NEUTRAL LABELING REACTANTS AND CONJUGATES DERIVED THEREOF

FIELD OF THE INVENTION

[0001] This invention relates to novel neutral derivatives of diethy- lenetriaminepentaacetic acid which allow introduction of the said derivatives to bioactive molecules.

BACKGROUND OF THE INVENTION

[0002] The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.

[0003] Because of its excellent metal chelating properties diethy- lenetriaminepentaacetic acid (DTPA) is one of the most widely used organic ligands in magnetic resonance imaging (MRI) and positron emission tomography (PET) [Aime, S., Botta, M., Fasano, M. and Terrano, E. 1998, Chem. Soc. Rev., 27, 19, Caravan, P., Ellison, J.J., McMurry, T.J. and Lauffer, R.B., 1999, Chem. Rev., 99, 2293, Woods, M., Kovacs, Z. and Sherry, A.D., 2002, J. Supramol. Chem., 2, 1]. Indeed, the first FDA approved contrast agent in clinical use is the Gd ³⁺ DTPA chelate [Runge, V.M., 2000, J. Magn. Res. Imaging, 12, 205.]. The corresponding ¹¹¹ In and ⁶⁸ Ga chelates, in turn, are suitable for PET applications [Anderson, CJ. and Welch, M.J., 1999, Chem. Rev. 99, 2219], while Eu ³⁺ , Tb ³⁺ , Sm ³⁺ and Dy ³⁺ chelates can be used in applications based on dissosiation enhanced lanthanide fluorescence immunoassay (DEL- FIA) [PCT WO 03/076939A1]. ^99m Tc DTPA in turn, is suitable for single positron emission computed tomography (SPECT) [Lorberboym, M., Lampl, Y. and Sadeh, M., 2003, J. Nucl. Med 44, 1898, Galuska, L., Leovey, A., Szucs- Farkas, Z., Garai, I., Szabo, J., Varga, J. and Nagy, E.V., 2002, Nucl. Med. Commun. 23, 1211]. Bioactive molecules labeled with ¹¹¹ In or ^117m Sn DTPA may find applications as target-specific radiopharmaceuticals [Volkert, W.A. and Hoffman, TJ. , 1999, Chem. Rev. 99, 2269]. [0004] In several applications, covalent conjugation of DTPA to bioactive molecules is required. Most commonly this is performed in solution by

allowing an amino or mercapto group of a bioactive molecule to react with isothiocyanato, haloacetyl or 3,5-dichloro-2,4,6-triazinyl derivatives of the label molecule. Several bifunctional DTPA derivatives are currently commerically available. Also solid phase methods for the introduction of DTPA to synthetic oligonucleotides [US 6,949,639] and oligopeptides [Fl 20055653] have been demonstrated.

[0005] The net charge of DTPA chelates is most commonly -2, which may cause problems in several applications. The commonly used MRI contrast agent Gd-DTPA (Magnevist) distributes thorough the extracellular and intravascular fluid spaces, but does not cross an intact blood-brain barrier. Naturally, bioactive molecules labeled with this type of chelates have lower cell permeability than the corresponding intact molecules [Rogers, B. E., Anderson, CJ. , Connett, J. M., Guo, LW., Edwards, W.B., Sherman, E.L., Zinn, K.R., Welch, M.J., 1996, Bioconjugate Chem. 7, 511]. This diminishes the suitability of DTPA chelates to in vivo applications. Furthermore, the negatively charged chelates may bind unselectively to positively charged binding sites of target molecules, such as antibodies, via electrostatic interactions which may result in low recoveries [Rosendale, B. E., Jarrett, D.B., 1985, Clin. Chem., 31 , 1965]. Naturally, all these above mentioned problems will be even more serious when the target molecule is labeled with several charged chelates [Peuralahti, J., Suonpaa, K., Blomberg, K., Mukkala, V.-M., Hovinen, J. 2004, Bioconjugate Chem. 15, 927].

