, e.g., ribozymes, which cleave the phosphodiester bond of RNS (see Science 261, 709 (1993)). The in vivo stability of RNS derivatives can be increased by partial or complete substitution of the 2'-hydroxyl group by other substituents. Such substituents are, alkoxy groups, especially the methoxy group (see, Chem.

Pharm. Bull. 13, 1273 (1965), Biochemistry 10, 2581, a hydrogen atom, a fluorine atom (see Can. J. Chem. 46, 1131 (1968)) or an amino group (see, e.g., J. Org. Chem. 42, 714 (1977)). Several of these substituents, as well as others, can also be introduced at the 2'-position using the methods disclosed in U.S. application Ser. No. 08/264,029, filed June 22, 1994. Other possibilities for stabilizing the internucleotide bond are the replacement of one or two oxygen atoms in the phosphodiester bridge while forming phosphorothioates (Trends Biochem. Sci. 14, 97 (1989)) or phosphorodithioates Chem. Soc., Chem. Commun. 591 (1983) and Nucleic Acids Res. 12, 9095 (1984)) and the-use' of alkylphosphonates instead of phosphodiesters (Ann. Rep. N. Y. Acad. Sci. 5i02, 220 (1988)).

The stabilization can be achieved in that the hydroxyl groups in 2'-position of the ribose units, independently of one another, are modified. Such a modification can be achieved by a replacement of this hydroxyl group by an OR group, a halogen atom, especially a fluorine atom, a hydrogen atom or an amine radical, especially by an amino group. Radical R of the alkoxy group stands, in this case, for a straight-chain or branched alkyl radical with 1 to 20 C 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 C atoms, such as cyclopentyl or cyclohexyl, which optionally contain 1-2 hydroxy groups, and optionally is interrupted by 1-5 oxygen atoms. The stabilization is also increased because the present hydroxyl groups in 3'and 5'-positions optionally are etherified.

Another stabilization of the polynucleotide takes place in that the phosphodiesters being used as internucleotide bond are replaced partially or completely, and independently of one another, by phosphorothioates, phosphorodithioates or alkylphosphonates, especially preferably by lower alkylphosphonates, such as, methyl phosphonate. These internucleotide bonds can also be linked to the terminal radicals in and 5'-positions or else also connect or 5'-5'-positions. The phosphodiester bond makes possible further linkages by hydroxyalkyl radicals, which are present on nitrogen or carbon atoms of the nucleobases, thus, for example, two thymidines can be linked by the hydroxyalkyl chains present in 3-position or two purine bases by the radicals present in 8-positions. The linkage can also take place to hydroxyl groups in or or 5 '-position.

The modified internucleotide bonds can optionally occur preferably at the ends of the polynucleotide, and they are especially preferably bound to the thymidine.

According to the invention, oligonucleotide radicals N used are not limited to specific oligonucleotide sequences. But preferred are those oligonucleotides that bond specifically with high bonding affinity to taiget structures with the exception of nucleic acid.

A process for identifying suitable oligonucleotides, which are required as initial substances for the conjugates according to the invention, is described in U.S. Patent 5,270,163. This process, termed SELEX, can be used to make a nucleic acid ligand to any desired target molecule.

The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligandenriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule.

The basic SELEX method has been modified to achieve a number of specific objectives.

a~ -11- U.S patent No. 5,496,938, filed October 21, 1992, describes methods for obtaining improved nucleic acid ligands after SELEX has been performed. U.S patent No. 5,705,337, filed March 8, 1995, describes methods for covalently linking a ligand to its target.

The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics.

Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base substitutions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed September 8, 1993, that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2 '-positions ofpyrimidines. U.S. patent application Ser. No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino 2'-fluoro and/or 2 '-O-methyl U.S. patent application Ser. No. 08/264,029, filed June 22, 1994, describes oligonucleotides containing various 2'-modified pyrimidines.

The SELEX method encompasses combining selected oligoneucleotides with other selected oligoneucleotides and non-oligoneucleotide functional units as described in U.S patent No. 5,637,459 filed August 2, 1994, and No. 5,683, 867, filed April 28, 1994, respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligoneucleotides with the desirable properties of other molecules.

In its most basic form, the SELEX process may be defined by the following series of steps: 1) A candidate mixture of nucleic acids of differing sequence is prepared. The candidate mixture generally includes regions of fixed sequences each of the .members of the candidate mixture contains the same sequences in the same location) and regions of randomized sequences. The fixed sequence regions are selected either: to assist in the amplification steps described below, to mimic a sequence known to bind to the target, or to enhance the concentration of a given structural arrangement of the nucleic acids in the candidate mixture. The randomized sequences can be totally randomized the probability of finding a base at any position being one in four)-or only partially randomized the probability of finding a base at any location.can be selected at any level between 0 and 100 percent).

2) The candidate mixture is contacted with the selected target under conditions favorable for binding between the target and members of the candidate mixture.

Under these circumstances, the interaction between the target and the nucleic acids of WO 96/02274 PCTIEP95/02539 -12the candidate mixture can be considered as forming nucleic acid-target pairs* between the target and those nucleic acids having the strongest affinity for the target.

3) The nucleic acids with the highest affinity for the target are partitioned from those nucleic acids with lesser affinity to the target. Because only an extremely small number of sequences (and possibly only one molecule of nucleic acid) corresponding to the highest affinity nucleic acids exist in the candidate mixture, it is generally desirable to set the partitioning criteria so that a significant amount of the nucleic acids in the candidate mixture (approximately 5-50%) are retained during partitioning.

4) Those nucleic acids selected during partitioning as having the relatively higher affinity to the target are then amplified to create a new candidate mixture that is enriched in nucleic acids having a relatively higher affinity for the target.

By repeating the partitioning and amplifying steps above, the newly formed candidate mixture contains fewer and fewer unique sequences, and the average degree of affinity of the nucleic acids to the target will generally increase. Taken to its extreme, the SELEX process will yield a candidate mixture containing one or a small number of unique nucleic acids representing those nucleic acids from the original candidate mixture having the highest affinity to the target molecule.

The SELEX patents and applications describe and elaborate on this process in great detail. Included are targets that can be used in the process; methods for partitioning nucleic acids within a candidate mixture; and methods for amplifying partitioned nucleic acids to generate enriched candidate mixture. The SELEX patents and applications also describe ligands obtained to a number of target species, including both protein targets where the protein is and is not a nucleic acid binding protein.

Therefore, the SELEX process can be used to provide high affinity ligands of a target molecule.

Target molecules are preferably proteins, but can also include among others carbohydrates, peptidoglycans and a variety of small molecules. As with conventional proteinaceous antibodies, nucleic acid antibodies (oligonucleotide ligands) can be employed to target biological structures, such as cell surfaces or viruses, through specific interaction with a molecule that is an integral part of that biological structure.

Oligonucleotide ligands are advantageous in that they are not limited by self tolerance, as are conventional antibodies. Also nucleic acid antibodies do not require animals or cell cultures for synthesis or production, since SELEX is a wholly in yjtro process. As is well-known, nucleic acids can bind to complementary nucleic acid sequences. This property of nucleic acids has been extensively utilized for the detection, quantitation and isolation of nucleic acid molecules. Thus, the methods of the present invention are not intended to encompass these well-known binding capabilities between nucleic acids. Specifically, the methods of the present invention related to the use of nucleic WO 96/02274 PCT/EP95/02539 -13acid antibodies are not intended to encompass known binding affinities between nucleic acid molecules. A number of proteins are known to function via binding to nucleic sequences, such as regulatory proteins which bind to nucleic acid operator sequences. The known ability of certain nucleic acid binding proteins to bind to their natural sites, for example, has been employed in the detection, quantitation, isolation and purification of such proteins. The methods of the present invention related to the use of oligonucleotide ligands are not intended to encompass the known binding affinity between nucleic acid binding proteins and nucleic acid sequences to which they are known to bind. However, novel, non-naturally-occurring sequences which bind to the same nucleic acid binding proteins can be developed using SELEX. In particular, the oligonucleotide ligands of the present invention bind to such target molecules which comprise a three dimensional chemical structure, other than a polynucleotide that binds to said oligonucleotide ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein said oligonucleotide ligand is not a nucleic acid having the known physiological function of being bound by the target molecule.

It should be noted that SELEX allows very rapid determination of nucleic acid sequences that will bind to a protein and, thus, can be readily employed to determine the structure of unknown operator and binding site sequences which sequences can then be employed for applications as described herein. SELEX is dius a general method for use of nucleic acid molecules for the detection, quantitation, isolation and purification of proteins which are not known to bind nucleic acids. In addition.

certain nucleic acid antibodies isolatable by SELEX can also be employed to affect the function, for example inhibit, enhance or activate the function, of specific target molecules or structures. Specifically, nucleic acid antibodies can be employed to inhibit, enhance or activate the function of proteins.

The oligonucleotides used in the conjugates according to the invention are Sobtained in a preferred embodiment according to the process described below.

Thus, suitable oligonucleotides can be obtained in that a mixture of oligonucleotides containing random sequences is brought together with the target structure, and certain oligonucleotides exhibit an increased affinity to the target structure relative to the mixture of the oligonucleotides, the latter are separated from the remainder of the oligonucleotide mixture, then the oligonucleotides with increased affinity to the target structure are amplified to obtain a mixture of oligonucleotides that exhibits an increased portion of oligonucleotides that bond to the target structures.

In the process, first a DNA strand is produced in a preferred way by chemical synthesis. On-the 3'-end, this DNA strand has a known sequence, which is used as promoter for an RNA polymerase and at the same time is complementary to a primer WO 96/02274 PCT/EP95/02539 -14sequence for the polymerase chain reaction (PCR). In an especially preferred embodiment, in this case,. the promoter for the T7 RNA-polymerase is involved.

Then, a random sequence is synthesized on the promoter. The random sequence can be obtained in that the suitable four bases are fed in the same ratio to the synthesis machine. Thus, completely random DNA sequences result. In a preferred embodiment, the length of the random sequence is about 15 to 100 nucleotides.

Another DNA sequence, which can be used for the polymerase chain reaction (PCR), is synthesized on this DNA piece with the random sequence.

After synthesis of this DNA strand, the latter is transcribed in a complementary RNA strand with the help of an RNA polymerase. In a preferred embodiment, the T7 RNA polymerase is used in this case. In the transcription, nucleotides that are modified are offered to the RNA polymerase. In an especially preferred embodiment, the ribose is modified in 2'-position. In this case, a substitution of the hydrogen atom :or the hydroxyl group by an alkoxy group, preferably methoxy, amino or fluorine, can 'be involved. The RNA oligonucleotides produced in this manner are then introduced in the selection process.

In the selection process, the RNA oligonucleotides are brought togethcr with the ta,Get structure. Target structure is understood to mean a structure on whir-- the oligonucleotide is to bond specifically and with high affinity.

Such structures are, macromolecules, tissue structures of higher organisms, such as animals or humans, organs or parts of organs, cells, especially tumor cells or tumors.

In this connection, the target structure must not absolutely be in pure form, it can also be present on a naturally occurring organ or on a cell surface. Stringency may applied to the selection process by the addition of polyamino (tRNA, heparin), plasma or whole blood to the SELEX reaction.

If an isolated protein is involved here, the latter can be bound to a solid phase, for example, a filter. In the selection, an excess of the target structure relative to the RNA mixture is used. In the incubation, the specific oligonucleotide molecules bond on the target structures, while the unbound oligonucleotides are separated from the mixture, for example by washing.

Then, the oligonucleotide molecules are separated from the target molecules or removed by washing with suitable buffers or solvents.

With the help of the reverse transcriptase, the RNA oligonucleotide found is transcribed in the complementary DNA strand.

Since the DNA strand obtained exhibits primer sequences (or promoter sequences) on.both ends, an amplification of the DNA sequences found can be performed simply with the help of the polymerase chain reaction.

S- !Z WO 96/02274 PCT/EP95/02539 The DNA oligonucleotides amplified in this way are then again transcribed with the help of the RNA polymerase in RNA oligonucleotides and the thus obtained RNA oligonucleotides can be used in a further selection step (as described above).

After separating the bonding RNA oligonucleotides, obtained in the second selection step, from the target molecules, the latter are again transcribed in DNA with the help of the reverse transcriptase, the thus obtained complementary DNA oligonucleotides are amplified with the help of the polymerase chain reaction and then transcribed again with the help of the RNA polymerase to the RNA oligonucleotides, which are available for a further selection step.

It has turned out that the desired high specificities and high bonding affinities can be obtained if the selection steps are repeated several times. Rarely will the desired oligonucleotide sequence be obtained as early as after one or two selection steps. As soon as the desired specificity and bonding affinity between target structure and oligonucleotide is obtained, the oligonucleotide(s) can be sequenced and as a result, the sequence of the specifically bonding oligonucleotides can be determined.

Especially advantageous in this process is that this process can be used not only with suitable proteins, butalso in vivo. But the above-mentioned selection process can also be perfonricd on purified target structures. But it is essential, especially for the in vivo diagnosis, that the specificity of the oligonucleotides is provided for the target structure in the living environment. Therefore, the selection processes can also be performed on cells or cell cultures, on tissues or tissue sections, on perfused organs and even on living organisms.

In this case, it is advantageous that the modified oligonucleotides can withstand the degradation by the almost omnipresent RNAs. As a result, the desired oligonucleotide sequences are themselves accumulated in selection processes on living organisms, since corresponding naturally occurring oligonucleotides would be degraded by the RNAs.

Oligonucleotide radical N can exhibit one or more connecting components B, or substituents B-K, which can be selected independently of one another. Claimed are oligonucleotide conjugates, which contain 1 to 10 identical or 2 to 10 different connecting components B. Especially preferred are oligonucleotide conjugates with one or two connecting components B.

Connecting component B connects oligonucleotide radical N with a complexing agent or complex K.

Advantageously, polydentate, open-chain or cyclic complexing ligands with O, S and N can be used as donor atoms.

As examples for complexing-agent radicals K, there can be mentioned the polyaminopolycarboxylic acids reduced by a hydrogen atom, a hydroxy group and/or SUEST T" WO 96/02274 PCT/IEP95/02539 -16an acetic acid group, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, trans-1,2-cyclohexanediaminetetraacetic acid, 1,4,7,10-tetraazacyclododecanetetraacetic acid, 1, 4 7 -triazacyclononanetriacetic acid, 1,4,8,11tetraazatetradecanetetraacetic acid, 1,5, 9 -triazacyclododecanetriacetic acid, 1,4,7,10tetraazacyclododecanetriacetic acid and 3,6,9,15-tetraazabicyclo-[9,3,1]-pentadeca- 1(15),11,13-trienetriacetic acid.

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

To illustrate the varied possibilities for complexing agents K according to this invention, reference is made to figures 1 to 3, in which some advantageous structures are compiled. These figures are meant as a selection and do not limit this invention in any way to the represented complexing agents.

Complexing agent K can contain all radioactive isotopes, usually used in nuclear medicine for diagnostic and therapeutic purposes, in the form of their metal ions. Stable isotopes, which are excited by external radiation to emit diagnostic or therapeutic radiation, or isotopes, which are converted by radiation from outside to radioactive isotopes, also can be used.

Isotopes suitable according to the invention are selected from the elements of atomic numbers 5, 21-29, 31, 39, 42-44, 49, 57-83 or For use of the compound according to the invention as a radiopharmaceutical agent, the complexing agent contains a radioactive element. All radioactive elements which are able to achieve a therapeutic or diagnostic effect in vivo or in vitro are suitable for this purpose. Preferred are radioactive isotopes of the elements copper, bismuth, technetium, rhenium or indium. Especially preferred are 99Tc-complexes.

If the compounds of general formula I according to the invention contain positron-emitting isotopes, such as, Sc-43, Sc-44, Fe-52, Co-55, Ga-68 or Cu- 61, the latter can be used in positron emission tomography (PET).

If the compounds of general formula I according to the invention contain gamma-radiation-emitting isotopes, such as, Tc-99m or In-11l, they can be used in single photon emission tomography (SPECT).

The compounds according to the invention can be used also in radiotherapy in the form of their complexes with radioisotopes, such as, Ir-192.

The compounds according to the invention can also be used in radioimmunotherapy or radiation therapy. The latter are distinguished from the corresponding diagnosis only by the amount and type of the isotope used. In this case, the purpose is the destruction of tumor cells by high-energy shortwave radiation with a smallest possible range. Suitable P-emitting ions are, for example, Sc-46, Sc-47, Sc- 48, Ga-72, Ga-73, Y-90, Re-186 or Re-188. Suitable a-emitting ions exhibiting small WO 96/02274 PCT/EP9502539 -17half-lives are, for example, At-209, At-211, Bi-211, Bi-212, Bi-213 and Bi-214, and Bi-212 is preferred. A suitable photon- and electron-emitting ion is 158 Gd, which can be obtained from 157 Gd by neutron capture.

If the agent according to the invention is intended for use in the variant of radiation therapy proposed by R. L. Mills et al. (Nature 336, 787 (1988)), the central ion must be derived from a M6ssbauer isotope, such as, for example, 57 Fe or 15 1 Eu.

Those carboxylic acid groups that are not required for complexing the metal ions of the elements of atomic numbers 21 to 29, 31, 39, 42 to 44, 49, 57 to 83 or can optionally be present as salts of an inorganic or organic base, such as alkali- or alkaline-earth metal hydroxides and carbonates, especially sodiurn and potassium hydroxide, or ammonia and alkylamines, or amino acid or as ester or amide.

Further, compounds that are excited by neutrons to emit particles and/or radiation can be used. Especially effective in this case is gadolinium.

Advantageously, those compounds can also be used that contain the isotope In such cases, K can have the structure 2(CH2 xC-

B

i H in which x \0/

CH

stands for a whole number from 1 to The invention further relates to processes for the production of the conjugates according to the invention.

