Metal locenes and conjugates derived thereof

Field of the invention

This invention relates to novel titanocene derivatives tethered to reporter groups which can be introduced into biospecific binding reactants.

Background of the invention

Oligonucleotides, DNA and RNA can be transformed statistically by bisulfite-catalyzed transamination of cytosine residues [Hayatsu, H., 1976, Biochemistry, 15, 2677, Draper, D. E., Gold, L., 1980, Biochemistry, 19, 1774, Molander, J., Hurskainen, P., Hovinen, J., Lahti, M., Lδnnberg, H., 1993, Bio- conjugate Chem., 4, 362, Adarichev, V.A., Kalachnikov, S. M., Kiseliova, A.V., Dymshits, G. M., 1998, Bioconjugate Chem., 9, 651], by electrophilic substitution of C8 of guanine residues [Tchen, P., Fuchs, R. P. P., Sage, E., Leng, M., 1984, PNAS, 81 , 3466] or by using label molecules tethered to platinum, which coordinates at N7 of guanine residues [US 5,985,566]. All these methods have their drawbacks. Attachment of linkers or labels to critical positions of DNA weaken hybridization properties of the oligomer. Although platinum predominantly coordinates at N7 of guanine residues, the reactivity of the platinated label is dependent on the base sequence of the target molecule as well as on the label attached. Furthermore, not only do N7 of guanine residues react but also other bases such as N1 of adenine residues [Lippert, B. 2000, Coordination Chemistry Rev., Lippert B., 1989, Progr. Inorg. Chem., 37, 1 , Lippert B., Cisplatin, Chemistry and Biochemistry of a Leading Anticancer Drug/; Ed.

B. Lippert, Wiley-VCH, 1999, Perspectives on Bioinorganic Chemistry, Vol. 4, Eds R. W. Hay, J. R. Dilworth and K. B. Nolan, JAI Press, 1999]. Although coordination of the platinum label at N7 of guanine allows formation of natural Watson-Crick base pairs, the bulky substituents definitely change the three dimensional structure of the oligomer, especially in the cases where the label attached is an organic chromophore with intercalating properties. Furthermore, it has been shown that in certain cases platinated oligonucleoti- des are extremely labile as soon as the platinated oligonucleotide is paired with its complementary strand [Anin, M. -F., Gaugheron, F; Leng, M, 1992, Nucleic Acids Res., 20, 4825]. This, in turn, results in the formation of abasic sites and loss of genetic information and the label.

Because of the importance of post-translational protein phosphory- lation in regulating cell signalling, several methods for protein mapping have

been developed [Ahn, N. G., Resing, K. A., 2001 , Nat. Biotechnol, 19, 317, Adam, G, Sorensen, E, Gravatt, B., 2002, MoI. Cell. Proteomics, 1.10, 781]. One of them is based on derivatization of phosphopeptides by phosphate elimination and subsequent Michael addition in the presence of mercaptans or amines. This method has been successfully exploited in enrichment of phosphopeptides to solid phase [Knight, Z.A., Schilling, B, Row, R. H., Kenski, D. M., Gibson, B.W., Shokat, K.M., 2003, Nat. Biotechnol. 21 , 1047] and derivatization of phosphopeptides with various tether groups [Goshe, M. B., Conrads, TP. , Panisko, E.A., Agnell, N. H. Veenstra, T.D., Smith, R.D., 2001 , Anal. Chem. 73, 2578, Jaffe, H. Veeranna, H., Pant, H. C, 1998, Biochemistry, 37, 16211 , Adamczyk, M., Gebler, J. C, Wu. J., 2001 , Rapid Commun. Mass Spetrom. 15, 1481]. The final characterization and quantitation is most commonly performed by mass spectrometry with or without proteolytic digestion. It has been shown recently that phosphopeptides can be derivatized even with amino and mercapto-functionalized reporter groups, such as organic dyes [Mattila, K., Siltainsuu, J., Balaspiri, L., Ora, M., Lδnnberg, H, 2005, Org. Bio- mol. Chem. 3, 3039]. Accordingly, by using this kind of labels, mass spectro- metric analyses can be avoided. Although several promising results based on this approach has been demonstrated in literature, the method always involves phosphate elimination under highly basic conditions. This causes limitations to the substrate and the label.