[0006] Several of the above mentioned problems can be avoided by neutralizing the net charge of the chelate by substituting two of the DTPA ace- tates with carboxamido functions. Indeed, several this type of chelators have been synthesized [Hanaoka, K., Kikuchi, K., Urano, Y., Narazaki, M., Yokawa, T., Sakamoto, S., Yamaguchi, K., Nagano, T. 2002, Chem.Biol. 9, 1027., Feng, J., Sun, G., Pei, F., Liu, M. 2003, Bioorg. Med. Chem. 11 , 3359]. The non-ionic derivative, Gd[DTPA-bis(ethylamide)] [Konings, M.S., Dow, W.C., Love, D.B., Raymond, K.N., Quay, S.C., Rocklage, S.M. 1990, Inorg. Chem. 29, 1488], called as gadodiamide (Omniscan) is currently in clinical use. Its

osmolality is 40% of that of Gd-DTPA [Lunby, B., Gordon, P., Hugo, F., 1996, Eur. J. Radiol. 23, 190].

[0007] It is known that if one of the acetic acid groups of DTPA is used for conjugation, the resulting chelate is less stable than the parent DTPA molecule [Paul-Roth, C. and Raymond, K.N. 1995, Inorg. Chem. 34, 1408, Li, W.P., Ma, D.S., Higginbotham, C, Hoffman, T., Ketring, A.R., Cutler, CS. and Jurisson, S. S. 2001 , Nucl. Med. Biol. 28, 145.]. This may be a serious problem especially in in vivo applications if toxic metal ions have to be used. This has to be taken in account when modifying the metal chelating part of the DTPA mo- lecule.

[0008] Several of the above mentioned problems can be avoided by using neutral derivatives of the macrocyclic chelator 1 ,4,7,10- tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA) instead of DTPA in the biomolecule conjugation. However, DOTA is not suitable to all applications. Because of its slow kinetics of chelate formation, the use of DOTA is problematic in applications where short-living radioisotopes are required. In DELFIA assays, in turn, where the chelate has to be rapidly dissociated in acidic conditions, the lanthanide(lll) DOTA chelates are too stable.

SUMMARY OF THE INVENTION [0009] The main object of the present invention is to provide DTPA derivatives, where two of the DTPA acetates are substituted with amides. These chelates do not suffer from the disadvantages of the charged DTPA acetates. Furthermore, the chelating properties of the ligands are practically intact. Accordingly, these new chelates are highly suitable for magnetic resonance imaging (MRI), positron emission tomography (PET), single positron emission computed tomography (SPECT) and dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) as well as target-specific radiopharmaceuticals.

[0010] Thus, the present invention concerns a chelate of a formula (I) suitable for labeling of bioactive molecules,

R

( coo-

N-

-N X-A-^ ^COO ^"

N^ M ³⁺

< * [0011] ^co ° ^" (I)

[0012] wherein,

[0013] -A- is a linker;

[0014] R is -CONH ₂ , -CONHR ¹ or -CONR ¹ R ² where R ¹ and R ² , same or different are formed from one to ten moieties, each moiety being selected from the group consisting of phenylene, alkyl containing 1-12 carbon atoms, 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.

[0015] X is a reactive group for conjugation of the chelate to a bios- pecific reactant, wherein said reactive group -X- is selected from amino, ami- nooxy, haloacetamido, the said halide being preferably bromide or iodide, isot- hiocyanato, 3,5-dichloro-2,4,6-triazinylamino, maleimido, a thioester or an acti- ve ester of a carboxylic acid,

[0016] and M is a metal.

[0017] According to another aspect, the invention concerns a bio- specific binding reactant conjugated with the chelate according to this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 shows reversed phase HPLC trace of a thyroxine conjugate labeled with a neutral DTPA-Eu(III) chelate (crude reaction mixture). The peak at fo 28.14 min is the desired product as judged on ESI-TOF MS analysis. [0019] Figure 2 shows the titration curves of thyroxine (T ₄ ) labeled with various chelates. Open diamonds: 0.35 nM T ₄ labeled with the conven-

tional chelate used in AutoDELFIA Neonatal T ₄ kit/ 0.40 nM Ab; open squares: 0.20 nM T ₄ -DTPA/ 0.35 nM Ab; filled diamonds: 0.35 nM 13/ 30 nM Ab; filled squares: 0.35 nM 13 / 0.35 nM Ab. The structure of T ₄ -DTPA is shown in Chart 2.

DETAILED DESCRIPTION OF THE INVENTION

[0020] According to a preferable embodiment, the linker -A- is formed from one to ten moieties, each moiety being selected from the group consisting of phenylene, alkyl containing 1 -12 carbon atoms, 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.