Thus, conjugates in which connecting component B is bound on the 5'-end of the oligonucleotide can be obtained by reaction of the oligonucleotide with a phosphoramidite derivative (Tetrahedron 49, 1925-1963 (1993)). To this end, the group of the oligonucleotide is reacted with a phosphoramidite of general formula PR'(NR2")OR'". In this case, R' stands for an alkyl, alkoxy or arylalkoxy group, optionally containing N, NO 2 Si or SO2, with 1 to 20 C atoms, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, methoxy, ethoxy, propyloxy, butyloxy, benzyloxy or phenylethoxy, which optionally can be substituted. As substituents, especially cyano and nitro groups are used. Advantageously, for example, methoxy, P-cyanoethoxy or nitrophenylethoxy groups can be used. Especially preferred are P-cyanoethoxy groups. R" is a C 1

-C

4 alkyl radical, and ethyl and propyl radicals are especially suitable. Preferred are isopropyl radicals. R' is an alkyl or arylalkyl group, optionally containing S, O, N, CN, NO 2 or halogen, with 1 to 20 C atoms. Preferably, protected amino and thioalkyl radicals as well as protected amino and thiooxaalkyl radicals are used. Especially preferred are 6-amino-hexyl, 6-thiohexyl, 3,6,9-trioxa- 11-amino-undecyl and 3,6-dioxa-8-amino-octanyl groups. As protective groups, WO 96/02274 PCT/EP95/02539 -18generally usual N- or S-protective groups can be used. For example, trifluoroacetyl, phthalimido and monomethoxytrityl groups are suitable.

In an especially preferred embodiment of this invention, 3-cyanoethyl-N,Ndiisopropylamino-6-(trifluoroacetamido)-l-hexyl-phosphoramidite is used as phosphoramidite derivative.

In another preferred embodiment of this invention, p-cyanoethyl-N,Ndiisopropylamino-(3,6,9-trioxa-11-phthalimido-l-undecyl)-phosphoramidite is used as phosphoramidite derivative.

In another embodiment of the invention, connecting component B is bound on the 3'-end of oligonucleotide N in a way analogous to the one described above by a phosphorus-containing group.

The above-described reaction between oligonucleotide and phosphoramidite can take place as solid-phase reaction, and the oligonucleotide is still on the column of an automatic synthesizer. After an oligonucleotide of the desired sequence has been obtained and exposure of the 5'-hydroxy group of the oligonucleotide has taken place, with trichloroacetic acid, it is reacted with the phosphoramidite and the reaction product is oxidized and released. Then, the thus obtained oligonucleotide derivative is coupled on the terminal amino or thiol group with the complexing agent or complex K optionally by another linker group. The radical bound in the first step by the phosphorus-containing group on the oligonucleotide then forms, together with the optionally present additional linker group, connecting component B.

The linkage between oligonucleotide and the complexing agent can also take place so that the free 5'-hydroxyl group of the oligonucleotide is reacted with a complexing agent or complex, which terminally carries a bondable phosphorus radical.

Such a one can be described by the formula a) O-Ra

K-B-P

'R b

R

in which Ra stands for a C 1

-C

6 alkyl radical, which optionally carries a cyano group in p-position, Rb stands for a secondary amino group and K and B have the indicated meaning or the formula b) C'I Me'"ri-ri rrr- rt WO 96/02274 PCT/EP95/02539 -19- 0

II

K-B-P-O-Rc

H

in which Rc stands for a trialkylammonium cation and K and B have the mentioned meaning, or the formula c) O S II

I

o 0 R d

R

in which Rd stands for an aryl radical, optionally substituted with one or more halogen atom(s) and/or one or more nitro group(s), or a Ci-C 6 alkyl radical, which optionally is.substituted in P-position with a cyano group, and K, B and Rc have the mentioned meaning, and when using a radical of formula an oxidation step to phosphate takes place after completion of the coupling reaction. In both cases, radical -ORa or -ORc optionally can be cleaved off in a hydrolysis.

The linkage of the oligonucleotide derivative by the linker with the complexing agent or complex K can take place also as a solid-phase reaction on the column of an automatic synthesizer. The compound according to the invention can then be isolated from the solid vehicle by detaching.

The linkage of the oligonucleotide with the linker can take place not only by the 5'-OH group of the sugar of the terminal nucleotide, but also by other functional groups, which can be generated from the 5'-OH group, such as, an amino or carboxy group. Such nucleotides carrying amino or carboxy groups are known and can be produced easily. The synthesis of a 5'-deoxy-5'-aminouridine is described in J.

Med. Chem. 22, 1273 (1979) as well as in Chem. Lett. 6, 601 (1976). deoxyuridine is available as described in J. Med. Chem. 21, 1141 (1978), or Nucleic Acids Symp. Ser. 9, 95 (1981).

The linkage with the complexing agent then takes place by a linker carrying a carboxylic acid or amino group in a way known to one skilled in the art. The linker then forms connecting component B together with the -NH-CH 2 or the -CO-4' group.

It can be pointed out that the distribution of the conjugates according to the invention into a nucleotide radical, a connecting component and a complexing agent or complex takes place purely formally and thus independently of the actual synthetic I1ICTrII mr- rr #I WO 96/02274 PCT/EP95/02539 structure. Thus, in the above-mentioned case, the group -NH-CH 2 or -CO-4' is considered as belonging to connecting component B, while the oligonucleotide reduced in 4'-position by a CH 2 -OH group is designated as oligonucleotide radical N.

A process for the production of conjugates, in which the connecting component to the phosphodiester or phosphorothioate bridges reduced by the OH groups takes place, consists in that first two sugar units are linked to a dinucleotide (see, e.g., Chem. Lett. 1305 (1993)). In this case, there first results a triester of formula 0

II

3 5 o-U-v in which U stands for a corresponding alkylene radical and V stands for a protected amino or sulfur group. After cleavage, of the amino protective group, the complexing agent can optionally be linked, in a way known to one skilled in the art, Sby a linker with the amino group in the form of an amide bond. The linker then forms connecting component B together with group O-U-V' (in which V' stands for a group -NH).

An alternative process consists in that the phosphotriester passing through intermediately by reaction with 1,5-diaminopentane) is subjected to an aminolysis (see Biochemistry 22, 7237 (1988) or J. Am. Chem. Soc. 10, 4470 (1988)).

The thus obtained compounds of formula

O

3

NH-U-NH

2 can be linked as described above with the complexing agent optionally by a linker.

For coupling purposes, dinucleoside-phosphate-monothiotriesters are also suitable (see J. Am. Chem. Soc. 111, 9117 (1983) and Nucl. Acids Res. 20, 5205 (1992)).

The nucleobases offer an especially great variety to link the complexing agents with the nucleotides. A linkage by amino groups in 2-position in the purines and in 4position in the pyrimidines can take place directly. But it is often more advantageous first to modify the purines or pyrimidines and to link these derivatized bases with the complexing agents (optionally by additional linkers). Suitable derivatized nucleobases are described, in Biochemie [Biochemistry] 71, 319 (1989), Nucl. Acids Res. 16, 4937 (1988) or Nucleosides Nucleotides 10, 633 (1991).

An alternative process for linking by the nucleobases consists in the palladiumcatalyzed coupling of bromine or iodine nucleobases with functionalized radicals 01 ID!TITI 1r-- cnurr-r ft WO 96/02274 PCT/EP95/02539 -21- (Biogenic and Medical Chemistry Letter V, 361 (1994)). By these functionalized radicals, the complexing agent can then optionally be linked with the nucleobase by another linker according to known methods. As functionalized radicals in of the pyrimidine and in 8-position of the purine, an acrylic ester or an allylamine can be mentioned as examples (see Nucl. Acids Res. 14, 6115 (1986) and Nucl. Acids Res. 16, 4077 (1988)). Another alternative process for preparing 5-position modified pyrimidines, especially for introducing functional groups such as carbonyl, alkenyl or aryl groups at the 5-position, and an improved palladium catalyst capable of coupling modifying groups at the 5-position of pyrimidines is described in U.S. patent application Ser. No. 08/076,735, filed June 14, 1993. The halogen derivatives used as precursor can be obtained as described, in Biophys. J. 44, 201 (1983), J. Am.

Chem. Soc. 86, 1242 (1964) or Chem. Commun. 17 (1967).

The production of the metal complexes according to the invention from the metal-free oligonucleotide conjugates takes place as disclosed in DE 34 01 052, by the metal oxide or a metal salt (for example, the nitrate, acetate, carbonate, chloride or sulfate) of the desired metal isotope being dissolved or suspended in water and/or a lower alcohol (such as methanol, ethanol or isopropanol) and reacted with the solution or sspension of the equivalent amount of the oligonucleotide conjugate contairing the complexing agent and then, if desired, present acidic hydrogen atoms being substituted by cations of inorganic and/or organic bases or amino acids or free carboxylic acid groups being converted to amino acid amides.

The neutralization of possibly still present free acid groups takes place with the help of inorganic bases (for example, hydroxides, carbonates or bicarbonates) of, for example, sodium, potassium, lithium, magnesium or calcium and/or organic bases, such as, among others, primary, secondary and tertiary amines, such as, for example, ethanolamine, morpholine, glucamine, N-methyl- and N,N-dimethyl-glucamine, as well as basic amino acids, such as, for example, lysine, arginine and ornithine, or of amides of originally neutral or acidic amino acids.

The production of the pharmaceutical agents according to the invention takes place also in a way known in the art, by the oligonucleotide conjugates according to the invention optionally by adding the additives usual in galenicals being suspended or dissolved in aqueous medium and then the suspension or solution optionally being sterilized or sterilized by filtration. Suitable additives are, for example, physiologically harmless buffers (such as, for example, tromethamine), additives of complexing agents (such as, for example, diethylenetriarinepentaacetic acid) or if necessary electrolytes, such as, for example, sodium chloride or if necessary antioxidants, such as, for example, ascorbic acid, or, especially for oral forms of administration, mannitol or other osmotically active substances.

nI IneP riri r'I WO 96/02274 PCT/EP9502539 -22- If suspensions or solutions of the agents according to the invention in water or physiological salt solution are desired for enteral administration or other purposes, they can be mixed with one or more adjuvant(s) usual in galenicals (for example, methyl cellulose, lactose, mannitol) and/or surfactant(s) (for example, lecithins, Tween

TR

MyrjTR).

The pharmaceutical agents according to the invention preferably contain 0.1 p mol/l to 3 mmol/l of the oligonucleotide conjugates according to the invention and are generally dosed in amounts of 0.01 nmol/kg 60 gmol/kg. They are intended for enteral and parenteral administration.

In nuclear medical in vivo use, the labeled compounds generally are dosed in amounts smaller than 10- 10 mol/kg of body weight, and the exact dose can vary greatly as a function of the body region studied but especially also as a function of the respectively selected method of study. Starting from an average body weight of kg, the amount of radioactivity for diagnostic uses is between 40 and 1100 MBq, preferably 200-800 MBq, for therapeutic uses 1-500 MBq, preferably 10-100 MBq per administration. The administration takes place normally intravenously, intraarterially, interstitially, peritoneally or intratumorally, and the intravenous administration is pre.trred. In general, 0.1 to 20 ml of the agent in question is administered pe study.

This invention further relates to a process for detecting target structures. In this case, one or more of the above-described compounds are brought together with the sample to be studied in vivo or in vitro. In this case, oligonucleotide radical N bonds specifically and with high bonding affinity to the target structure to be detected.

If the target structure is present in the sample, it can be detected there based on the signal. The process is especially suitable for a noninvasive diagnosis of diseases.

In this case, one or more of the above-described compounds is administered in vivo and it can be detected by the signal whether the target structure, on which oligonucleotide radical N bonds specifically and with high affinity, is present in the organism to be studied.

But in addition to the mere detection of target structures in samples to be studied, the latter can also be specifically destroyed. In this respect, the compounds of this invention are suitable especially in radiotherapy, in cancer therapy.

Another embodiment of this invention comprises a diagnosis kit for in vivo detection of target structures, which contains one or more of the above-mentioned compounds.

The conjugates and agents according to the invention meet the many requirements that are to be made on a pharmaceutical agent for radiotherapy and diagnosis. They are distinguished especially by a high specificity or affinity relative to nr rnClrrrt(-L CII C-- WO 96/02274 PCT/EP95/02539 -23the target structure in question. Relative to known oligonucleotide conjugates, the conjugates according to the invention exhibit an especially high in vivo stability. This was achieved by a substitution of the 2'-hydroxyl group and the incorporation of modified thymidine sequences on the terminal hydroxyl groups of the nucleotides.

Surprisingly, the specificity of the oligonucleotide is not significantly impaired either by this modification or by the coupling with the complexing agent. Other advantages are the controllable pharmacokinetics as well as the necessary compatibility.

Brief Description of the Drawings Various other objects, features and attendant advantages of the present invention will be more fully appreciated as the same becomes better understood when considered in conjunction with the accompanying drawings, wherein: Figure 1 shows a selection of cyclic complexing agents K, which can be used advantageously for this invention. marks the bonding site on connecting component B.

Figures 2 and 3 show a selection of open-chain complexing agents K, which can be used advantageously for this invention.

The following examples are to illustrate these inventions in more detail.

The polynucleotides described in the examples contain modified compounds.

They mean: A, U, C, G the nucleotides contain a 2'-OCH 3 group the internucleotide bond is a methyl phosphonate the internucleotide bond is a thiophosphonate the internucleotide bond is a dithiophosphonate -24- Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.

The entire disclosures of all applications, patents and publications, cited above and below, are hereby incorporated by reference.

WO 96/02274 PCT/EP95/02539

EXAMPLES

Example 1 a) 5'-(6-Amino-hexyl-phosphoric acid ester) of the '-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T-3' The 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUA-3', identified according to the SELEX process, with the modification of a sequence T*T*T*T*T-3' placed upstream is produced in the usual way in an automatic synthesizer of the Pharmacia company (see Oligonucleotides and Analogues, A Practical Approach, Ed. F. Eckstein, Oxford to University Press, Oxford, New York, Tokyo, 1991), and the oligonucleotide is also present on the column of the solid vehicle. By reaction with trichloroacetic acid solution in dichloromethane, the 5'-hydroxy group is opened. The loading of the column is about 10 mg of 35mer-oligonucleotide. To join the linker, the column is reacted with an acetonitrile solution of 50 pmol of 1-cyanoethyl-N,N-diisopropylamino-6- (trifluoroacetamido)-l-hexyl-phosphoramidite (produced according to Nucl. Acids.

Res. 16, 2659-2669 (1988)) in the presence of tetrazole. The oxidation of the formed phosphite to the completely protected phosphotriester takes place with iodine in tetrahydrofuran. Then, the column is washed in succession with methanol and water.

To remove the modified oligonucleotide from the solid vehicle, the contents of the column are conveyed in a multivial, mixed with 5 ml of 30% ammonia solution, the vessel is sealed and shaken overnight at 55 0 C. It is then cooled to 0°C, centrifuged, the vehicle is washed with 5 ml of water and the combined aqueous phases are subjected to a freeze-drying.

For purification, the solid material is taken up in 2 ml of water, mixed with 2 ml of 0.5 M ammonium acetate solution and mixed with 10 ml ethanol, it is allowed to stand overnight at -20 0 C, centrifuged, the residue is washed with 1 ml of ethanol 0 C) and finally dried in a vacuum at room temperature. 8 mg of the title compound is obtained as colorless powder.

b) 10-[5-(2-Carboxyphenyl)-2-hydroxy-5-oxo-4-aza-pentyl]- 1,4,7-tris(carboxymethyl)- 1,4,7,10-tetraazacyclododecane g (144.3 mmol) of 1, 4 7 -tris(carboxymethyl)-1,4,7,10tetraazacyclododecane (D03A) is dissolved in 250 ml of water and the pH is adjusted to 13 with SN sodium hydroxide solution. Then, a solution of 38.12 g (187.6 mmol) of N-(2,3-epoxypropyl)-phthalimide in 100 ml of dioxane is instilled within one hour, stirred for 24 hours at 50 0 C and the pH is kept at 13 by adding 5N sodium hydroxide solution. The solution is adjusted to pH 2 with 10% hydrochloric acid and evaporated to dryness in a vacuum. The residue is dissolved in some water and purified on an ion WO 96102274 PCT/EP95/02539 -26exchange column (Reillex(R) POly-( 4 -vinyl)-pyridine, it is eluted with water). The main fractions are concentrated by evaporation in a vacuum, and the residue is given a final purification by chromatography on RP-18 (LiohroPrep(R)/mobile solvent: gradient of tetrahydrofuranlmethanol/water). After concentration by evaporation of the main fractions, 63.57 g (71 of theory) of an amorphous solid is obtained.

Water content: 8.5 Elementary analysis (relative to the anhydrous substance): Cld: C 52.90 H 6.57 N 12.34 Fnd: C 52.65 H 6.68 N 12.15 c) lO-(3-Axnino-2-hydroxy-propyl)-1 ,4 7 -tris(carboxy-methyl)-1.,4 tetraazacyclododecane g (88.1 mmol) of the title compound of example lb is refluxed in 300 ml of concentrated hydrochloric acid for 24 hours. It is evaporated to dryness, the residue is dissolved in some water and purified on ail ion exchange column (Reillex poly-.

(4-vinyl)-pyridine (it is eluted with water)). The main fractions are evaporated to dryness.

Yield: 3 9 g (95 of theory) of .5 vitreous solid.

Water content: 10.3% Elementary anialysis (relative to the anhydrous substance): Cld: 0 48.68 H 7.93 N 16.70 Fnd: C 48.47 H18.09 N 16.55 d) 1 O-[ 7 4 Nitrophenyl).2.hydroxy.5oxo.7(carboxymethyl)4azaheptyl]-l,4,7..

tris(carboxymethyl)-1 ,4 9.84 g (41.8 minol) of 3-(4-nitrophenyl)-glutaric anhydride Org. Chem. 2!k, 3856 (1961)) is added to 14.62 g (34.86 mmol) of the title compound of example lc) in 200 ml of dimethylformamide/20 mnl of triethylamine and stirred overnight at room temperature. It is evaporated to dryness 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 Elementary analysis (relative to anhydrous substance): Old: C051.37 H16.47 N 12.84 Fnd: 0 51.18 H 6.58 N 12.67 e) 1 O-[ 7 4 -Aminophenyl)-2..hydroxy.5.oxo..7.(carboxy..methyl)4a za..heptyl]..1,4,7tris(carboxymothyl)-1 l0-tetraazacyclododecane.