Titanocene dichloride and its analogues have been shown to have significant activity against a various cancer cell lines [Kδpf-Maier, P., Kδpf, H. Chem. Rev., 1987, 87, 1137, Clarke, M.J., Zhu, F., Frasca, D. R., 1999, Chem. Rev., 99, 2511 , Harding, M. M., Mokshdi, G., 2000, Curr. Med. Chem., 7, 1289, Yang, P., Guo, M., 1999, Coord. Chem. Rev., 185, 189, Melendez, E., 2002, Crit. Rev. Oncol/Hematol, 42, 309, Sthrohlfendt, K., Pampillon, C, Sweeney, N., Tacke, M., 2006, Chemiedozententagung, A41]. In contrast to c/s-platin, titanocene dichlorides form complexes even with the phosphodiester backbone. Recently, a versatile method for the synthesis of functional ti- tanocenes have been reported [Gansauer, A., Franke, D., Lauterbach, T., Nie- ger, M., 2005, J. Am. Chem. Soc, 127, 11622]. There, a titanocene tethered to an acid chloride was allowed to react with various alcohols and amines giving rise to a serie of titanocene conjugates. One of the obstacles in the use of titanocens has been their very low aqueous solubility. Accordingly, several attempts to increase their hydro-

philicity have been reported. For example US 5,296,237 teaches to mix metal- locenes with polyols and various additives to give rise to water-soluble metal- locene-complexes. Although these metallocene-complex compositions can be used as cytostatics in cancer therapy, they are not suitable for analytical pur- poses. Causey, P.W., Baird, M.C., 2004, Organometallics, 23, 4486, in turn, discloses titanocene dichloride derivatives containing alkylammonium pendant to one or both cyclopentanedienyl rings. Although the positively charged pendant arm significantly enhances the water-solubility of these complexes, the pendant limits the further derivatization of these complexes, e.g. attachment of the conjugate group essential for bioanalytical purposes. A further attempt to increase hydrophilicity can be found in Boyles, J. R., Baird, M. C, Camping, B. G., Jain, N., 2001 , J. Inorg. Biochem., 84, 159.

The fluorescent labels used for biomolecule derivatization are most commonly organic dyes. Although organic chromophores can be utilized in several applications, these labels suffer from many commonly known drawbacks such as Raman and Raylegh scattering, low water solubility and concentration quenching. Instead, the unique properties of lanthanide(lll) chelates, such as high water solubility and strong long decay-time luminescence, make them ideal labels for numerous assays. Furthermore, large Stokes shift and very sharp emission bands enable the simultaneous use of four lanthanides (i.e. Eu, Tb, Sm, Dy) in the analysis. Time-resolved fluori- metric assays based on lanthanide chelates have found increasing applications in diagnostics, research and high throughput screening. The heterogeneous DELFIA ^® (Dissociation-Enhanced Lanthanide Fluorescence Immuno- assay) technique is applied in assays requiring exceptional sensitivity, robustness and a multi-label approach. Development of highly luminescent stable chelates extends the use of time resolution to homogeneous assays, based on fluorescence resonance energy transfer (TR-FRET), fluorescence quenching (TR-FQA) or changes in luminescence properties of a chelate during a binding reaction.

Summary of the invention

The main object of the present invention is to provide titanocene derivatives which allow labelling of biospecific binding reactants. These derivatives can be used in diagnostic and therapeutic applications. Accordingly, they are suitable for in vitro diagnostics, molecular imaging, and they can be used as radiopharmaceuticals.

The major advantages of the present invention are: (i) The present titanocenes are derivatives of metal chelates with high water solubility. Accordingly, they do not suffer from the hydrophobi- city of titanocene dichloride. (ii) The present labelling reactants do not suffer from concentration quenching, and thus they are highly suitable for multilabelling. (iii) Since they bind to the biomolecule statistically, the labelling degree can be controlled simply by the ratio of the reactants. (iv) The present reporter groups can be used in the labelling of oligonuc- leotides, LNA, DNA, RNA, phosphopeptides and phosphoproteins.

Thus, the present invention concerns a labelling reactant of a formula (I) suitable for labelling of biospecific binding reactants

G ¹ L ¹

Ar G ² -L ² -ArTi-X

Cl (I)

wherein L ¹ and L ² are a linkers, same or different or not present; G ¹ and G ² are conjugate groups, same or different, or not present; X is Cl or

-A-G ³ , wherein A sulphur of oxygen and G ³ is conjugate group, and wherein Ar is an aromatic ring; provided all of the conjugate groups G ¹ , G ² and G ³ are not simultaneously absent.