[0021] R is -CONH ₂ , -CONHR ¹ or -CONR ¹ R ² where R ¹ and R ² , same or different are formed from one to ten moieties, each moiety being se- lected from the group consisting of phenylene, alkyl containing 1-12 carbon atoms, 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. [0022] Where X is an active ester of a carboxylic acid, said ester is preferably an λ/-hydroxysuccinimido, p-nitrophenol or pentafluorophenol ester.

[0023] According to a preferable embodiment the metal M is a metal suitable for use in bioaffinity assays such as a lanthanide or a metal suitable for use in positron emission tomography (PET), single positron emission tomo- graphy (SPECT) or magnetic resonange imaging (MRI).

[0024] A preferable metal to be used in MRI is gadolinium. However, also lanthanides, particularly europium (III), but also other lanthanides such as samarium (III) and dysprosium (III) are useful in MRI applications. In PET and SPECT applications a radioactive metal isotope is introduced into the chelating agent just before use. Particularly suitable radioactive isotopes are Ga-66, Ga-67, Ga-68, Cr-51 , ln-1 1 1 , Y-90, Ho-166, Sm-153, Lu-177, Er-169,

Tb-161 , Tc-98m, Dy-165, Ho-166, Ce-134, Nd-140, Eu-157, Er-165, Ho-161 , Eu-147, Tm-167 and Co-57.

[0025] Suitable metals for use in bioaffinity assays are lanthanides, especially europium (III), samarium (III), terbium (III) or dysprosium (III). The biospecific binding reactant to be labeled is, for example, an oligopeptide, protein, oligosaccaride, polysaccaride, phospholipide, PNA, LNA, antibody, hapten, drug, receptor binding ligand or lectine. Most preferably, the biospecific binding reactant is an oligopeptide.

[0026] The invention will be illuminated by the following non- restrictive Experimental Section.

EXPERIMENTAL SECTION

[0027] The invention is further elucidated by the following examples.

The structures and synthetic routes employed in the experimental part are depicted in Schemes 1-3. Experimental details are given in Examples 1 -14. Comparison of the stabilities of one of the neutral DTPA chelates and the parent DTPA acetate in DELFIA Enhancement Solution ^® and in DELFIA Inducer ^® is shown in Example 15. Structure of the parent DTPA acetate is shown in Chart 1. Example 16 shows the suitability of thyroxine labeled with neutral DTPA Eu(III) chelate in DELFIA based T4-assay. The properties of the new conjugate are compared with the corresponding DTPA acetate as well as with the conventional chelate used in AutoDELFIA ^® Neonatal T4 kit Structure of the thyroxine tracer labeled with DTPA acetate is shown in Chart 2.

Procedures

[0028] Adsorption column chromatography was performed on co- lumns packed with silica gel 60 (Merck) or neutral aluminum oxide (Aldrich; 150 mesh, Brockmann I). 17-α-hydroxyprogesterone 3-CMO and L-thyroxine were purchased from Steraloids and Sigma, respectively. All dry solvents were from Merck and they were used as received. HPLC purifications were performed using a Shimadzu LC 10 AT instrument equipped with a diode array de- tector, a fraction collector and a reversed phase column (LiChrocart 125-3 Pu- rospher RP-18e 5 μm). Mobile phase: (Buffer A): 0.02 M triethylammonium

acetate (pH 7.0); (Buffer B): A in 50 % (v/v) acetonitrile. Gradient: from 0 to 1 min 95% A, from 1 to 21 min from 95% A to 100% B. Flow rate was 0.6 ml_ min ^"1 . NMR spectra were recorded on a Bruker 250 spectrometer operating at

250.13 MHz for H. The signal of TMS was used as an internal reference. ESI- TOF mass spectra were recorded on an Applied Biosystems Mariner instrument. Time-resolved fluorometer VICTOR ² V was a product of PerkinElmer LAS.

Examples

[0029] Example 1. The synthesis of 3-(4-nitrobenzyl)-4-oxo-1 ,9- diphenyl-2,5,8-triazanona-1 ,8-diene, 2.