WO 96/02274 PCr/EP95/02539 -27- 21.0 g (32.07 mmol) of the title compound of example Id) is dissolved in 250 ml of methanol and 5 g of palladium catalyst (10% Pd on C) is added. It is hydrogenated overnight at room temperature. The catalyst is filtered off and the filtrate is evaporated to dryness in a vacuum.

Yield: 19.63 g (98% of theory) of a cream-colored solid Water content: 0.8% Elementary analysis (relative to anhydrous substance): Cld: C 53.84 H 6.35 N 12.60 Fnd: C 53.73 H 6.45N 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 of example le) is dissolved in 200 ml of water and 6.64 g (57.8 mmol) of thiophosgene in 50 ml of chloroform is added.

It is stirred for 1 hour at 50 0 C. It is cooled to room temperature, the organic phase is separated and the aqueous phase is shaken out twice with 100 ml of chloroform. The aqueous phase is evaporated to dryness and the residue is absorptively precipitated in 100 ml of isopropanol at room temperature. The solid is filtered off and washed with ether. After drying overnight in a vacuum (40 0 12.74 g (97% of theory) of a cream-colored solid is obtained.

Water content: 3.1% Elementary analysis (relative to anhydrous substance): Cld: C 52.24 H 6.35 N 12.60 S 4.81 Fnd: C 52.37 H 6.44 N 12.48 S 4.83 g) Conjugate of 5-(6-amino-hexyl-phosphoric acid ester) of the 35mer oligonucleotide 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T-3' and 10-[7-(4isothiocyanato-phenyl)-2-hydroxy-5-oxo-7-(carboxymethyl)-4-azaheptyl]-1,4,7tris(carboxymethyl)- 1,4,7,10-tetraazacyclododecane 8 mg of the oligonucleotide obtained in example la) is dissolved in 2.5 ml of a NaHCO 3 /Na 2

CO

3 buffer (pH 8.0) and mixed with 1 mg of 10-[7-(4isothiocyanatophenyl)-2-hydroxy-5-oxo-7-(carboxymethyl)-4-aza-heptyl]-1,4,7-tris- (carboxymethyl)-1,4,7,10-tetraazacyclododecane (title compound of example If). It is stirred for 5 hours at room temperature, the pH is adjusted to 7.2 by adding 0.01 M hydrochloric acid and the solution is subjected to an ultrafiltration through a membrane with the exclusion limit 3,000 (Amicon YM3) and then a freeze-drying. 7 mg of the desired conjugate is obtained.

WO 96102274 PCT/EP95/02539 -28h) 11 1 ndiumn complex of the thiourea conjugate of 5-( 6 -amino-hexyl-phosphoric acid ester) of the 35iner oligonucleotide 3 and 10-[7-(4isticaaohnl--yrx--xo7(abxmty)4aaetl ,4,7tris(carboxymethyl)- 1,4,7, 1 -tetraazacyclododecane gil of an 111 indium(Il) acetate solution (3 50 liCi), (produced from indium(HI) chloride in 2 M sodium acetate solution and adjustment of the pH to 4. 0 with 0. 1 M hydrochloric acid) is added to 135 gtl of a solution of 1 mg of the title compound of example 1g) in MES buffer, pH 6.2 (MES 2(morpholino)ethylsulfonic acid). The pH is brought :o 4.2 by adding 0.0O1 M hydrochloric acid. It is stirred for 1 hour at 37'C at pH 4. 2. It is brought to pH 6 with 2 M sodium acetate solution and 10 gil of P. 0. 1 M Na-;EDTA th1,'enediatnineteuaacttic acid disodiuai salt is added to coinplax c 'idium. I I final pur.i f icatior. of thus obtained ;abeled conjugate (1h) talkes place, by HPLC 'V1usioii chromatography: :TSK-400/MES-huffer). T he f aceiols containing thL: 1 abeled ccnjugate. are diluted with physiological common saltz olution, adjusted 1op .2 VvIth 0.01 M sodium hydroxide solution and filtered. A thtus produced solu~iU:n then represents a suitable preparation for radiodiagnosis.

-Example 2 a) Conjugate of 5 (6-amino-l -hexyl-phosphoric acid ester) of the oligonucleotide 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T- 3 and N-2aio3(-sticaaohey)poy]tasccoeae1 ,2-diarnine- N,N'-N',N",N"-pentaacetic acid 8 mg of the oligonucleotide obtained in example la) is dissolved in 2.5 ml of a NaHCO 3 /Na 2

CO

3 buffer (pH1 8.0) and 1 mg of N-[2-amino-3-(pisothiocyanatophenyl)propyl-trans.cyclohexe-.1 .2-diamine-N,N',N',N" ,Nt pentaacetic acid is added (produced according to Bioconjugate Chem. 1, 59 (1990)).

It is stirred for 5 hours at room temperature, then adjusted to pH 7.2 with 0. 1 M hydrochloric acid and the solution is subjected to an ultrafiltration through a membrane with the exclusion limit 3,000 (Amnicon YM3). After freeze-drying, 6 mg of thiourea conjugate 2a) is obtained.

b) Bismuth-212 complex of the conjugate of 5'( 6 -amino-l-hexyl-phosphoric acid ester) of the 35mer oligonucleotide

CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T-

3 and N-[2-amino-3- WO 96/02274 PCT/EP95/02539 -29- 4 -isothiocyanatophenyl)-propyl]-trans-cyclohexane-l,2-diamine-N,N'-N' ,N pentaacetic acid A 21 2 bismuth-tetraiodide solution in 0.1 M of hydroiodic acid is brought to pH 4 with 2 M acetic acid. An aliquot of this solution of the activity of about 3 mCi is added to 1 mg of the title compound of example 2a), dissolved in 0.5 ml of 0.02 M MES-buffer and 0.5 ml of 0.15 M sodium chloride solution is added. It is stirred for minutes at room temperature. It is brought to pH 6 with 2 M sodium acetate solution and 20 gl of a 0.01 M Na2EDTA solution is added. It is stirred for minutes. The purification of the complex takes place by HPLC (exclusion chromatography: TSK-400/MES-buffer). The radioactive conjugate fractions are combined, diluted with physiological common salt solution, and adjusted to pH1 7.2 with 0.01 M sodium hydroxide solution. After filtration, a preparation suitable for radiotherapy is obtained.

Example 3 S a) Idiu -11 complex of the conjugate of 5'(6-ami- -hexyl-pho c acid ser) of ti 35mer oligoniuleotide 'c a C AUi (GGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T-3' and N-[2-amini-3- (4-isothiocyanatophenyl)-propyl]-trans-cyclohexane-,2-diamine-N,N'-N',N pentaacetic acid ml of a 15 pl of an llindium(I) acetate solution (350 pCi) (produced from indium(III) chloride in 2 M sodium acetate solution and adjustment of the pH to with 0.1 M hydrochloric acid) is added to 0.5 ml of a solution of 1 mg of the title compound of example 2a) in MES-buffer, pH 6.2 (MES 2 -(N-morpholino)ethylsulfonic acid). The pH is brought to 5.0 by adding 0.01 M hydrochloric acid. It is stirred for 1 hour at 37 0 C at pH 5.0. It is brought to pH 6 with 2 M sodium acetate solution and 10 gl of a 0.1 M Na 2 EDTA ethylenediamine-tetraacetic acid disodium salt is added to complex excess 11 indium. The final purification of thus obtained labeled conjugate (lh) takes place by HPLC (exclusion chromatography:

TSK-

4 0 0/MES-buffer). The fractions containing the labeled conjugate are diluted with physiological common salt solution, adjusted to pH 7.2 with 0.01 M sodium hydroxide solution and filtered. A thus produced solution then represents a suitable preparation for radiodiagnosis.

RI IRTITI IT cucct-n, s, WO 96/02274 PCr/]EP95/02539 Example 4 a) Conjugate of 5'-(6-alnino-l-hexyl-phosphoric acid ester) of the oligonucleotide 5'-CUCAUGCAGCGCAAGACGAUAGCUACAUAT*T*T*T*T- 3 and 2 4 -isothiocyanato-benzyl)-diethylenetriamneN, N,N' ,N "-pentaacetic acid 8 mg of the oligonucleotide obtained in example la) is dissolved in 2.5 ml of a NaHiC0 3 /Na 2

CO

3 buffer (pH 8.0) and 1 mg of 2 4 -isothiocyanato-benzyl)-diethylen-e.

triamine-N,N' ,N",N"-pentaacetic acid is added (produced according to: Bioconjugate Chem. 2,187 (1991)). It is stirred for 5 hours at room temperature, then adjusted to pH 7.2 with 0.01 M hydrochloric acid and the solution is subjected to an ultrafiltration through a membrane with the exclusion limit 3,000 (Amicon YM 3).

After-freeze-drying, 6 mg of the thiourea conjugate is bxidned.

b) Ytriun-0comnplex of the- conjugate o -(-amino- I-hexyl-phiosphoric acid ester) of the 35iner ofigonuc~eotidf. 1' .CUCAUiGC AGC%'CCAAGAC(:14AAUAGCUACAUAT*T*.T*T*T-3' and 24,1isothiocyaato~bepyl)-diehy,)efltim N ,N 11- "-ntacetic acid so A ution of :ytitgdissolved' in 0.05 M 6nrnn acdtt sol ution (abou 380 i~i~ i aded o 1~mgf te thiourea derivative: of exaple a n05m t00 M ammot um acetate solution of 'pH adjusted to p11 '5.2 with 3 M acetic acid and stirred for 1 hour at room temperature. It is adjusted to pH4 7.0 With 0.01- M sodium hydroxide solution and the conjugate is purified by HPLC .(TSK-400/MES-buffer).

he main fractions are combined, diluted with physiological conmnon salt snlution and brought to pH 7.2 with 0.01 M sodium hydroxide solution. After filtration, a preparation suitable for the radiotherapy is obtained.

Example a) 6 -Mercapto-1-hexyl-phosphoric acid ester) of the 3 9 -T*T*T*T*TAGGAGGAGGAGGGAGAGCGCAUGAGAUU-3' (modified ligand for serine protease) The 5'-AGGAGGAGGAGGGAGAGCGCAAAUGAGAUU..3' (seq. no. 13 of US Patent No. 5,270,163) identified according to the SELEX process, modified in the sugar units and by a 5'-linked sequence of 5 tymidines, is produced in the usual way in an automatic synthesizer of the Pharmacia company (see Oligonucleotides and Analogues, A Practical Approach, Ed. F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1991), and the oligonucleotide is also present on the column of the solid vehicle. By reaction with trichloroacetic acid solution in dichloromethane, the WO 96/02274 PCT/EP95/02539 -31group is opened. The loading of the column is about 10 mg of oligonucleotide. To join the linker, the column is reacted with a solution of 50 ±mol of P-cyanoethyl-N,N-diisopropylamino-S-trityf-6-mercapto)-phosphoramidite in acetonitrile in the presence of tetrazole. The oxidation of the formed phosphite to the completely protected phosphotriester takes place with iodine in tetrahydrofuran. Then, the column is washed in succession with methanol and water. To remove the modified oligonucleotide from the solid vehicle, the contents of the column are conveyed in a multivial, mixed with 5 ml of 30% ammonia solution, the vessel is sealed and shaken overnight at 55 0 C. It is then cooled to 0°C, centrifuged, the vehicle is washed with ml of water and the combined aqueous phases are subjected to a freeze-drying.

For purification, the solid material is taken up in 2 ml of water, mixed with 2 ml of 0.5 M ammonium acetate solution and mixed with 10 ml ethanol, it is allowed to st4nd overnight at -20 0 C, centrifuged, the residue is washed with 1 ml of ethanol finally dried in a vacuum at room temperature: 9 mg.pf the S-tritylated title compound isobtained. To cleave the trityl protective group, the product is dissolved in 0.5 ml of water, mixed with 0.1 ml of 1 l silver nitte solution and stirred for i ihour atroom temperature. Then, it is mixed with 0.1 Wm 1 M dithiothreitol soluti6n. After 15 minut;it is centrifuged, and the s: upernatant solution is extracted several times wit ethyl acet te. After the freeze- 0 drying. 8 mg of the desired title compound is obtained frotm the aqueous solution.

4-Benzyloxy-N-methanesulfonyl-phenylalanine-methylester 19.58 g of methanesulfonic acid chloride is instilled in 50 g of 4-benzy~bxyphenylalanine-methylester-hydrochloride in 300 ml of pyridine at o0C and stirred for 3 hours at 0°C. It is evaporated to dryness in a vacuum and the residue is dissolved in 500 ml of dichloromethane. It is shaken out twice with 300 ml of 5N hydrochloric acid each, dried on magnesium sulfate and concentrated by evaporation in a vacuum.

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-methylester and 1.2 1 of 1,2-diaminoethane are stirred for 3 hours at 80 0 C. The residue is evaporated to dryness and absorptively precipitated with 200 ml of water, the precipitate is suctioned off, washed neutral with water and dried overnight at 60 0

C.

Yield: 37.68 g of a cream-colored, amorphous powder.

CZI IP(ZTITI IT W:r7 P111 P 9MF~ WO 96102274 PCT/EP95/02539 -32d) 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 triazaheptan-3-one and 4.76 g of triethylamine in 200 ml of chloroform is mixed at 0 0 C with a solution of 10.27 g of di-tert-butyl-dicarbonate in 50 ml of chloroform. It is stirred for 5 hours at room temperature, shaken with 5 sodium carbonate solution and water, dried on magnesium sulfate and concentrated by evaporation in a vacuum.

The residue is recrystallized from 100 ml of methanol.

Yield: 20.19 g of a foamy solid.

e) 2-(4-Hydroxybenzyl)-l1-methanesulfonyl-7-(tert-butyloxycarbonyl)-1 ,4,7triazabeptan-3-one g of 2-(4-benzyloxybenzyl)-l1-miethanesulfonyl-7-(tert-butyloxycarboniyl)- 41,4,7-triazaheptan-3-one, dissolved in 300 mld of dihooehnis stirred with 4'g -of-palladium-carbon overnight under a hydrogen atmosphere. It is fitered and .the solution is concentrated by evaporation i a vacuum.

Yield:' 16.17 g of a vitreous foam, which solidifies after a few minutes.

f) '2-[4-(3-Oxapropionic acid-benzylester)-benzyl]-1-methaesulfonyl-7-(tertbutyloxycarbonyl)-1 ,4,7-triazaheptan-3-one g of 2-(4-hydroxybenzyl)-1-methanesulfonyl-7-(tert-butyloxycarbonyl)- 1 ,4,7-triazaheptan-3-one, 8.56 g of,bromoacetic acid-benzyl ester and 13.18 g of potassium carbonate are refluxed in 300 ml of acetonitrile for 24 hours. It is filtered and evaporated to dryness in a vacuum. The residue is dissolved in 200 ml of dichioromethane, shaken out twice with 50 ml of water each. The organic phase is dried on magnesium sulfate and concentrated by evaporation in a vacuum. The residue is chromatographed with dichloromethane-hexane-acetone (20/10/1) as eluent.

Yield: 10.06 g of foamy solid a) 2-[4-(3-Oxapropionic acid-benzylester)-benzyl]- 1-methanesulfonyl- 1,4,7triazaheptan-3-one g of 2-[4-(3-oxapropionic acid-benzylester)-benzyl)-l1-methanesulfonyl-7- (tert-butyloxycarbonyl)-1 ,4,7-triazaheptan-3-one is stirred for 1 hour at room temperature with 100 ml of trifluoroacetic acid. It is evaporated to dryn ess in a vacuum.

Yield: 9.2 g of vitreous foam, which solidifies while standing.

h) 9-Chloro-l1-methanesulfonyl-2-[4-(3-oxapropionic acid-benzylester)-benzyl]-l ,4 ,7triaza-3 ,8-d lone WO 96102274 PCT/EP95/02539 -33- 9 g of 2-[4-(3-oxapropionic acid-benzylester)-benzyl]-l1-methanesulfonyl-1 7triazaheptan-3-one and 1.78 g of triethylamine are dissolved in 200 ml of chloroform.

At 0 0 C, 1.98 g of chioroacetyl chloride, dissolved in 20 ml of chloroform, is instilled within 30 minutes and then stirred for 2 hours at 0 0 C. It is washed with 100 ml of 5 hydrochloric acid, twice with 50 ml of water each, dried on magnesium sulfate and evaporated to dryness in a vacuum. The residue is chroniatographed on silica gel with dichloromethane-ethyl acetate (20/1) as eluent.