The conjugate groups can be, for example, a luminescent or non- luminescent lanthanide(lll) chelates, labels suitable for SPECT or PET applications, biotin or a spin label.

According to another aspect, the invention concerns a biospecific binding reactant conjugated with the labelling reactant according to this invention.

The target (the biospecific binding reactant) can be, for example, a synthetic oligonucleotide, DNA or RNA as well as a phosphopeptide or a phos- phoprotein. The molecule of formula (I) reacts statistically, and the degree of labelling can be adjusted by controlling the ratio of the reaction components. The molecule of formula (I) reacts with phosphoproteins and phosphopeptides in physiological pH with no need of phosphoelimination at highly basic conditions.

Detailed description of the invention

According to a preferable embodiment, the linkers -L ¹ , and L ² -, same or different, are formed from one to ten moieties, each moiety being selected from the group consisting of phenyl, alkyl containing 1-12 carbon at- oms, ethynediyl (-C≡C-), ethylenediyl (-C=C-); ether (-O-), thioether (-S-), amide (-CO-NH- and -NH-CO- and -CO-NR and -NR-CO-), carbonyl (-CO-), ester (-COO- and -OOC-), disulfide (-SS-), diaza (-N=N-) or a tertiary amine (-NR-), where R represents an alkyl containing less than 5 carbon atoms.

According to a particularly preferable embodiment, Ar is cyclopenta- 1 ,3-diene.

According to a preferable embodiment, the conjugate groups G ¹ -G ³ are reporter groups, such as metal chelates. Especially preferred are luminescent or non-luminescent lanthanide(lll) chelates, particularly chelates of europium (Eu), terbium (Tb) or gadolinium (Gd). Particularly preferable lanthanide (III) chelates for this purpose are luminescent chelates based on triazacycloalkanes, 2-(3-(pyridin-2-yl)-1 H-pyrazol-1 -yl)pyridine and terpyridine and nonluminescent chelates based on pyridine-2,6-diyl-bis(methylenenitrilo) tetrakis(acetic acid) and diethylenetriaminepentaacetic. Also other metal chelates can be used. As examples can be mentioned gadolinium chelates for use in MRI applications and chelates of radioactive metal isotopes suitable for use in PET applications. One of the conjugate groups G ¹ -G ³ can also be a group useful for immobilizing the target biomolecule to as solid support.

According to a particularly preferable embodiment, the conjugate groups G ¹ -G ³ are metal chelates comprising terpyridine, 2-(3-(pyridin-2-yl)-1 H- pyrazol-1-yl)pyridine or diethylene triamine bound to a group of formula (II)

wherein R ¹ is methylene or 1-butynyl and is bound further to L ¹ , L ² or A of formula (I) and R ² is a single bond or alkyl, where the alkyl is methylene or ethylene, and is bound further to the terpyridine, the 2-(3-(pyridin-2-yl)-1 H- pyrazol-1-yl)pyridine or the diethylene triamine.

Schematic presentation of the preparation of the novel labelling reactant

Synthetic strategy for the preparation of illustrative titanocene derivatives of lanthanide(lll) chelates is described in Figure 1. A representative synthetic procedure is the following: 1. Reaction of the titanocene acid chloride [1 ; synthesized as disclosed in Gansauer, A., Franke, D., Lauterbach, T., Nieger, M., 2005, J. Am. Chem. Soc, 127, 11622] with the ligands (2a; synthesized as disclosed in Mukkala, V. -M., Helenius, M., Hemmila, I., Kankare, J., and Takalo, H. 1993, HeIv. Chim. Acta 76, 1361 ; 2b; synthesized as disclosed in Peuralahti, J., Ha- kala, H., Mukkala, V.-M., Hurskainen, P., Mulari, O., and Hovinen, J. 2002 Bio- conjugate Chem., 13, 870; 2c, synthesized as disclosed in US 5,859,215; 2d, synthesized as disclosed in Corson, D. T., and Meares, C. F., 2000, Bioconju- gate Chem., 11 , 292) in dichloromethane in the presence of sodium hydride.

2. Removal of the /-butyl groups with trifluoroacetic acid followed by treatment with lanthanide(lll) chloride as disclosed in Mukkala, V.-M., Helenius, M., Hemmila, I., Kankare, J., and Takalo, H. 1993, HeIv. Chim. Acta 76, 1361 gives rise to the desired titanocene derivatives 4a-d.