[0030] 2-(4-nitrobenzyl)-3-oxo-1 ,4,7-triazaheptane (1 ) (4.6 g, 18.2 mmol), disclosed in Corson, D.T., Meares, C.F., 2000, Bioconjugate Chem. 1 1 , 292, was dissolved to EtOH (45 ml_) and the solution was cooled on an ice bath. Benzaldehyde (3.7 ml_, 36.5 mmol) was added dropwise and mixture was stirred at ice bath for an hour. Stirring was continued for an additional hour at RT. Solution was dried over Na ₂ SO ₄ filtered and evaporated to dryness. ESI-TOF MS for C ₂₅ H ₂₅ N ₄ O ₃ ⁺ (M+H) ⁺ : calcd, 429.19; obsd 429.20.

[0031] Example 2. The synthesis of 3-(4-nitrobenzyl)-1 ,9-diphenyl- 2,5,8-triazanonane 3. [0032] Compound 2 (1.8 g, 4.2 mmol) was dissolved to dry THF (40 ml_) and deaerated with argon. The solution was cooled on an ice-water bath, and BH ₃ -THF-complex (1 M, 40 ml_) was added dropwise. The solution was allowed to warm to RT and then refluxed overnight. The solution was cooled on ice-water bath and the excess of borane was destroyed by careful addition of water. When foaming had ceased the solution was evaporated to dryness. The residue was dissolved in 20% aq. HCI and refluxed for 3 h, and then stirred overnight at RT. The solution was evaporated to dryness. The residue was partitioned between cone, aqueous ammonia and dichloromethane. The aqueous phase was extracted twice with dichloromethane. The combined or- ganic layers were dried over Na ₂ SO ₄ . Purification was performed on neutral

AI ₂ O ₃ (eluent, from 0 to 3 % methanol (v/v) in CH ₂ CI ₂ ). ESI-TOF MS for C ₂₅ H ₃ IN ₄ O ₂ ⁺ (M+H) ⁺ : calcd, 419.24; obsd 419.23.

[00331 Example 3. The synthesis of 2,5,8-tris(te/ιf- butoxycarbonylmethyl)-3-(4-nitrobenzyl)-1 ,9-diphenyl-2,5,8-triazanonane, 4. [0034] Compound 3 (2.7 g, 6.45 mmol) was dissolved in dry DMF

(15 ml_). Bromoacetic acid terf-butyl ester (4.8 ml_, 32.3 mmol) and DIPEA (9.01 ml_, 51.6 mmol) were added and mixture was stirred overnight at RT. The mixture was filtered and the filtrate was evaporated to dryness. Purification was performed on silica gel (eluent, petroleum ether, bp 40-60 "C: ethyl aceta- te 10:1 , v/v). Yield was 3.9 g (79 %). ¹ H NMR (CDCI ₃ ): δ 8.04 (2H, d, J 8.6); 7.37-7.25 (4H, m); 7.20 (2H, d, J 8.6); 7.14-7.02 (6H, m); 3.76 (1 H, d, J 13.4); 3.74 (2H, s); 3.66 (1 H, d, J 13.7); 3.35-3.27 (3H, m); 3.20 (2H, s); 3.19 (1 H, d, J 16.1 ); 3.04-2.95 (2H, m); 2.89-2.62 (2H, m); 2.38 (1 H, dd, J 8.9 and 12.8); 1.46 (9H, s); 1.44 (18H, s). ESI-TOF MS for C ₄₃ H ₆₁ N ₄ O ₈ ⁺ (M+H) ⁺ : calcd, 761.45; obsd 761.41.

[0035] Example 4. 2,5,8-tris(te/ιf-butoxycarbonylmethyl)-3-(4- aminobenzyl)-1 ,9-diphenyl-2,5,8-triazanonane, 5.