Yield: 6.97 g of waxy solid i) 9-Chloro-l1-methanesulfonyl-2-[4-(3-oxapropionic acid-benzylester)-benzyl]- 1,4,7triaza-3 ,8dione g of 9-chioro-l1-methanesulfonyl-2-[4-(3-oxapropioni'c acid-benzylester)benzyll-1,4,7-triaza-3,8-dione, dissolved in 150 ml of dichioromethane, is stirred. with 2 g of palladium-carbon overnight under a hydrogen atmosphere. -It is filtered and the solution is co-icentrated by,~ evaporation in a vacuum.- Ylield:. 5,33 g of vitreous solid J) 10-Acetyl-2-[4-(3-oxapropionic acid)-benzyl]-1 -(metha nesulfonyl)-l 0-thia- 1,47triazadecane-3,8.-dionje 5 g of 9-chloro-1-methaesulfonyl-2-[4-(3-oxapropionic acid-benzylester)benzyl]-1,4,7-triaza-3,8-dione, dissolved in 80 ml of chloroform,, is refluxed with 1.98 g of triethylamine and 0.74 g of thioacetic, acid for 19 minutes. The solution is poured in 200 ml of ice-cold hydrochloric acid, the organic phase is separated, dried on magnesium sulfate and concentrated by evaporation in a vacuum. After chromatography on silica gel with hexane-ethyl acetate 4.64 g of the desired compound is obtained as vitreous solid.

k) 10-Acetyl-2-[4-(3-oxapropionic acid-(2 ,5-dioxo-pyrrolidin- 1-yl)-ester)-benzyl]- 1- (methanesulfonyl)-1O-thia-1 ,4,7-triazadecane-3 ,8-dione 4 g of 10-acetyl-2-[4-(3-oxapropionic acid)-benzyl]-l1-.(methanesulfonyl)-10thia-1 ,4,7-triazadecane-3,8-dione, 1.91 g of dicyclohexylcarbodjirnide, 4 g of Nhydroxysuccinimide and 30 mg of 4-dimethylaminopyridine are stirred for 24 hours in ml of chloroform at room temperature. It is then mixed with 20 ml of diethyl ether, filtered, and the residue is concentrated by evaporation in a vacuum. The residue is chromatographed on silica gel with dichloromethane-dioxane (10/1) as eluent.

Yield: 3.47 g of a cream-colored solid Elementary analysis: Q1 IOCTITI IT= ourr-r t, WO 96/02274 -3-PCTfEP9SIO2539 Cid: C 46.15 H 4.93 N 9.78 S11.20 Fnd: C 46.03 H 4.83 N 9.64 S 11.05 1) 10-Acetyl-2-{4-[3-oxapropiornc acid-(6-maleimido-hexanoyl)-hydrazide-belzyl} -1methaesulfonyl-10-thia-l ,4,7-triazadecane-3 ,8-dione 3 g of 10-acetyl-2-114-(3-oxapropioflic acid-(2,5-dioxo-pyrrolidin-1 -yl)-ester)bezl--mtaeufnl-0ti-,,-raaeae38din and 1.17 g of 6-malcimidocaproic acid hydrazide (Science 2U1, 212 (1993)) are. stirred in 40 ml of dimethylformamide for 5 hours at 60PC. With vigorous stirring, 100 ml of water is instilled and filtered off from the precipitated precipitate. It is dried in a vacuum and -the residue is purified by a FLASH chromatography on a silica gel column with dic-hloromethane/dioxane (10/1) as eluent. 2.8 g of the title compound is obtained as white solid.

Elementary analysis (relative to anhydrous substance): Cld: C. 49.25 H 5.61 N 12.30 .S 9.39 Fnd: C 49.33 H 5.95 N 12.43 S 9M1 in) Conjuigate of 5 '-(6-mercapto-l-hexyl-phosphori~c acid ester) of the oligonucleotide 5-'..T*T*T*T*TAGGAGGAGGAGGGAGAGCGCAJ4AUGAGAUU~o-.i' .,and 10-acetyl-2-{4-II3-oxapropiolic acid7(6-rnaleimiido)-hexanoyl)-hydrazidel-bel)l- 1-methanesulfonyl-lO-thia- 1,4,7-triazadecane-3 ,8-dione mng of the thiol-conitaining oligonucleotide produced according to example is mixed in 2 ml of phosphate buffer (pH 7.4) with 1 mng of the maleijuide derivative, produced according to example 51), dissolved in 0.2 mil of dimethylformamide. It is allowed to stand for 2 hours at room temperature, and the solution is subjected to an ultrafiltration through a membrane with the exclusion limit 3,000 (Amicon YM 3) and then a freeze-drying. 5 mg of the desired conjugate is obtained.

n) Technetium-99m complex of the conjugate of 5'-(6-Mercapto-1-hexyl-phosphoric acid ester) of the 3 and lO-acetyl- 2-{4-[3-oxapropionic acid-(6-maleimido)-hexaI1oyl)-hydrazide]-be1zyl1-1 methanesulfonyl- 10-thia- 1,4,7-triazadecane-3 ,8-dione 1 ml of a solution of potassium-D-glucarate (12 mg/ml) and tin(II) chloride (100 .ig/ml) in 0.2 M NaHCO 3 are freezed-dried in a vial and then mnixed with [Tc- 99m]-sodium pertechnetate solution (1 ml, 1 mnCi) from an Mo-99/Tc-99m generator.

After standing at room temperature for 15 minutes, an aliquot is removed and mixed with the same, volume of the conjugate of example 5m) (1 mg/mi) dissolved in 0.2 M SUBSTITUTE SHEET (RULE 26) WO 96/02274 PCT/EP95/02539 NaHC0 3 solution. After 15 minutes, both thin-layer chromatography and HPLC showed that 98% of the radioactivity was taken up by the conjugate.

Example 6 a) Conjugate of 5'-(6-amino-l-hexyl-phosphoric acid ester) of the oligonucleotide 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T-3' and S-benzoyl MAG 3 -2,3,5,6-tetrafluorophenylester 3 mg of S-benzoyl-MAG 3 -2,3,5,6-tetrafluorophenylester (produced according to to US 4,965,392), dissolved in 0.2 ml of dimethylformamide, is added to 8 mg of the title compound of example la), dissolved in 0.5 ml of 0.1 M phosphate buffer (pH 7), and stirred for 3 hours at room temperature. It is diluted with water and the solution is subjected to an ultrafiltration (Amicon YM 3, exclusion limit 3,000). After freezedrying, 5 mg of conjugate 6a) is obtained.

9 Technetium complex of the conjugate of 5'-(6-amino-1-hexyl-phosphoric acid ester) of the 35mer-oligonucleotide CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T-3' and S-benzoyl

MAG

3 -2,3,5,6-tetrafluorophenylester 1 mg of the title compound of example 6a) is dissolved in 200 p.l of water and mixed with 1 ml of 0.1 M phosphate buffer of pH 8.5. 200 pl of 99technetium-(V)gluconate solution (about 15 mCi) is added to this mixture and allowed to stand for minutes at room temperature. The tracer yield (determined by HPLC) is about Example 7 a) Conjugate of the 5'-(6-amino-1-hexyl-phosphoric acid ester) of the oligonucleotide 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T-3' Sand the 99mtechnetium complex of 2,3,5,6-tetrafluorophenyl-4,5bis(mercaptoacetamido)-pentanoic acid ester 3 mg of the title compound of example la), dissolved in 0.6 ml of phosphate buffer, is added to the 99 mtechnetium complex of 2,3,5,6-tetrafluorophenyl-4,5-bis- (mercaptoacetamido)-pentanoic acid ester (produced according to: J. Nucl. Med. 32, 1445 (1991)) about 100 mCi, dissolved in 2 ml of phosphate buffer, pH 7.2. It is adjusted to pH 10 with 1.0 M potassium carbonate buffer and stirred for 20 minutes at room temperature. For final purification, the solution is added to a Sephadex column (Pharmacia) and eluted with 75 mmol of sodium chloride solution. The main fractions 0,I MUM iT;:.qHP;:T (PI 11 r- %Z WO 96/02274 PCTIEP95102539 -36are combined, diluted with physiological conmmon salt solution and filtered. The thus obtained solution can be used for radiodiagnostic studies.

Example 8 a) Conjugate of 5'-(6-mercapto-1-hexyl-phosphoric acid ester) of thle oligonucleotide 5 f-T*T*T*T*TAGGAGGAGGAGGGAGAGCGCAAAUGAGAUU-.

3 and '(N-maleimrido)-3-oxapentyl-{2-[3-carboxybenzoyl)-thio]-acetyl}glycylglycyiglycinate mg of the thio-contaiing oligonucleotide produced according to example is mixed under N 2 in 2 ml of phosphate buffer (pH1 7.4) with 1 mg of mal .eimido)-,3-oxapentyl-{2-[3-carboxy-benzoyl)tioI-acetyl}-glycyiglycylglycinate (produced according to Bioconj. Chem. 431 (1990)), dissolved in 0.2 ml of dixnethylfor mamide. It is allowed to stir for 2 hours at room temperature and the solution is -subjected to an ultrafiltration, through a membrane (Amicon YM 3) and. then, a freeze-drying. 5.5 mg of the; desired, conjugate is obtained.

b) Technietium-99m complex of the conjugate 5'-(6-mercapto-1 -hexyl-phosphoric acid, ester) of the' 35mer-oligonticleotide T*T*T*T*TAGGAGGAGGAGGGAGAGCGCAAAUGAGAUU-3 'and 5' (Nrnaleimido)-3 .oxapentyl- {2-[3-carboxybenzoyl)-Ithio]-acetyl}-glycylglycylglycinate 1 ml of a solution of potassium-D-glucarate (12 mg/mi) and tin(fl) chloride (100 g.g/ml) in 0.2 M NaHCO 3 is freeze-dried in a vial and then mixed with [Tc- 99m]-sodium pertechnetate solution (1 nil, 1 mCi) from an Mo-99ITc-99m generator.

After standing at room temperature for 15 minutes, an aliquot is removed and mixed with the same volume of the conjugate of example 8a) (1 mg/mi) dissolved in 0.2 M NaHCO 3 solution. The substance can be isolated by freeze-drying. The tracer yield determined by HPLC is 96%.

Example 9 a) Conjugate of 5' -(6-mercapto-l1-hexyl-phosphonic acid ester) of the oligonucleotide 5'-T*T*T*T*TAGGAGGAGGAGGGAGAGCGCAAAUGAGAUU-3' and 1 -[6-(2-vinyl-6-hexyloxymethyl)-pyridine]- 1,4,8,1 1-tetraazacyclotetradecane A solution of 4 mg of the thio-containing oligonucleotide, produced according to example 5a), in 2 ml of phosphate buffer (pH 7.4) is mixed under N 2 with 1 mg of 1 -[6-(2-vinyl-6-hexyl-oxymethyl)-pyridine]-1 1 1-tetraazacyclotetradecane Q1 TMIZTITI IT Z Quccr tr)i I, r nrx WO 96/02274 PCT/EP95/02539 -37- (produced according to EP 0 588 229), dissolved in 0.5 ml of dimethylfornmide. It is allowed to stir for 4 hours at 35 0 C, mixed with 10 ml of ethanol and the product is isolated by centrifuging. The purification takes place by reversed-phase chromatography on a 1 x. 25 cm. column with a 50 mmol triethylammonium acetate (pH 7) acetonutrile gradient. The combined fractions are freeze-dried, dissolved in 1 m-l of water and desalted on a Sephadex G-10- column. The title compound (about 4 mg) is isolated by freeze-drying.

b) Tc-99mn complex of the conjugate of 6 -mercapto-l-hexyl-phosphoric acid ester) of the 3 5mer-oligonucleotide T*T*T*T*TAGGAGGAGGAGGGAGAGCGCAAAUGAGAUU-3 and 1 -1 6 -(2-vinyl- 6 -hexyloxymethyl)-pyridinel. 1,4,8, 11 -tetraazacyclotetradecane The procedure -is performed as described in example 8b). -The tracer yield, determined by HPL.," is 92 Example a) Conjugate of 6 -mercapto-l-hexyl-phosphoric acid ester) of the oligonucleotide 3 'and N-[4-hydroxy-3-(1 ,4,8,11 -tetraazacyclotetradec5yl)Ibezyl-2-( 6 vinyl prdin 2 -ylmethoxy)-acetmide A solution of 6.5 mg of the thiol-containing oligonucleotide, produced according to example 5a), in 2 ml of phosphate buffer (pH 8.0) is mixed with 1.2 mg of N-[4-hydroxy-3{j ll-tetraazacyclotetradecy)bezl2-(6vinyl-pyridn 2 ylmethoxy)-acetanmjde (produced according to J. Chem. Soc., Chem. Commun. 156 (1988)), dissolved in 0. 1 ml of dimethylformaxnide. It is stirred for 4 hours under N2 at 350C2, mixed with 10 ml of ethanol and the product is isolated by centrifuging. The purification takes place by reversed-phase chromatography on a 1 x 25 cm column with a 50 mmol triethylamrininum acetate (pH 7 )/acetonitrile gradient. The combined fractions are desalted on a Sephadex-G-10 column. By freeze-drying, 5 mg of the title compound is obtained as white powder.

b) Copper-64 complex of the conjugate of 6 -mercapto-1-hexyl-phosphopjc acid ester) of the 35mer oligonucleotide

T*T*T*T*TAGGAGGAGGAGGGAGAGCGCAA.AUGAGAUU-

3 and N-[4hydroxy-3-(1 ,4 11l-tetraaza-cycloteradec5syl).bel 2(6viylypyidn 2 ylmethoxy)-acetamide vi it) c-ri-, i rr, WO 96102274 PCT/EP95/02539 -38- 1 mg of the conjugate obtained according to 10a) is incubated in 1 ml of phosphate buffer (pH 8) With 6CUCI 2 (0.2 mCi). The tracer yield, determined after 1 hour by HPLC, is 98% The product is isolated by freeze-drying.

Example 11 a) Conjugate of 5 '-(6-mercapto- 1-hexyl-phosphoric acid ester) of the oligonucleotide 5'-T*T*T*T*TAGGAGGAGGAGGGAGAGCGCAAUGAGAUU- 3 and 1,4,7,10-eraaylddcae2[5aa8mlimd--x)otn]1,4,7,10tetraacetic acid 1 mg of 1,4,7, lO-tetraazacyclododecane2[(-aza8malein-id-6-oxo)-octaneJ 1,4,7, 1O-tetraacetic acid (produced. according to J. Chem. Soc., Chem. Conimun. 796, (1989)) is added to a solution of 5 mg of the thiol-containing oligonucleotide, produced according to example 5a), in, 2 ml of phosphate buffer (pH 8) under N 2 It is stirred for 3 hours at 35 0 C, mixed with 10 nil of isopropyl alcohol and the product is isolated by centrifuging. The purification takes place by reversed-phase chromatography on a 1 x 25 cm. column with a 25 ninol triethylainmonium acetate (pH 7)/acetonitrile gradient. The combined fractioUS -are gently concentrated by evaporation in a vacuum, dissolved in a little water and desalted with the help of a Sephadex-G-.10 column. By freeze-drying, 4 mng of the title icompound is obtained as white powder.

b) Yttrium-90 complex of the conjugate of 5 6 -mercapto-1-hexyl-phosphonic acid ester) of the 35mer oligonucleotide 5f-T*T*T*T*TAGGAGGAGGAGGGAGAGCGCAAAJUGAGAUU-3, and 1,4,7,10teraaylddcn--(-za8mliio6oo-cae 7, 10-tetraacetic acid (1 mCi), dissolved in 1 ml of 0.05 M amnmonium acetate solution, is mixed with 1 mig of the conjugate produced according to example 11 la) and heated for 1 hour to 85 0 C. The tracer yield, determined by HPLC, is 95 The product is isolated by freeze-drying.

Example 12 a) Conjugate of 5 '-(6-mercapto-1-hexyl-phosphonic acid ester) of the oligonucleotide 5 '-T*T*T*T*TAGGAGGAGGAGGGAGAGCGCAAJUGAGAUU..3' and 1,4 7 -triazacyclononane-2-[(5-aza-8-maleirnido6oxo)-octane].1 ,4,7-triacetic acid 1 mg of 1,4,7,10tizccooae2[5aa--aemd--x)otn] 1 ,4,7-triacetic acid (produced according to J. Chem. Soc., Chem. Conunun. 794, q1 IRSTITI ITP qWPr:T (PI 1i r: 1)rn WO 96/02274 PCT/EP95/02539 -39- (1989)) is added to a solution of 5 mg of the thiol-containing oligonucleotide, produced according to example 5a), in 2 ml of phosphate buffer (pH 8) under N 2 It is stirred for 6 hours at 35 0 C, mixed with 10 ml of isopropanol and the product is isolated by centrifuging. The purification takes place by reversed-phase chromatography on a 1 x 25 cm column with a 25 mmol triethylammonium acetate (pH 7)/acetonitrile gradient. The combined fractions are gently concentrated by evaporation in a vacuum, dissolved in a little water and desalted with the help of a Sephadex G-10 column. By freeze-drying, 3 mg of the title compound is obtained as white powder.

b) Gallium-67 complex of the conjugate of 5'-(6-mercapto-l-hexyl-phosphonic acid ester) of the 35mer oligonucleotide T*T*T*T*TAGGAGGAGGAGGGAGAGCGCAAAUGAGAUU-3' and 1,4,7triazacyclononane-2-[(5-aza-8 -maleimido-6-oxo)-octane]-1,4,7-triacetic acid 20 mg of the conjugate produced according to example 12a) is dissolved in ml of 0.1 M citrate buffer of pH 4.5 and mixed with 0.1 ml of a gallium-67-citrate solution (0.2 mCi). It is allowed to stand for 2 hours at room temperature and the product is desalted on a Sephadex-G-10 column. After freeze-drying, 17 mg of the title compound is obtained as white powder.

Example 13 a) Phosphitylation of 5'-0-(4,4'-dimethoxytrityl)-5-(prop-2-en- 1-one)-2'-deoxyuridine mg of 4-dimethylaminopyridine, 3 ml of diisopropylethylamine and 962 tl (4.31 mmol) of 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite are added in succession to a stirred solution of 2.1 g (3.59 mmol) of 5'-0-(4,4'-dimethoxytrityl)-5- (prop-2-en-l-one)-2'-deoxyuridine (produced according to Nucleosides Nucleotides 13, 939-944, (1994)) in 50 ml of tetrahydrofuran. After about 30 min, a white precipitate is formed. It is filtered, the solution is concentrated by evaporation in a vacuum and the residue is spread between dichloromethane and 5 sodium bicarbonate solution. The dichloromethane phase is dried on magnesium sulfate and concentrated by evaporation in a vacuum. The residue is purified by quick chromatography on silica gel, and it is eluted with dichloromethane/hexane/diisopropylethylamine (80:18:2). 1.8 g of the desired compound is obtained as a white foam.

Elementary analysis: Cld: C 64.28 H 6.29 N 7.14 P 3.95 Fnd: C 64.02 H 6.60 N 7.21 P 4.09 Q1 IP(ZTI1 t CUCC Int ir nrn WO 96/02274 PCTIEP95/02539 b) Conjugate of the 36mer oligonucleotide U*T*T*T*T*TCUCAUGGAGCCAAGACGAAUAGCUACAUA-3' and 10-(4-aza-2hydroxy-5-imino-8-mercapto-octane-1,4,7-tris(carboxymethyl)-1,4,7,10tetraazacyclododecane 5-(prop-2-en-1-one)-2'-deoxyuridine The 30mer-oligonucleotide identified according to the SELEX process is produced with the modification of a 5'-linked sequence 5'-T*T*T*T*T in the usual way in an automatic synthesizer of the Pharmacia company (see Oligonucleotides and to Analogues, A Practical Approach, Ed. F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1991), and the oligonucleotide is also present on the column of the solid vehicle. 'By reaction with trichloroacetic acid solution in dichloromethane, the group is opened. The load on the column is about 10 mg of the oligonucleotde. The 5hydroxy group is reacted in the presence of tetrazole with the phosphoramidite obtaind according to example 13a). Then, the phosphite is.

Sconverted to the phosphtriester by treatment with iodine solution and the terminal DMT radical istleaived -rteaction with trichloroacetic acid solution in l dichldioroiethane. For' additioin of a thiol groiup to the a,-unsaturated carbonyl system present on the terminal 2'%deoxyuridine, it is reacted with a solution of 10-(4-aza-2a) hydroxy-5-imino-8-inercapto-octane)-1,4,7-tris(carboxymethyl)-1,4,7, 0-tetraazacyclododecane*) in tetrahydrofuran and washed in succession with methanol and water. To remove the modified oligonucleotide from the solid vehicle, the contents of the column are conveyed in a multivial, mixed with 5 ml of 30% ammonia solution, the vessel is sealed and shaken overnight at 55 0 C. It is then cooled to GoC, centrifuged, the vehicle is washed with 5 ml of water and the combined aqueous phases are subjected to a freeze-drying.

For purification, the solid material is taken up in 2 ml of water, mixed with 2 ml of 0.5 M ammonium acetate solution and mixed with 10 ml of ethanol, it is allowed to stand overnight at -20 0 C, centrifuged, the residue is washed with 1 ml of ethanol 20 0 C) and finally dried in a vacuum at room temperature.

6 mg of the title compound is obtained as 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: 15.7 ml of IN sodium hydroxide solution and 480 mg (3.49 mmol) of 2iminotetrahydrothiophenehydrochloride are added to a solution of 1.46 g (3.49 mmol) of 10-(3-amino-2-hydroxy-propyl)-1,4,7-tris-(carboxymethyl)-1,4,7,10tetraazacyclododecane (see example Ic) in a mixture of 50 ml of water and 50 ml of C I TCTIT I IT c2U r Mri II r nr~ WO 96/02274 PCT/EP95/02539 -41methanol and stirred for 3 hours at room temperature, concentrated by evaporation in a vacuum to about 1/4 of the initial volume and mixed with stirring with an anion exchanger (IRA 410) until a pH of 11 is reached. The solution is filtered and mixed with stirring in small portions with enough cation exchanger IRC 50 until a pH of is reached. After filtering, the solution is freeze-dried. 1.39 g of the desired substance is obtained as white powder with a water content of 4.9%.

Elementary analysis (relative to the anhydrous substance): Cld: C 48.45 H 7.74 N 16.14 S 6.16 Fnd: C 48.30 H 7.98 N 16.05 S 6.44 c) Yttrium-90 complex of the conjugate of the 36mer oligonucleotide U*T*T*T*T*TCUCAUGGAGCCAAGACGAAUAGCUACAUA-3' and 10-(4-aza-2- Shydroxy-5-imino-8-mercapto-octane)-L,4,7-tris(carboxymethyl)-1,4,7,10- S' tetrazacyclododecane 15, 5-(prop-2-en--one)-2'-deoxyuridine A solution of 9yttrium, dissolved in 0.05 M ammonium acetate solution (about 380 mCi), is added to 1 mg of the thio derivative of example 13b) in 0.5 ml of 0.05 M aninanium acetate solution of pH 6, adjusted to pH 5.2 with 3 M acetic acid and stirred for 1 hour at room temperature. It is adjusted to pH 7.0 with 0.01 M sodium 20 hydroxide solution and the conjugate is purified by HPLC (TSK-400/MES-buffer).

The main fractions are combined, diluted with physiological common salt solution 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-triphenylmethyl mercaptoacetic acid and 1.83 g 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 mmol) of dicyclohexylcarbodiimide, dissolved in 50 ml of absolute dichloromethane, is instilled with ice cooling. It is stirred for 1 hour at 0°C and for 18 hours at room temperature. It is filtered, concentrated by evaporation and chromatographed on silica gel (eluent: CH 2 C12/MeOH: 10%-30%).

Yield: 3.56 g (77.0% of theory), white powder Elementary analysis: Cld: C 67.51 H 5.67 N 6.06 0 13.84 S 11.20 Fnd:. C 67.37 H 6.02 N 5.91 S 6.73 MIRUTITIPH1-FPrT (PI11 F 9M WO 96/02274 FCT/EP95102539 -42b) [S-(Triphenylmethyl-mercaptoacetyl)-glycyl-glycinamidyl -6-hexanol 4.63 g (10 mmol) of S-(triphenyl-methyl-mercaptoacetyl)-glycyl-glycine methyl ester, produced under example 14a), is heated to 100 0 C in 11.72 g (100 mmol) of 6aminohexanol/50 ml of 1,4-dioxane under argon atmosphere for 2 hours. Then, the reaction batch is poured on a mixture of 100 ml of dichloromethane and 100 ml of water. With stirring and ice cooling, a pH of 6 is adjusted by 10 M hydrochloric acid, the organic phase is separated and dried on sodium sulfate. After evaporation of the solvent, the crude product is purified on silica gel (eluent: CH 2 Cl2/MeOH: Yield: 2.97 g (54.2% of theory), white powder Elementary analysis: Cld: C 67.98 H 6.81 N 7.67 O 11.68 S 5.85 Fnd: C67.72 H 6.93 N 7.93 S5.64 c) [S-(Triphenylmethyl-mercaptoacetyl)glycylglycinamidyl-6-hex-1-yl}diisopropylamide-O'-methyl-phosphorous acid diester 5.48 g (10 mmol) of the [S-(triphenylmethyl-mercaptoacetyl)-glycylglycinamidyl]-6-hexanol produced under example 14b) is dissolved in 100 ml of absolute dichloromethane. 5.17 g (40 mmol) of diisopropylethylamine is added under argon atmosphere and 3.95 g (20 mmol) of phosphorous acid mononethylester diisopropylamide chloride, dissolved in 50 ml of absolute dichloromethane, is instilled at o 0 C. It is stirred for 0.5 hour at 0 0 C and then for 2 hours at room temperature.

For working-up, it is mixed under ice cooling with 320 mg (10 mmol) of absolute methanol, and after concentration, chromatographed on silica gel (eluent:

CH

2 C1 2 /MeOH: 95% 5/5% triethylamine).

Yield: 1.98 g (27.9% of theory), colorless oil d) 5'-[(Mercaptoacetyl-glycyl-glycyl-amidyl)-6-hex-1-yl]-phosphoric acid ester of the 35mer oligonucleotide T*T*T*T*TCUCAUGGAGCGCAAGACGAAUAGCUACAUA-3' A 30mer-oligonucleotide, identified according to the SELEX process, with the modification of a 5'-linked sequence 5'-T*T*T*T*T is produced with the help of an automatic synthesizer of the Pharmacia company (see Oligonucleotides and Analogues, A Practical Approach, Ed. F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1991), and the oligonucleotide in protected form is also present on the column of the solid vehicle. The load on the column is about 15 mg of After cleavage of the 5'-DMT-protective group (trichloroacetic acid/dichlorornethane), WO 96/02274 PCT/EP95/02539 -43it is coupled according to the standard methods with the phosphoramidite described under example 14c). After oxidation with iodine in tetrahydrofuran, the conjugate is cleaved off from the vehicle. In this case, the material is mixed with 10 ml of ammonia solution, the vessel is sealed and shaken overnight at 55 0 C. It is cooled to 0 0 C, centrifuged, the vehicle is washed with 10 ml of water and the combined aqueous phases are subjected to a freeze-drying.

For purification, the material is taken up in 5 ml of water, mixed with 4 ml of M ammonium acetate solution and mixed with 20 ml of ethanol. For completion of the precipitation, it is cooled overnight (-20 0 centrifuged, the residue is washed with 1 ml of ethanol (-200C) and dried in a vacuum. 9 mg of a white powder is obtained.

For cleavage of the S-trityl protective group, the material is taken up in 5 ml of mmol triethylammonium acetate solution (pH 7.0) and incubated with 500 pl of 0.1 M silver nitrate solution for 30 minutes. Then, 500 1l of 0.14 M dithiothreitol solution is added and incubated for another 30 minutes. After centrifuging, the clear supernatant is desalted on Sephadex G 10. The fractions containing the product are freeze-dried. 4 mg of the conjugate is obtained as white powder.

e) Technetium-99m complex of the conjugate amidyl)-6-hex-l-yl]-phosphoric acid ester of the 35mer oligonucleotide '-T*T*T*T*TCUCAUGGAGCGCAAGACGAAUAGCUACAUA-3' 1 mg of conjugate 14d) is dissolved in 1 ml of 0.1 M disodium hydrogen phosphate buffer (pH After adding 10 mg of disodium tartrate, it is mixed with sodium pertechnetate solution (1 mCi) and then with 10 ml of tin(II) chloride solution (5 mg of SnCl 2 /1 ml of 0.01 M HCI). The tracer yield (about 93%) is determined by HPLC.

Example a) [S-(Triphenylmethyl-mercaptoacetyl)-glycyl-glycinamidyl]-6-hex-1-yl toluenesulfonic acid ester 5.48 g (10 mmol) of the [S-(triphenylmethyl-mercaptoacetyl)-glycylglycinamidyl]-6-hexanol produced under example 14b) is 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 stirred for 24 hours at room temperature. Then, it is concentrated and chromatographed on silica gel (eluent:

CH

2 C1 2 /MeOH: 95:5).

Yield: .4.32 g (61.5% of theory), colorless oil IN IPITITI Tr CUC-r mi it r nen\ WO 96/02274 PCT/EP95/02539 -44- Elementary analysis: Cld: C 65.03 H 6.18 N 5.99 O13.68 S 9.14 Fnd: C 64.93 H 6.32 N 5.78 S 8.87 b) 5'-[(Mercaptoacetyl-glycamidyl)-6-hex-1-yl]-phosphoric acid ester of the oligonucleotide 5'-T*T*T*T*TCUCAUGGAGCGCAAGACGAAUAGCUACAUA-3, A 3 0mer-oligonucleotide, identified according to the SELEX process, with the modification of a 5'-linked sequence 5'-T*T*T*T*T is produced with the help of an automatic synthesizer of the Pharmacia company (see Oligonucleotides and Analogues, A Practical Approach, Ed. F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1991), and the oligonucleotide in protected form is also present on the column of the solid vehicle. The load on the column is about 15 mg of 3 After cleavage of the 5'-DMT-protective group (trichloroacetic acid/dichloromethane), it is coupled according to the standard methods with S-trityl-6-mercapto-hexylphosphoramidite. After oxidation with iodine in tetrahydrofuran, the trityl-protected compound is cleaved off from the vehicle, isolated and purified (see example 14d).

The cleavage of the S-trityl protective group, the isolation of the SH-groupcarrying oligonucleotide and the purification take place as described under example 14d) (6 mg).

For coupling, the SH-group-carrying 35mer-oligonucleotide (6 mg) in 500 pl of 0.1 M sodium carbonate solution is mixed under argon atmosphere with 100 mg of toluenesulfonic acid ester 15a), dissolved in 500 pl of dimethylformamide. After minutes, it is neutralized and diluted with water to a volume of 5 ml. After the centrifuging, the clear supernatant is freeze-dried.

For cleavage of the S-trityl groups, the procedure is performed as described under 14d). After purification, 4 mg of the conjugate is obtained.

c) Technetium-99m complex of the conjugate glycinamidyl)-6-hex-l-yl]-phosphoric acid ester of the 35mer oligonucleotide 5'-T*T*T*T*TCUCAUGGAGCGCAAGACGAAUAGCUACAUA-3' 1 mg of the conjugate described under example 15b) is dissolved in 1 ml of 0.1 M disodium hydrogen phosphate buffer (pH After adding 10 mg of disodium tartrate, it is mixed with sodium pertechnetate solution (1 mCi) and then with 10 pl of tin(n) chloride solution (5 mg of SnCl 2 /1 ml of 0.01 M HC1). The tracer yield (about is determined by HPLC.

.q1IRTITi ITP q4C'r:Tt I~ it C ni WO 96102274 PCT/EP95/02539 Example 16 a) N-[3-Thia-5-(triphenylmethylmercapto)-1 -oxo-pent- l-yl]-S-tripheniylmethyl-cysteine methyl ester 2.69 g (10 rnrol) of N-[ 3 -thia-5-(triphenylmethylmercapto)1..oxo.pent-1 -ylj-Striphenylmethyl-cysteine methyl ester (production according to DE 43 10 999) is dissolved together with 5.58 g (20 mruol) of triphenyhuethyl chloride in 100 ml of absolute dichloromethane. After adding 2.02 g (20 mmol) of trietbylamine, it is allowed to stir overnight under argon atmosphere at room temperature. For workingup, the organic phase is washed three times in each case with 1 citric acid solution, saturated sodium bicarbonate solution and with water. After drying on sodium sulfate, it is concentrated by evaporation and purified on silica gel (eluent: C11 2 C1 2 /Me OH: 95:5).

Yield: 4.53 g (60.1 of theory) of colorless oil Elementary analysis: Old: C 73.27 H 5.75 N 1.86 06.37 S 12.76 Fnd: C073.31 H 5.48 N 1.63 S 12.49.

b) N4[3-Thia-5-(triphenylriethylmercaptay 1-oxo-pent-1 -ylI-S-triphenylmethyl-N'-(6hydroxy-hex-1 -yl)cys,-teinaniide 7.54 g (10 mniol) of the N-[ 3 yl]-S-cysteine methyl ester described under example 16a) is heated to 100PC in 11.72 g (100 mmol) of 6-aminohexanol/50 ml of 1,4-dioxane under argon atmosphere for 2 hours. Then, the reaction batch is poured on a mixture of 100 ml of dichioromethane and 100 ml of water. With stirring and ice cooling, it is adjusted to a pH of 6 with M hydrochloric acid, the organic phase is separated and dried on sodium sulfate.

After evaporation of the solvent, the -crude product is purified on silica gel (eluent:

CH

2 Cl 2 /MeOH: 5 Elementary analysis: Cld: C072.99 H 6.49 N 3.34 05.72 S11.46 Fnd: C072.73 H 6.62 N 3.11 S11.17 c) {[N-[3-Thia-5-(triphenylmethylmercapto)-l1-oxo-pent-1 -yl]-S-triphenylmethylcysteinyl}-2-amino-eth- 1-yl }-diisopropylamide-Q'-methylphosphorous acid diester 8.39 g (10 nunol) of the N-[3-thia-5-(tripheniylmethylmercapto)-l -oxo-pent- 1ylI-S-triphenylmethyl-N '-(6-hydroxy-hex- 1-yl)cysteinamide, produced under example 16b), is dissolved in 200 ml of absolute dichioromethane. 5.17 g (40 mmol) of diisopropylethylamine is added under argon atmosphere and 3.95 g (20 mmol) of phosphorous acid monomethylester-diisopropylamide-chloride, dissolved in 50 ml of IZ? ?DQTITI ITr cucr: /ni r n' WO 96/02274 PCTEP95102539 -46absolute dichloromethane, is instilled at 0°C. It is stirred for 0.5 hour at 0°C and then for 1.5 hours at room temperature. For working-up, it is mixed, with ice cooling, with 320 mg (10 mmol) of absolute methanol and after concentration, chromatographed on silica gel (eluent: CH 2

CI

2 /MeOH: 95:5/5% (triethylamine)).

Yield: 5.37 g (53.7% of theory) of yellowish oil d) 5'-[N-(3-Thia-5-mercapto-1 -oxo-pent-1 -yl]-cysteine-N'-(6-hydroxy-hex-1 -yl)amide]-phosphoric acid ester of the 35mer oligonucleotide 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T-3' A 30mer-oligonucleotide, identified according to the SELEX process, is produced with the modification of a sequence T*T*T*T*T-3' placed upstream with the help of an automatic synthesizer of the Pharmacia company (see Oligonucleotides and Analogues, A Practical Approach, Editor F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1991), and the oligonucleotide in protected form is also present on the column df the solid vehicle. The load on the column is about 15 mg of After cleavage of the 5'-DMT-protective group (trichloroacetic acid/dichloromethane), it is coupled according to.the standard methods with phosphoramidite (f6c) and then oxidized.

The cleavage from the vehicle of the base protective groups and the purification of the bis-S-trityl-protected conjugate take place as described under 14d) (about 12 mg).