[0036] Compound 4 (3.76 g, 4.94 mmol) was dissolved in anhydrous methanol (75 ml_). Pd/C (10%, 0.22 g) and sodium borohydride (0.23 g) were added, and the mixture was strirred for 0.5 h at RT and filtered through Celite. The filtrate was neutralized with 1 M HCI and concentrated in vacuo. The residue was suspended in dichloromethane, washed with sat. NaHCO ₃ and dried over Na ₂ SO ₄ . Purification was performed on silica gel (eluent petroleum ether, bp 40-60 ⁰ C: ethyl acetate: triethylamine, from 10:1 :1 to 5:1 :1 , v/v/v)). ¹ H NMR (CDCI ₃ ): δ 7.30-7.15 (10H, m); 6.90 (2H, d, J 8.3); 6.57 (2H, d, J 8.3); 3.82 (1 H, d, J 13.9); 3.72 (1 H, d, J 13.9); 3.70 (2H, s); 3.52 (2H, s); 3.36 (1 H, d, J 17.1 ); 3.26 (2H, s); 3.25 (1 H, d, J 13.9); 3.16 (2H, s); 2.91-2.83 (2H, m); 2.71-2.60 (6H, m); 2.43 (1 H, dd, J 8.8 and 14.9); 1.45 (9H, s); 1.43 (9H, s); 1.41 (9H, s). ESI-TOF MS for C ₄₃ H ₆₃ N ₄ O ₆ ⁺ (M+H) ⁺ : calcd, 731.47; obsd 731.42.

[00371 Example 5. The synthesis of 2,5,8-tris(te/ιf- butoxycarbonylmethyl)-3-(4-te/ιf-butyloxycarbonylaminobenzy l)-1 ,9-diphenyl- 2,5,8-triazanonane, 6.

[0038] Di-terf-butyldicarbonate (0.68 g, 3.01 mmol) was dissolved in acetonitrile (25 ml_) containing triethylamine (420 μl_, 3.01 mmol). Compound 5 (2.00 g, 2.74 mmol; predissolved in 8 ml_ of acetonitrile) was added drop- wise, and the reaction was allowed to proceed for 2 h at RT. All volatiles were removed in vacuo. Purification was performed on silica gel [eluent petroleum ether, bp 40-60 ⁰ C: ethyl acetate: triethylamine, 10:1 :1 v/v/v)]. ESI-TOF MS for C ₄₈ H ₇ IN ₄ O ₈ ⁺ (M+H) ⁺ : calcd, 831.53; obsd 831.46.

[00391 Example 6. The synthesis of 1 ,4,7-tris(te/ιf- butoxycarbonylmethyl)-2-(4-terf-butyloxycarbonylaminobenzyl) -1 ,4,7- triazaheptane, 7.

[0040] Compound 6 (2.00 g, 2.41 mmol) was dissolved in anhy- drous methanol (40 ml_) and deaerated with argon. Pd/C (10%; 150 mg) and ammonium formate (0.76 g, 12.03 mmol) were added, and the mixture was heated at reflux for 15 min, before being filtered through Celite and concentrated. Purification was performed on silica gel [eluent petroleum ether, bp 40- 60 ⁰ C: ethyl acetate: triethylamine, 5:1 :1 (v/v/v)]. ESI-TOF MS for C ₃₄ H ₅₉ N ₄ O ₈ ⁺ (M+H) ⁺ : calcd, 651.43; obsd 651.40.

[0041] Example 7. The synthesis of 1 ,7-bis(aminocarbonylmethyl)- 1 ,4,7-tris(te/ιf-butoxycarbonylmethyl)-2-(4-te/ιf-butyloxyc arbonylaminobenzyl)- 1 ,4,7-triazaheptane, 8.

[0042] Compound 7 (0.50 g, 0.77 mmol) was dissolved in dry ace- tonitrile (5 ml_). lodoacetamide (0.26 g, 1.54 mmol) and potassium carbonate

(0.42 g, 3.07 mmol) were added, and the mixture was heated at reflux for 5h, before being filtered and concentrated in vacuo. Purification was performed on silica gel [eluent petroleum ether, bp 40-60 ⁰ C: ethyl acetate: triethylamine,

2:5:1 (v/v/v)]. ESI-TOF MS for C ₃₈ H ₇₁ N ₄ Oi ₀ ⁺ (M+H) ⁺ : calcd, 765.48; obsd 765.45.

[0043] Example 8. The synthesis of 2-(4-aminobenzyl)-1 ,7- bis(aminocarbonylmethyl)-1 ,4,7-tris(carboxymethyl)-1 ,4,7-triazaheptane, 9.

[0044] Compound 8 (0.10 g, 0.13 mmol) was dissolved in TFA (5 ml_), stirred for 4 h at RT and concentrated. It was used for the next step without further purification.