For cleavage of the S-trityl protective groups, the material is taken up in 5 ml of 50 mmol triethylammonium acetate solution (pH 7.0) and incubated with 500 gl of 0.1 M silver nitrate solution for 30 minutes. Then, 500 1l of 0.14 M dithiothreitol solution is added and incubated for another 30 minutes. After centrifuging, the clear supernatant is desalted on Sephadex G 10. The fractions containing the product are freeze-dried. 5 mg of white powder is obtained.

e) Technetium-99m complex of the conjugate 5'-[N-(3-thia-5-mercapto-l-oxo-pent-lyl]-cysteine-N'-(6-hydroxy-hex-l-yl)-amide]-phosphoric acid ester of the oligonucleotide 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T-3' 1 mg of the conjugate described under example 16d is dissolved in 1 ml of 0.1 M disodium hydrogen phosphate buffer (pH After adding 10 mg of disodium tartrate, it is mixed with sodium pertechnetate solution (1 mCi) and then with 10 ml of tin(II) chloride solution (5 mg of SnCI 2 /1 ml of 0.01 M HC1). The tracer yield (about 96%) is determined by HPLC.

n( InCITt~! lre WO 96/02274 PCT/EP95/02539 -47- Example 17 a) 3 -Thia-5-(triphenylmethylmercapto)-l-oxo-pent-1-yl]-S-triphenylmethyl-N'- (6-hydroxy-hex-l-yl)cystein-imidyl}-p-toluenesulfonic acid ester 8.39 g (10 mmol) of the N-[ 3 yl]-S-triphenylmethyl-N'-(6-hydroxy-hex-l-yl)cysteinamide, produced under example 16b, is 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 stirred for 20 hours at room temperature. Then, it is concentrated and chromatographed on silica gel. (Eluent: CH 2 C1 2 /MeOH: 97:3).

io Yield: 6.44 g (67.0% of theory) of yellowish oil Elementary analysis: Cld: C 72.47 H 6.29 N 2.91 0 8.32 S 10.01 Fnd: C 72.19 H 6.47 N 2.68 S 9.83 b) 5'-[(Mercaptoacetyl-glycyl-glycinamidyl)-13-tridec-7-thio- 1-yl]-phosphoric acid ester of the 35mer oligonucleotide CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T-3' A 30mer-oligonucleotide, identified according to the SELEX process, is produced with the modification of a sequence T*T*T*T*T-3' placed upstream with the help of an automatic synthesizer of the Pharmacia company (see Oligonucleotides and Analogues, A Practical Approach, Editor F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1991), and the oligonucleotide in protected form is also present on the column of the solid vehicle. The load on the column is about 15 mg of After cleavage of the 5'-DMT-protective group (trichloroacetic acid/dichloromethane), it is coupled according to the standard methods with S-trityl-6-mercaptohexyl-phosphoramidite.

After oxidation with iodine in tetrahydrofuran, the trityl-protected compound is cleaved off from the vehicle, isolated and purified (see example 14d).

The cleavage of the S-trityl protective group, the isolation of the SH-groupcarrying oligonucleotide and the purification take place as described under example 14d) (7.5 mg).

For coupling, the SH-group-carrying 30mer-oligonucleotide (7 mg) in 550 pl of 0.1 M sodium carbonate solution is mixed under argon atmosphere with 180 mg of toluenesulfonic acid ester 17a), dissolved in 500 gl of dimethylformamide. After minutes, it is neutralized and diluted with water to a volume of 5 ml. After the centrifuging, the clear supernatant is freeze-dried. For cleavage of the S-trityl groups, the procedure is performed as described under 14d). After purification, 4.3 mg of conjugate is obtained.

Of IOCTITI IT M CL.rr im, to r hn, WO 96/02274 PCT/EP95/02539 -48c) Technetium-99m complex of the conjugate glycinamidyl)-13-trideca-7-thio-l-yl]-phosphoric acid ester of the oligonucleotide 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T-3' 1 mg of the conjugate described under example 17b) is dissolved in 1 ml of 0.1 M disodium hydrogen phosphate buffer (pH After adding 10 mg of disodium tartrate, it is mixed with sodium pertechnetate solution (1 mCi) and then with 10 pl of tin(II) chloride solution (5 mg of SnCl 2 /1 ml of 0.01 M HCI). The tracer yield (about 93%) is determined by HPLC.

Example 18 a) Conjugate of 6 -amino-l-hexyl-phosphoric acid ester) of the oligonucleotide 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T-3' and N-(tetrahydro-2-oxo-thiophen-3-yl)-thiodiglycolic acid monoamide A 30mer-oligonucleotide, identified according to the SELEX process, is produced with the modification of a sequence T*T*T*T*T-3' placed upstream with the help of an automatic synthesizer of the Pharmacia company (see Oligonucleotides and Analogues, A Practical Approach, Editor F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1991), and the oligonucleotide in protected form is also present on the column of the solid vehicle. The load on the column is about 15 mg of After cleavage of the 5'-DMT-protective group (trichloroacetic acid/dichloromethane), it is coupled according to the standard methods with N-trifluoroacetylaminohexylphosphoramidite.

After oxidation with iodine in tetrahydrofuran, the amino group-carrying oligonucleotide is cleaved off from the vehicle and isolated and purified as described under Ib) (9.3 mg).

For coupling with the N-(tetrahydro-2-oxo-thiophen-3-yl)-thiodiglycolic acid monoamide (DE 43 11 023), 5 mg of the amino group-carrying oligonucleotide is dissolved in 1 ml of 2 M sodium carbonate solution. After adding 100 mg of the thiolactone derivative, it is incubated for 4 hours at room temperature. Then, it is neutralized and the desalting is achieved by ultrafiltration through a membrane with an exclusion limit of 3,000 (Amicon YM3). After lyophilization, it is subjected to a freeze-drying. 6.3 mg of the desired conjugate is obtained.

b) Technetium-99m complex of the conjugate 6 -amino-l-hexyl-phosphoric acid ester) of the 35mer oligonucleotide CI I In C"T'I'm I-rr- WO 96/02274 PCT/EP9502539 -49- CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T-3' and N-(tetrahydro- 2 -oxo-thiophen-3-yl)-thiodiglycolic acid monoamide 1 mg of the SH-group-carrying conjugate described under example 18a) is dissolved in 1 ml of 0.1 M disodium hydrogen phosphate buffer (pH After adding 10 mg of disodium tartrate, it is mixed with sodium pertecmetate solution (1 mCi) and then with 10 gl of tin(II) chloride solution (5 mg of SnCI2/1 ml of 0.01 M HCI). The tracer yield is determined by HPLC.

Example 19 a) 6 -Amino-l-hexyl-phosphoric acid ester) of the 32mer-oligonucleotide S'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT-3'-3'T5 The 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUA-3', identified according to the SELEX process, with the modification of a thymidine sequence placed upstream, which is bound by 5'-position on the vehicle, is produced in the usual way in an automatic synthesizer of the Pharmacia company (see Oligonucleotides and Analogues, A Practical Approach, Editor F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1991), and the oligonucleotide is also present on the column of the solid vehicle. By reaction with trichloroacetic acid solution in dichloromethane, the 5'-hydroxy group is opened. The load on the column is about 10 mg of the 3 2mer-oligonucleotide. To join the linker, the column is reacted with an acetonitrile solution of 50 pmol of -cyanoethyl-N,N-diisopropylamino-6-(trifluoroacetamido)- 1-hexyl-phosphoramidite (produced according to Nucl. Acids. Res. 16, 2659-2669 (1988)) in the presence of tetrazole. The oxidation of the formed phosphite to the completely protected phosphotriester takes place with iodine in tetrahydrofuran. Then, the column is washed in succession with methanol and water. To remove the modified oligonucleotide from the solid vehicle, the contents of the column are conveyed in a multivial, mixed with 5 ml of 30% ammonia solution, the vessel is sealed and shaken overnight at 550C. It is then cooled to 0°C, centrifuged, the vehicle is washed with ml of water and the combined aqueous phases are subjected to a freeze-drying.

For purification, the solid material is taken up in 2 ml of water, mixed with 2 ml of 0.5 M ammonium acetate solution and mixed with 10 ml of ethanol, it is allowed to stand overnight at -20 0 C, centrifuged, the residue is washed with 1 ml of ethanol 20 0 C) and finally dried in a vacuum at room temperature.

8 mg of the title compound is obtained as colorless powder.

C4 1OCTITI ?Tf7 IL!!-rr Il WO 96/02274 PCT/EP95/02539 b) Conjugate of 6 -amino-1-hexyl-phosphoric acid ester) of the 32mer oligonucleotide 5'-CUCAUGGAGCGCAAGACGAJLUAGCUACAUAT_3'-3'T-5 and lO-[ 7 4 -isothiocyanatopheny)2hy drxy-5-oxo7(carboxymethyl)azahpl- 1 4 ,7-tris-(carboxymethyl)-1 lO-tetraazacyclododecane 8 mng of the oligonucleotide obtained in example 19a) is dissolved in 2.5 ml of a NaHCO 3 /Na 2

CO

3 buffer (pH 8.0) and mixed with 1 mng of 10-[7-(4isothiocyantophenyl)2hydroxy5oxo7(crboxymethyl)azaheptI)...l 4 ,7-tris- (carboxymethyl)41,4,7,10-tetraazcycldodecane (title compound of example If). It is stirred for 5 hours at room temperature, the pH1 is adjusted to 7.2 by adding 0.01 Mv hydrochloric acid and the solution is subjected to an ultrafiltration through a membrane with the exclusion limit 3,000 (Amicon YM3) and then a freeze-drying. 7 mng of the desired conjugate is obtained.

c) 1 tt lndiumn complex of the conjugate of 6 -axnino-1-hexyl-phosphoric acid ester) ,of the 32mer oligonucleotide and lO-[ 7 4 -isothiocyanatophenyl)2hydroxy5ox7(caboxmethyl)-4 aza-heptyl]-1 4 7 -:tris-(carboxymethyl)-1 O-tetraazacyclododecane g! ef an ttt indium(III) acetate solution (350 J.LCi), (produced from, t indiurn(III) chloride in 2 M sodium acetate solution and adjustment of the pH to 4. 0 with 0. 1 M hydrochloric acid) is added to 135 gI of a solution of 1 mig of the title compound of example 19b) in MES buffer, pH 6.2 (MES

-N

morpholino)ethylsulfonic acid). The pH is brought to 4.2 by adding, 0.01 M hydrochloric acid. It is stirred for 1 hour at 37 0 C at pH1 4.2. It is brought to pH 6 with 2 M sodium acetate solution and 10 gI of a 0. 1 M Na 2 EDTA solution (Na 2

EDTA

ethylenedianiine-tetraacetic acid disodium salt) is added to complex excess indium. The final purification of thus obtained labeled conjugate (1h) takes place by HPLC (exclusion chromatography: TSK-400/MES-buffer). The fractions containing the labeled conjugate are diluted with physiological common salt solution, adjusted to pH 7.2 with 0.01 M sodium hydroxide solution and filtered. A thus produced solution then represents a suitable preparation for radiodiagnosis.

Example a) S 6 -Amino-1-hexyl'phiosphoric acid ester) of the 35mer oligoniucleotide 3 The 3 0mer-oligonucleotide 5'-TAGGAGGAGGAGGGAGAGCGCAAAUGAGAUU-3' (seq. no. 13 of US Patent No. 5,270,163), identified according to the SELEX process, is produced in the usual WO 96/02274 PCTIEP95/02539 -51way in an automatic synthesizer of the Pharmacia company (see Oligonucleotides and Analogues, A Practical Approach, Ed. F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1991), and the oligonucleotide is also present on the column of the solid vehicle. The four final thymidines are bound by phosphorodithioates to the thymidine present on the 5'-end according to the technique described by G. Blaton et al. in "Oligonucleotides and Analogues," pp. 109-135. For this purpose, first the hydroxy group is opened by treatment with trichloroacetic acid. Then, the 3'-hydroxy group of a 5'-DMTO-thyridine is converted with tris-pyrrolidinophosphine and tetrazole in the diamidite, which is converted to the phosphorothioamidite by adding 2,4dichlorobenzyl mercaptan. This compound is activated with tetrazole and reacted with the 5'-hydroxy group of the oligonucleotide to thiophosphatetriester. The oxidation to phosphorodithioate takes place with elementary sulfur in a solution of carbon disulfide, pyridine, triethylamine 95:95:10. The benzyl groups are not yet removed. In an analogous way, 3 additional thymidines are also bound. The load on the column is about 10 mg of the 35mer-oligonucleotide. Then, a 5'-hydroxy group is again opened.

To join the linker; the column is reacted with an acetonitrile solution of 50 I mol of 2-cyanoethyl-N,N'diisopropylamino-6-(trifluoroacetamido)-1-hexyl phosphoramidite (lit. in example in the presence of tetrazole. The oxidation of the formed phosphite to the phosphotriester takes place with iodine in tetrahydrofuran. Then, the column is washed in succession with methanol and water. Then, the protective groups of the dithionate bonds are removed by a solution of thiophenol/triethylamine/dioxane 1:2:2, which takes place in 2 hours. Thereupon, the column is washed in each case with 3 times its volume with methanol, then washed with ether and dried. To remove the modified oligonucleotide from the solid vehicle, the contents of the column are conveyed in a multivial, mixed with 5 ml of 30% ammonia solution, the vessel is sealed and shaken overnight at 55 0 C. It is then cooled to 0°C, centrifuged, the vehicle is washed with 5 ml of water and the combined aqueous phases are subjected to a freeze-drying.

For purification, the solid material is taken up in 2 ml of water, mixed with 2 ml of 0.5 M ammonium acetate solution and mixed with 10 ml of ethanol, it is allowed to stand overnight at -20 0 C, centrifuged, the residue is washed with 1 ml of ethanol 0 C) and finally dried in a vacuum at room temperature.

8 mg of the title compound is obtained as colorless powder.

b) Conjugate of 5'-(6-amino-l-hexyl-phosphoric acid ester) of the oligonucleotide 5'-T***T***T***T***TAGGAGGAGGAGGGAGAGCGCAAAUGAGAUU-3 and WO 96/02274 PCTAEP95/02539 -52- 10-[7-(4-isothiocyanato-phenyl)-2-hydroxy-5-oxo-7-(carboxymethyl)-4-aza-heptyl]- 1,4,7-tris(carboxymethyl)- 1,4,7, 8 mg of the oligonucleotide obtained in example 20a) is dissolved in 2.5 ml of a NaHCO 3 /Na 2

CO

3 buffer (pH 8.0) and mixed with 1 mg of 10-[7-(4isothiocyanatophenyl)-2-hydroxy-5-oxo-7-(carboxymethyl)-4-aza-heptyl]-1,4,7-tris- (carboxymethyl)-1,4,7,10-tetraazacyclododecane (title compound of example 1if). It is stirred for 5 hours at room temperature, the pH is adjusted to 7.2 by adding 0.01 M hydrochloric acid and the solution is subjected to an ultrafiltration through a membrane with the exclusion limit 3,000 (Amicon YM3) and then a freeze-drying.

7 mg of the desired conjugate is obtained.

c) 11'Indium complex of the conjugate 6 -amino-1-hexyl-phosphoric acid ester) of the 35mer oligonucleotide 5'-T***T***T***T***TAGAGGAGGAGGGAGAGCGCAAAUGAGAUU-3 and 10-[7-(4-isothiocyanatophoniyl)-2-hydroxy-5-xo-7-(carboxymethyl)4-azaTheptyl]- '-1,4,7-tris(carboxymethyl)- pl of an Illindium(Il) acetate solution (350 .pCi), @(produced from 1 1 indium(III) chloride in 2 M sodium acetate solution and adjustment of the pH to with 0.1 M hydrochloric acid) is added to 135 gl of a solution of 1 mg of the title compound of example 20b) in MES buffer, pH 6.2 (MES 2-(Nmorpholino)ethylsulfonic acid). The pH is brought to 4.2 by adding 0.01 M hydrochloric acid. It is stirred for 1 hour at 37 0 C at pH 4.2. It is brought to pH 6 with 2 M sodium acetate solution and 10 pl of a 0.1 M Na 2 EDTA ethylenediaminetetraacetic acid disodium salt is added to complex excess 11 1indium. The final purification of thus obtained conjugate (1h) takes place by HPLC (exclusion chromatography: TSK-400/MES-buffer). The fractions containing the labeled conjugate are diluted with physiological common salt solution, adjusted to pH 7.2 with 0.01 M sodium hydroxide solution and filtered. A thus produced solution then represents a suitable preparation for radiodiagnosis.

Example 21 a) 5'-(6-Amino-1-hexyl-phosphoric acid ester) of the 35mer oligonucleotide '-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT**T**T* The 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUA-3', identified according to the SELEX process, with the modification of a sequence placed upstream, is obtained by first a sequence of 5 thymidines connected by cyanoethyl- I I WO 96/02274 PCT/EP95/02539 -53phosphate groups being produced on the vehicle by By reaction of this compound with a 0.5 M solution of tetraethylthiuram disulfide in acetonitrile, the sulfonation to phosphonothioate takes place within 15 minutes, which then with free group is starting material for the 35mer oligonucleotide. The entire synthesis takes place in the usual way in an automatic synthesizer of the Pharmacia company (see Oligonucleotides and Analogues, A Practical Approach, Editor F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1991), and the oligonucleotide is still present on the column of the solid vehicle. By reaction with trichloroacetic acid solution in dichloromethane, the 5'-hydroxy group is opened. The load on the column is about 10 mg of the 3 5mer-oligonucleotide. To join the linker, the column is reacted with an acetonitrile solution of 50 pmol of P-cyanoethyl-N,N-diisopropylamino-6- (trifluoroacetamido)-l-hexyl-phosphoramidite (produced according to Nucl. Acids.

Res. 16, 2659-2669 (1988)) in the presence of tetrazole. The oxidation of the phosphite formed in such a way to the completely protected phosphotriester takes place with iodine:in tetrahydrofuran. Then, the column is washed in successionewith methanol and water. To remove the modified oligonucleotide from the solid vehicle and to cleave the cyanoethyl groups, the contents of the column are conveyed in a multivial, mixed with 5 ml of 30% ammonia solution, the vessel is sealed and shaken overnight at 55 0 C. It is then cooled to 0°C, centrifuged, the vehicle is washed with ml of water and the combined aqueous phases are subjected to a freeze-drying.

For purification, the solid material is taken up in 2 ml of water, mixed with 2 ml of 0.5 M ammonium acetate solution and mixed with 10 ml of ethanol, it is allowed to stand overnight at -20 0 C, centrifuged, the residue is washed with 1 ml of ethanol 0 C) and finally dried in a vacuum at room temperature.

8 mg of the title compound is obtained as colorless powder.

b) Conjugate of 5'-(6-amino-l-hexyl-phosphoric acid ester) of the oligonucleotide 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT**T**T**T**T-3' and 10-[7- (4-isothiocyanatophenyl)-2-hydroxy-5-oxo-7-(carboxymethyl)-4-azaheptyl]-1,4,7tris(carboxymethyl)- ,4,7,10-tetraazacyclododecane 8 mg of the oligonucleotide obtained in example 20a) is dissolved in 2.5 ml of a NaHC0 3 /Na 2

CO

3 buffer (pH 8.0) and mixed with 1 mg of 10-[7-(4isothiocyanatophenyl)-2-hydroxy-5-oxo-7-(carboxymethyl)-4-aza-heptyl]-1,4,7-tris- (carboxymethyl)-l,4,7,10-tetraazacyclododecane (title compound of example if). It is stirred for 5 hours at room temperature, the pH is adjusted to 7.2 by adding 0.01 M hydrochloric acid and the solution is subjected to an ultrafiltration through a membrane with the exclusion limit 3,000 (Amicon YM3) and then a freeze-drying.

n' 'Do-riII~ r rrr r WO 96/02274 PCTIEP95/02539 -54- 7 mg of the desired conjugate is obtained.

c) 111'ndium complex of the conjugate 6 -amino-1-hexyl-phosphoric acid ester) of the 35mer oligonucleotide 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT**T**T**T**T-3' and 10-[7- (4-isothiocyanatophenyl)-2-hydroxy-5-oxo-7-(carboxymethyl)-4-aza-heptyl]-,4,7tris(carboxymethyl)- 1,4,7,10-tetraazacyclododecane tl of an 11 indium(I) acetate solution (350 (produced from 1 lindium(Ii) chloride in 2 M sodium acetate solution and adjustment of the pH to with 0.1 M hydrochloric acid) is added to 135 pl of a solution of 1 mg of the title compound of example 21b) in MES buffer, pH 6.2 (MES 2-(Nmorpholino)ethylsulfonic acid). The pH is brought to 4.2 by adding 0.01 M hydrochloric acid. It is stirred for 1 hour at 37 0 C at pH 4.2. It is brought to pH 6 with 2 M sodium acetate solution and 10 gl of a 0.1 M Na 2 EDTA ethylenediaminetetraacetic acid disodium salt is added to complex excess "lindium. The final purification of thus obtained conjugate (lh) takes place by HPLC (exclusion chromatography: TSK-400/MES-buffer). The fractions containing the labeled conjugate are diluted with physiological common salt solution, adjusted to pH 7.2 with 0.01 M sodium hydroxide solution and filtered. A thus produced solution then represents a suitable preparation for radiodiagnosis.

Example 22 a) N-(5-Mercapto-3-thia-l-oxo-pent-l-yl)-glycine methyl ester 12.56 g (0.1 mol) of glycine methyl ester hydrochloride, 13.42 g (0.1 mol) of and 10.12 g (0.1 mol) of triethylamine are dissolved under argon atmosphere in 500 ml of absolute dichloromethane. It is stirred for 24 hours at room temperature and the batch is then poured on 250 ml of 5% aqueous citric acid.

It is well-stirred, the organic phase is separated and dried on sodium sulfate. After evaporation of the solvent in a vacuum, the oily residue is chromatographed on silica gel (mobile solvent: dichloromethane/methanol, methanol 0-10%) Yield: 18.9 g colorless oil Elementary analysis: Cld: C 37.65 H 5.87 N 6.27 0 21.49 S 28.71 Fnd: C 37.43 H 6.02 N 6.12 S 28.48 b) N-[5-(Triphenylmethylmercapto)-3-thia-l-oxo-pent-1-yl]-glycine methyl ester WO 96/02274 PCT/EP95/02539 11.17 g (50 mmol) of N-(5-mercapto-3-thia-l-oxo-pent-l-yl)-glycine methyl ester (example 22a), 13.94 g (50 mmol) of triphenylmethyl chloride and 5.06 g mmol) of triethylamine are dissolved under argon atmosphere in 500 ml of absolute dichloromethane. It is stirred for 16 hours at room temperature and the batch is then poured on 150 ml of 5 aqueous citric acid. It is well-stirred, the organic phase is separated and dried on sodium sulfate. After evaporation of the solvent in a vacuum, the oily residue is chromatographed on silica gel (mobile solvent: dichloromnethane/methanol, 95:5).

Yield: 15.7 g colorless oil Elementary analysis: Cld: C 67.07 H 5.85 N 3.01 0 10.31 S 13.77 Fnd: C 67.01 H 6.11 N 2.93 S 13.49 c) N-[5-(Triphenylmethylmercapto)-3-thia-l-oxo-pent-1-yl]-glycine-N'-(6hydroxyhexyl)-amide 11.64 g (25 mmol) of N-[5-triphenylmethylmercapto)-3-thia-l-oxo-pent-l-yl]glycine methyl ester (example 22b) and 29.3 g (250 mmol) of 6-aminohexanol are .melted together under argor atmosphere for 16 hours at 100°C. After cooling down the reaction batch, it is taken up in 500 ml of dichloromethane and poured on 250 ml of 5% aqueous citric acid., Under ice cooling and stirring, it is adjusted to a pH of with concentrated hydrochloric acid. The organic phase is separated and dried on sodium sulfate. After evaporation of the solvent in a vacuum, the oily residue is chromatographed on silica gel (mobile solvent: dichloromethane/methanol, 0-10% methanol).

Yield: 7.7 g colorless oil Elementary analysis: Cld: C 67.60 H 6.96 N 5.09 0 8.72 S 11.64 Fnd: C 67.48 H 7.03 N4.92 S 11.43 d) N-Diisopropyl-O-cyanoethyl-0'-[7,10-diaza-8,11-dioxo-13-thia-15- (triphenylmethyl-mercapto)-pentadec-l-yl]-phosphorous acid amide 5.51 g (10 mmol) of N-[5-(triphenylmethylmercapto)-3-thia-l-oxo-pent-l-yl]glycine-N'-(6-hydroxyhexyl)-amide (example 22c) is dissolved in 50 ml of absolute dichloromethane and 50 ml of absolute pyridine. 4.73 g (20 mmol) of diisopropylamino-O-cyanoethyl-phosphorous acid chloride, dissolved in 50 ml of absolute dichloromethane, is instilled in the batch at room temperature. After 4 hours, it is poured on 250 ml of saturated, aqueous sodium bicarbonate solution, stirred and the organic phase is dried on magnesium sulfate. After evaporation of the solvent, the 0I IRITMTE SHFFT (RI 11E 28) WO 96/02274 PCT/EP95/02539 -56oily residue is chromatographed on silica gel (dichloromethane/methanoltriethylamine 98:1:1).

Yield: 4.32 g colorless foamy oil Elementary analysis: Cld: C 63.97 H 7.38 N 7.46 0 8.52 P 4.12 S 8.54 Fnd: C 63.81 H 7.41 N 7.22 P 3.97 S 8.31 e) 5'-[N-(3'-Aza-8'-mercapto-l' ,4'-dioxo-6'-a-oct-l' -yl)6aminohexyl-phosphoric acid ester] of 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T.T.T.-3' A 30mer-oligonucleotide identified according to the SELEX process, with the modification of a sequence placed upstream, is produced with the help of an automatic synthesizer of the Pharmacia company (see Oligonucleotides and Analogues,

A

Practical Approach, Ed. F. Eckstein, Oxford University Press, Oxford, New York, Tokyo, 1931), and the oligonucleotide in protected form is still present on the column of the solid vehicle. The load on the column is about 15 mg of 3 After cleavage of the 5'-DMT-protective group, it is coupled according to the standard methods with the phosphoramidite described under example 22d. After oxidation with iodine in tetrahydrofuran, the conjugate is cleaved off from the vehicle. For this purpose, the material is mixed with 10 ml of 30% ammonia solution and shaken overnight at 55 0 C. It is cooled to 0OC, centrifuged, the vehicle is washed with 10 ml of water and the combined aqueous phases are subjected to a freeze-drying. For purification, the material is taken up in 5 ml of water, 4 ml of 0.5 mol ammonium acetate is added and mixed with 20 ml of ethanol. For completion of the precipitation, it is cooled overnight (-20 0 centrifuged, the residue is washed with 1 ml of ethanol (-20 0 C) and dried in a vacuum. 9.5 mg of white powder is obtained. For cleavage of the S-trityl protective group, the material is taken up in 5 ml of 50 mmol triethylammonium acetate solution (pH=7) and incubated with 500 pl of 0.1 M silver nitrate solution for minutes. Then, 500 pl of 0.14 M dithiothreitol solution is added and incubated for another 30 minutes. After centrifuging, the clear supernatant is desalted on Sephadex G 10. The fractions containing the product are freeze-dried. 3.5 mg of white powder is obtained.

f) Tc-99m Complex of 5'-[N-(3'-aza-8'-mercapto-l 4 '-dioxo-6'-thia-oct-.'-yl)-6aminohexyl-phosphoric acid ester] of CUACAUAT*T*T*T*T*-3' 1 mg of the conjugate described under example 22e) is dissolved in 1 ml of 0.1 M disodium hydrogen phosphate buffer (pH After adding 10 mg of disodium SUBSTITUTE SHEET (RULE 26) WO 96/02274 PCT/EP95/02539 -57tartrate, it is mixed with sodium perteclmetate solution (1 mQi and then with 10 pa Of tin(II) chloride solution (5 mg/i Ml of 0.01 M HQI. The tracer yield (about 95%) is determined by HPLC.

Example 23 a) S -Aza-7 '-hydroxycarboxy-9'-mercapto. ,5 -dioxo-3 'thia-nonl'yl6aminohexylphosphoric acid ester] of 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*TTT-3 First, a solution of the N-hydroxysuccnimid ester of N-(2-oxotetrahydrothiophen3yl)-thodigycolic acid monoamide (DE 43 11 023) in 500 jgd of absolute DMF is produced. For this purpose, 24.9 mg 1 mmol) of N-( 2 -oxo-tetra.

hydrothyophen3yl)thiodiglycolic acid monoamide and 1 5 mg 1 mniol) of Nhydroxysuccinhimide are dissolved in 500 Ald of absolute DMF. At O 0 C, 19.2 mg 1 of EDC Is added to the batch. It is stirred for 30 minutes at 0 0

C.

mg. of the 6 -amino..hexyl-phosphoric'acid ester)- of CUCAUGGAGCGCAAGACGAAUAGCUACAUA*TTT-3 produced under example I s dissolved, in 1 ml of a sodium bicarbonate/sodium carbonate buffer (pH1 The DMF solution of the NHS ester prepared in advance is added, arid incubated for 16 hours at room temperature under argon atmosphere. Then, it is centrifuged, concentrated to a volume of 500 Ald and chromatographed on Sephadex After freeze-drying, 2 mg of the conjugate is obtained as white powder.

b) Tc-99m Complex of 5 '-IIN-(6'-aza-7 '-hydroxy-9 '-mercapto-l' ,5 '-dioxo-3 '-thia-nonl'-yl)- 6 -aminohexyl-phosphoric acid ester] of 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*TT-3 1 mg of the conjugate described under example 23a) is dissolved in 1 ml of 0. 1 M disodium hydrogen phosphate buffer. After adding 10 mg of disodjum tartrate, it is mixed with sodium pertechnetate solution (1 mCi) and then with 10 Ald of tin(II) chloride solution (5 mg/i ml of 0.01 M HQCI. The tracer yield is determined by IIPLC (about 92%).

Example 24 a -[-Mrataey)6aminohexylphosphoric acid ester] of CUCAUGGAGCGCALGACGA&AUAGCUACAUAT*T*TT-3 mg of the S-( 6 -amino-hexyl.phosphoric acid ester) of CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T produced under SUBSTITUTE SHEET (RULE 261 WO 96/02274 PCTIEP95/02539 -58example 1 is dissolved in 1 ml of a sodium bicarbonate/sodium carbonate buffer (pH Then, 23.1 mg (0.1 mmol) of S-acetylmercaptoacetic acid-NHS ester, dissolved in 500 jl of absolute DMF, is added to the batch and incubated for 17 hours at room temperature under argon atmosphere. Then, it is centrifuged, concentrated to a volume of 500 pl and chromatographed on Sephadex G-25. After freeze-drying, 3 mg of the conjugate is obtained as white powder.

b) Tc-99m Complex of 5'-[N-(mercaptoacetyl)-6-aminohexyl-phosphoric acid ester] of 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T*-3' 1 mg of the conjugate described under example 24a) is dissolved in 1 ml of 0 1 M disodium hydrogen phosphate buffer (pH After adding 10 mg of disodium tartrate, it is mixed with sodium pertechnetate solution (1 mCi) and then with 10 pl of tin(II) chloride solution (5 mg/1 ml of 0.01 M HCI). The tracer yield (about 97%) is determined by HPLC.

Example a) N-[2-(Triphenylmethylmercapt)-eth--yl]-thiodiglycolic acid monoamide 31.9 g (0.1 mol) of 2-(triphenylmethylmercapto)-ethylamine and 10.1 g (0.1 mol) of triethylamine are introduced in 500 ml of absolute dichloromethane. At 0°C a solution of 13.2 g (0.1 mol) of thiodiglycolic acid anhydride is instilled, stirred for I hour at 0GC and for 16 hours at room temperature. Then, it is poured on 250 ml of aqueous citric acid, well-stirred, the organic phase is separated and dried on sodium sulfate. After evaporation of the solvent in a vacuum, the residue is chromatographed on silica gel (mobile solvent: dichloromethane/methanol, 0-20% methanol).

Yield: 20.32 g colorless oil Elementary analysis: Cld: C 66.49 H 5.58 N 3.10 0 10.63 S 14.20 Fnd: C 66.21 H 5.73 N 2.98 S 14.02 b) 5'-[N-(1',5'-Dioxo-6'-aza-3'-thia-8'-mercapto-oct-l-yl)-aminohexylphosphoric acid ester] of 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T*-3, First, the NHS-ester of N-[ 2 -(triphenylmethylmercapto)-eth-1-yl]-thiodiglycolic acid monoamide (example 25a) is produced. For this purpose, 45.2 mg (0.1 mmol) of the previously mentioned acid is dissolved in 500 pl of absolute DMF (0.1 mmol) and mixed with 11.5 mg (0.1 mmol) of NHS. After cooling to OOC, 19.2 mg (0.1 mmol) of EDC is added and incubated for 30 minutes at 0°C.

SUBSTITUTE SHEET (RULE 26) WO 96/02274 PCT/EP95/02539 -59- A solution of 10 mg of the 5-( 6 -aminohexyl-phosphoric acid ester) of 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T*-3' in 1 ml of a sodium bicarbonate/sodium carbonate buffer (pH described under example 1, is mixed with 500 Al of the NHS-ester solution produced in advance. It is incubated for 16 hours at room temperature. Then, it is concentrated by evaporation to 500 il and chromatographed on Sephadex G-25. 6 mg of the S-trityl-protected compound is obtained. The cleavage of the S-trityl protective group, the isolation of the oligonucleotide carrying SH groups and the purification take place as described under example 22e. 4 mg of white lyophilizate is obtained.

c) Tc-99m Complex of 5'-[N-(1',5'-dioxo-6'-aza-3'-thia-8'-mercapto-oct-1-yl)aminohexylphosphoric acid ester] of S'-CUCAUGGAGCGCAAGACGAAUAG- CUACAUAT*T*T*T T*-3.

1 mg of the conjugate produced under example 25b is dissolved in 1 ml of 0.1 M disodium hydrogen phosphate buffer (pH After adding 10 mg of disodium tartrate, it is mixed with sodium pertechnetate solution (1 mCi) and then with 10 4l of tin(II) chloride solution (5 mg/1 ml of 0.01 M HCL). The tracer yield is determined by HPLC Example 26 a) N,N'-Bis-[2-(triphenylmt eethyhnercapto)-oxo-eth-1-yl]-3, 4 -diaminobenzoic acid methyl ester 3.32 g (20 mmol) of 3 4 -diaminobenzoic acid methyl ester and 13.38 g mmol) of S-triphenylmethyl-mercaptoacetic acid are dissolved in 200 ml of absolute dichloromethane. At 0 0 C, 8.25 g (40 mmol) of dicyclohexylcarbodiimide, dissolved in 100 ml of absolute dichloromethane, is instilled in the batch. It is stirred for 1 more hour at o'C and finally for 16 more hours at room temperature. It is filtered, shaken against 1% aqueous citric acid, the organic phase is dried on sodium sulfate and the solvent is evaporated in a vacuum. The residue is chromatographed on silica gel (mobile solvent: dichloromethane/methanol. 0-10%.methanol).

Yield: 10.2 g colorless oil Elementary analysis: Cld: C 75.16 H 5.30 N 3.51 0 8.01 S 8.02 Fnd: C 75.01 H 5.58 N 3.30 S 7.89 b) N,N'-Bis-[ 2 -(triphenylmethylmercapto)- 1-oxo-eth-1-yl]-3, 4 -diaminobenzoic acid SUBSTITUTE SHEET (RULE 26) WO 96/02274 PCT/IEP95/02539 7.99 g (10 mmol) of N,N'-bis-[2-triphenylmethylmercapto)-l-oxo-eth-1-yl]- 3 4 -diaminobenzoic acid methyl ester (example 26a) is mixed in 200 ml of dioxane, ml of water and 20 ml of methanol with 4 g (100 mmol) of sodium hydroxide. It is stirred for 5 hours at room temperature and the batch is poured on 300 ml of aqueous citric acid. It is extracted exhaustively with dichloromethane, the organic phase is dried on sodium sulfate. After evaporation of the solvent, the residue is chromatographed on silica gel (mobile solvent: dichloromethane/methanol, methanol 0-40%).

Yield: 3.56 g colorless oil Elementary analysis: Cld: C 74.97 H 5.14 N 3.57 0 8.15 S 8.17 Fnd: C 74.71 H 5.32 N 3.31 S 7.88 c) 5'-{N-[3',4'-Bis-(2"-mercaptoacetylamino)-benzoyl]-6-aminohexylphosphoric acid ester} of 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T*-3' First, the NHS-ester of N,N'-bis-[2-(triphenylmethylmercapto)-oxo-eth-l-yl]- 3 4 -diaminobenzoic acid (example 26b) is produced. For this purpose, 78.5 mg (0.1 mmol) of the acid is dissolved in 500 pl of absolute DMF and mixed with 11.5 mg (0.1 mmol) of NHS. After cooling to OOC, 19.2 mg (0.1 mmol) of EDC is added and incubated for 30 minutes at 0°C. A solution of 10 mg of the 6 -aminohexyl-phosphoric acid ester) of 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T*-3' in 1 ml of a sodium bicarbonate/sodium carbonate buffer (pH described under example 1, is mixed with 500 pl of the NHS-ester solution produced in advance. It is incubated for 17 hours at room temperature. Then, it is concentrated by evaporation to 500 ul and chromatographed on Sephadex G-25. 7 mg of the S-trityl-protected compound is obtained.

The cleavage of the S-trityl protective group, the isolation of the oligonucleotide carrying SH groups and the purification take place as described under example 22e. 3 mg of white lyophilizate is obtained.

d) Tc-99m Complex of 5'-{N-[3',4'-bis-(2"-mercaptoacetylamino)-benzoyl]-6aminohexylphosphorous acid ester) of CUACAUAT*T*T*T*T*-3' 1 mg of the conjugate described under example 26c) is dissolved in 1 ml of 0.1 M disodium hydrogen phosphate buffer (pH After adding 10 mg of disodium tartrate, it is mixed with sodium pertechnetate solution (1 mCi) and then with 10 pl of SUBSTITUTE SHEET (RULE 26) WO 96/02274 PCT/EP95/02539 -61tin(II) chloride solution (5 mg/1 ml of 0.01 M HCI). The tracer yield (about 91%) was determined by HPLC.

Example 27 a) N-[O-Acetyl-hydroxyaceyl]-glycyl-glycyl-glycine-tert-buyl ester 24.5 g (0.1 mol) of glycyl-glycyl-glycine-tert-butyl ester and 11.8 g (0.1 mol) of O-acetyl-glycolic acid are added together at 0 0 C in 500 ml of absolute dimethylformamide. A solution of 20.6 (0.1 mol) of dicylcohexylcarbodiimide in 500 ml of absolute dimethylformamide is instilled in the batch, stirred for 1 hour at 0 0

C

and finally overnight at room temperature. It is filtered and the filtrate is concentrated by evaporation on the oil pump. It is crystallized repeatedly from ethyl acetate/npentane.

Yield: 12.5 g white powder Elementary analysis: S Cid: C 48.69 H 6.71 N 12.17 032.43 Fnd: C 48.43 H 7.01 N 11.93 b) N-[O-Acetyl-hydroxyacety-glyylglycyl-glycine 3.45 g (10 mmol) of N[O-acetyl-hydroxyacetyl]-glycyl-glycylglycinetert butyl ester is stirred in 50 ml of trifluoroacetic acid for 15 minutes. Then, it is poured on 500 ml of absolute diethyl ester and the product is filtered off. It is recrystallized repeatedly from ethyl acetate/n-pentane.

Yield: 1.23 g white powder Elementary analysis: Cld: C 41.53 H 5.23 N 14.53 O 38.72 Fnd: C 41.31 H 5.51 N 14.32 Sc) 5-{N-[N'-(Hydroxyacetyl)-glycyl-glycylycyl]-6-aminohexylphosphoric acid ester) of 5'-CUCAUGGAGCCAAGACGAAUAGCUACAUAT*T*T*T*T*3' First, the NHS-ester of N-(O-acetylhydroxyacetyl)-glycyl-glycyl-glycine (example 27b) is produced. For this purpose, 28.9 mg (0.1 mmol) of the acid is dissolved in 500 gl of absolute DMF and mixed with 11.5 mg (0.1 mmol) of NHS.

After cooling to 0°C, 19.2 mg (0.1 mmol) of EDC is added and incubated for minutes at 0 0 C. A solution of 10 mg of the 6 -aminohexyl-phosphoric acid ester) of 5'-CUCAUGGAGCGCAAGACGAAUAGCUACAUAT*T*T*T*T3' in 1 ml of 1 M sodium carbonate solution, described under example 1, is mixed with 500 pl of the NHS-ester solution produced in advance. It is incubated for 18 hours at room SUBSTITUTE SHEET (RULE 26) WO 96/02274 PCT/EP95/02539 -62temperature. Then, it is concentrated by evaporation to 500 pl and chromatdgraphed on Sephadex G-25. After freeze-drying, 3 mg of the title compound is obtained.

d) Tc-99m Complex of 5-N-[N'-(hydroxyacetyl)-glycyl-glycyl-glycyl]-6aminohexylphosphoric acid ester} of CGAAUAGCUACAUAT*T*T*T*T*-3' 1 mg of the conjugate described under example 27c is dissolved in 1 ml of 0.1 M disodium hydrogen phosphate buffer (pH 10.5). After adding 10 mg of disodium tartrate, it is mixed with sodium pertechnetate solution (1 mCi) and then with 10 gl of tin(II) chloride solution (5 mg/l ml of 0.01 M HCI). The tracer yield is determined by HPLC The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

SUBSTITUTE SHEET (RULE 26)

I

Claims (10)

1. Oligoneucleotide conjugates consisting of an oligonecleotide radical Nand n substituents in which n is a number between and 10, B stands for a direct bond or a connecting component to the oligoneucleotide radical, and K means a complexing agent or complex of radioactive metal isotopes, or stable isotopes, which are converted by radiation from outside to radioactive isotopes, convert radiation from outside to radiation of different quality, different energy content and/or different wavelength, of elements of atomic numbers 5, 21-29, 31, 39, 42-44, 49, 57-83 or 85, characterized in that oligoneucleotide radical N exhibits a modification, which prevents or at least significantly inhibits the degradation by naturally occurring nucleases and in with oligoneucleotide bonds specifically with high bonding affinity to a target structure with the proviso that the oligoneucleotide radical N iL obtained by a process of identifying a nucleic acid ligand of a target from a candidate mixture of nucleic acids, said process comprising: a) contacting the candidate mixture with said target molecule; b) partitioning the nucleic acids havirg irncreased affinity to the target molecule relative to the candidate mixture; and c) amplifying the increased affinity,mixture of nucleic acids, whereby nucleic acid ligands of the target molecule are identified.

2. Compound according to claim 1, wherein the compound exhibits general formula (I) (I) in which N is an oligoneucleotide, which bonds specifically with high bonding affinity to a target structure and exhibits modifications that significantly reduce the degradation by naturally occurring nucleases, B is a chemical bond or a connecting component, which produces the connection between N and K, and K is a completing ligand, which can contain a signal-transmitting or therapeutically active element, and n is a number between 1 and with the proviso that the oligoneucleotide N is obtained by a process of identifying a nucleic acid ligand of a target from a candidate mixture of nucleic acids, said process comprising: a) contacting the candidate mixture with said target molecule; b) partitioning the nucleic acids having an increased affinity to the target molecule relative to the candidate mixture; and c) amplifying the increased affinity mixture of nucleic acids, whereby nucleic acid -ligands of the target molecule are identified. P:kWPDOCS\CM%\SPErI6M20 29// 99 -64-

3. Compound according to claim I or 2, in which N is an oigoneucleotide with 5 to 200 nucleotides, wherein a) the 1 '-position of the sugar unit, independently of one another, is occupied by the following groups: a group -OR, in which R is an alkyl radical with I to 20 carbon atoms, which optionally contains up to 2 hydroxyl groups and which optionally is interrupted by 1-5 oxygen atoms, a hydrogen atom, hydroxyl group, a fluorine atom, a amino group, and hydroxyl groups present in terminal positions 3' and independently of one another, optionally are etherified with radical R and/or b) the phosphodiesters, optionally being used as inteinucleotide bond, Sindependently of one another, are replaced by phosphorothioates, phosphorodithioates or alkylphosphonates, preferably methyl phosphonate, and/or c) the terminal adicals and 5S-positions are linked in an intramolecula manner,with one another by an internucleotide bond as described in b) and/or t .ontans n internucleotide bond as described in which link 3'-3 or -5'positions, and/or. e) it contains a phosphodiester bond as described in which connects, esterlike, two thymidines respectively by a C -Co hydroxyalkyl radical in 3- position or connects an analogously submitted thymidine radical, esterlike, with a hydroxyl group of another sugar in or or 5'-position and/or f) the terminal radicals in and 5'-positions contain internucleotide bonds optionally modified as described in b). nucleot Compound according to claim 3, wherein oligonuecleotid N comprises 15 to 100 nucleotides. Compound according to any of claims 1 to 4, wherein N is an oligonecleotide, which bonds specifically with high bonding affinity to a target structure and which can be obtained in that a mixture of oligonuecleotides containing random sequences is brought together with the target structure, wherein oligoneucleotides which exhibit an increased affinity to the target structure relative to the mixture of the oligonucleotides are separated from the remainder of the oligoneucleotide mixture, then the lionucleotides with increased affinity to the target structure are amplified to obtain a mixture of oligoneucleotides that exhibits an increased portion of oligoneucleotides that bond on the target structures.

6. Compound according to anyone of claims I to 5, wherein N is an olgioneuceotide, which xflb ih h n in N is an olgioneucleotide, which obta ecifially bonds with high bonding affinity to a target structure, and which can be obtained in that a) first, a DNA strand is produced by chemical synthesis, so that this DNA strand exhibits a defined sequence on 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 so that this DNA strand exhibits a defined DNA sequence on the 5'-end, which is complementary to a primer sequence for the polymerase chain reaction, and the sequence between the defined sequences contains a random sequence, and in that b) this DNA strand is transcribed in a complementary RNA strand with the help of an RNA-polymerase, and nucleotides are offered to the polymcrase, which are modified in the 2'-position of the ribose unit, and in that c) the RNA oligonucleotides, produced in this way, are brought together with the target structure on which the oligonucleotide specifically is to bond, and in that d) thoe oligonucleoides that have bound on the target structure are separated first together with the target structure from the nonbinding oligonucleoddes and then the bound oligoucleodde are separated again from the target structure, and in that th,. ese target-strture-specific RNA oligonucleotides are transcribed with the help ofrever transcriptase in a complementary DNA strand, and in that S ,4f) these DNA strands are amplified using the defined primer sequences with the polymerase chain reaction, and in that g) th DNA oligonucleotides amplified in this manner are then transcribed again th the help of the RNA-polymrase and with modified nucleotides in RNA- oligonucleotides, and in that h) above-mentioned selection steps c) to g) optionally are repeated often until the oligonucleotides, which are characterized by a high bonding affinity to the target structure, are sufficiently selected, and then the sequences of the thus obtained oligonucleotides optionally can be determined.

7. Compound according to claim 6, wherein the target structure is selected from among macromolecules, tissue structures of higher organisms, such as animals or humans, organs or parts of organs of an animal or human, cells, tumor cells or tumors.

8. Compound according to any one of claims 1 to 7, wherein connecting component(s) B is (are) bound to the 4'-end of oligoneucleotide radical N reduced in 4'-position by the CHI-OH group and/or WO 96/02274 PMFEP95025S9 b) to the 3 '-end of oligonucleotide radical N reduced in 3 1 -position, by a hydrogen atom and/or C) to the phosphodiester bridge(s), reduced by the OH group(s), between two nucleotidies each and/or d) to 1 to 10 nucleobase(s), which is (are) reduced by a hydrogen atom respectively in 8 -position(s) and/or the amino group(s) in a -oiif~

9. Compound according to claim 8, paragraph a) or wherein B has general formula X-Y-ZI, which is connected on the X side with the complexing agent or complex and on the Z side with the oligoneucleotice, in which X is a direct bond, an -NH or -S group, Y is a straigh-chain, branched..chain, -saturated or unsaturated C,-C akylene chain, which oTionally contains 1-2 cyclohexylene, 1-5 imino,. 1 -3 phenylene, 1-3 phenylenimim), 1-3 phenylenoxy, 1-3 hydroxyphenylene, 1-5 amido. 1-2 hydrazido, 1-5 carbonyl, 1-5 ethylenoxy, ureido, a tioureido, 1-2 carboxyalkylimino, 1-2 ester.rus 1-3 groups of Ar, in which Ar stands for a saturated Or unsaturated 5- or 6- mlembered rin, which optionally contains 1_2 heteom s, selected from nitogen, oxygen and sulfur and/or 1-2 carbonyl groups; 1-10 oxygen, and/or 1-5 sulfur atoms, and/or optionally is substituted by 1-1 hydroxy, 1-n2 mercapto, 1-5 oxo, 1-5 thioxo, 1-3 carboxy, 1-5 carboxy-CX- 4 -alkyl, 1-5 ester, 1-3 amino, 1-3 hydroXY-Cl-c 4 alkyl, 1-3 C,-C 7 -alkoxy groups, and Zi is -CONH-CHi.4. -NH-CO-4', -O-P(O)R-NH.C24'. CH 2 or in which 4' or 3' indicates the I.inkage to the terminal Sugar unit(s) and R' is a CI-C 4 alkyl or NR 2 R 3 grop, with R 2 2 ndaRd meaning hydrogen or C 1 -C 4 alkyl radicals. Compound according to claim 8, paragraph wherein B has general formula X-y-Z 2 which is connected on the X side with the complexing agent or complex and on the Z side with the oligonucleotide, in which Z 2 in the bridge linking two adjacent sugar units, 0 S 2_1 z2 1 Z 5' and/or ZP-0- I I P:\WPDGCS\CRN\SPECR620206.SPE 2911/99 -67- is the group -NR 2 or X is a direct bond, an -NH or -S group Y is a straight chain, branched chain, saturated or unsaturated C 1 -C, alkylene chain, which optionally contains 1-2 cyclohexylene, 1-5 imino, 1-3 phenylene, 1-3 phenylenimino, 1-3 phenylenoxy, 1-3 hydroxyphenylene, 1-5 amido, 1-2 hydrazido, carbonyl, 1-5 ethylenoxy, a ureido, a thioureido, 1-2 carboxyalkylimino, 1-2 ester groups, 1-3 groups of Ar, in which Ar stands for saturated or unsaturated 5- or 6- membered ring, which optionally contains 1-2 heteroatoms selected from nitrogen, oxygen and sulfure and/or 1-2 carbonyl groups; 1-10 oxygen, 1-5 nitrogen and/or 1-5 sulfur atoms, and/or optionally is substituted by 1-5 hydroxy, 1-2 mercapto, 1-5 oxo, 1-5 thioxo, 1-3 carboxy, 1-5 carboxy-C-C 4 alkyl, 1-5 ester, 1-3 amino, 1-3 hydroxy--C-C alkyl, 1-3 C-C 7 -alkoxy groups, and R 2 is hydrogen or C,-C 4 alkyl radicals. 1 Compound according to claim 8d), wherein B has general formula X.Y-.Z 3 in which 7Z stands for an -NH group or a direct bond to the nucelobaise, X is a direct bond, an -NH or -S group, and Y is a straight-chain, branched-chain, saturated or unsaturated Ci-C, alkylene chain which optionally contains 1-2 cyclohexylene, 1-5 imino, 1-3 phenylene, 1-3 phenylenimino, 1-3 phenylenoxy, 1-3 hydrophenylene, 1-5 amido, 1-2 hydrazido, 1-5 carbonyl, 1-5 ethylenoxy, a ureido, a thioureido, 1-2 carboxyalkylimino, 1-2 ester groups, 1-3 groups of Ar, in which Ar stands for saturated or unsaturated 5- or 6- membered ring, which optionally contains 1-2 heteroatoms selected from nitrogen, oxygen and sulfur and/or 1-2 carbonyl groups; 1-10 oxygen, nitrogen and/or 1-5 sulfur atoms, and/or optionally is substituted by 1-5 hydroxy mercapto, 1-5 oxo, 1-5 thioxo, 1-3 carboxy, 1-5 carboxy-C,-C 4 -alkyl, 1-5 ester, 1-3 amino, 1-3 hydroxy-C-C 4 alkyl, 1-3 Ci-C, -alkoxy groups.

12. Compounds according to any of the preceding claims, wherein the metal complex which acts as an imaging element, contains a radioactive isotope, selected from the elements copper, bismuth, technetium, rhenium or indium.

13. Process for detecting a target structure, wherein one or more of the compounds according to any one of the preceding claims are brought together with the sample to be studied in vivo or in vitro and based on the signal, it is detected whether P:\WDOC\CPWPEC620MSPE- 29IM 1Q -68- the target structure, on which oligonucleotide N bonds specifically with high bonding affinity, is present in the sample being studied. 1.Process for noninvasive diagnosis of diseases, wherein oine or more of the compounds according to anyone of claims, I to 12 is brought to togetheir with the target structure to be stdie in vivo and based on the. sigial,~ it is detected whether the target structure, on which oligonuecleotide, N specifically bonds,.. is present in the organism to be studied. Use of compound accordin~g to Anyone of claims I1 oil2 iraiodiagnosis and/or in radiotherapy.

16. Diagnosis kitfr invivo ndor mn vitro detection of target structures, wherein the 4agnsis, kt cxntains at east omlqqoipoiind, acording to ay one of claims I to 12,. DATED this 12th day o f March, 1999 SCHERING AG AND NEXSTAR PI*ARMACEUTICAILS By It Pate nt Attorneys. DAY.WES COLILSON- CAVE_