[0045] Example 9. The synthesis of the europium chelate of 2-(4- aminobenzyl)-1 ,7-bis(aminocarbonylmethyl)-1 ,4,7-tris(carboxymethyl)- 1 ,4,7- triazaheptane, 10.

[0046] Compound 9 was dissolved in water, and pH was adjusted to 6 with Na ₂ CO ₃ . Europium chloride (1.1 eq) was added, and the mixture was stirred for an hour at RT at pH 6. pH of the solution was rised to 8.5, and the europium hydroxide formed was removed by centrifucation. The product was isolated by precipitation upon addition of acetone. ESI-TOF MS for C ₂ IH ₂₈ EuN ₆ O ₈ ⁺ (M-H) ^" : calcd, 645.18; obsd, 645.11.

[0047] Example 10 . The synthesis of the europium chelate of 1 ,7- bis(aminocarbonylmethyl)-1 ,4,7-tris(carboxymethyl)-2-(4- isothiocyanatobenzyl)-1 ,4,7-triazaheptane, 11.

[0048] Compound 10 (30 mg, 0.046 mmol; predissolved in 200 μl_ of water) was added to the mixture of thiophosgene (15 μl_, 0.19 mmol), Na- HCO ₃ (20 mg) and chloroform (400 μl_), and the resulting suspension was stirred vigorously for 1 h at RT. The aqueous layer was separated, and washed with chloroform (2 -400 μl_). The product was isolated by precipitation from acetone. ESI-TOF MS for C ₂₂ H ₂₆ EuN ₆ O ₈ S- (M-H) ^" : calcd, 687.07; obsd, 687.01.

[0049] Example 1 1 . The synthesis of (5-aminopentylcarboxamido)- L-thyroxine, 12. [0050] L-thyroxine (40 mg, 0.05 mmol) was dissolved in the mixture of DMF (2.4 ml_) and TEA (320 μl_). Fmoc-aminohexanoic acid N- hydroxysuccinate (30 mg, 0.07 mmol) was added, and the mixture was stirred at RT for 1 h in dark. Piperidine (few drops) was added, and the reaction was allowed to proceed for 1 h, before being concetrated in vacuo. The residue was suspended in methanol. The precipitation was isolated by centrifugation and washed twice with methanol. ESI-TOF MS for C ₂ i H ₂₃ I ₄ N ₂ O ₅ ⁺ (M+H) ⁺ : calcd, 890.78; obsd, 890.73.

[0051] Example 12. Labeling of thyroxine derivative 12 with the isothiocyanate 11.

[0052] Compound 11 (15 mg, 17 μmol) was dissolved in the mixture of pyridine, water, and triethylamine (9:1.5:0.1 , v/v/v; 100 μl_). Compound 12 (15 mg, predissolved in 50 μl_ of water) was added, and the mixture was strir- red for 1 h at RT and concentrated. The residue was suspended in water and precipitated with acetone to yield the desired conjugate 13. Purification was performed on HPLC. ESI-TOF MS for C ₄₃ H ₄₈ Eu I ₄ N ₈ Oi ₃ S- (M-H) ^" : calcd, 1576.85; obsd, 1576.87. [0053] Example 13. Synthesis of the 17-α-hydroxyprogesterone derivative, 14.

[0054] 17-α-hydroxyprogesterone-3-CMO (0.10 g, 0.25 mmol) was dissolved in dioxane (4 mL). DCC (56 mg, 0.27 mmol) and N- hydroxysuccinimide (32 mg, 0.27 mmol) were added, and the reaction was al- lowed to proceed for 4 h at RT. DCU formed was removed by filtration, and the filtrate was concentrated in vacuo. The residue was redissolved in dioxane (7 mL). Glutamic acid (36 mg, 0.25 mmol; predissolved in 0.1 M NaHCO ₃ (7 mL) was added, and the mixture was strirred for 2 h at RT. The precipitation formed was removed by filtration, and the filtrate was concetrated in vacuo. Purifica- tion was performed on a preparative TLC plate (eluent, acetonitrile: water, 2:1 , v/v). ESI-TOF MS for C ₂₆ H ₃₅ N ₂ O ₈ - (M-H) ^" : calcd, 503.24; obsd, 503.28.

[0055] Example 14. Labeling of the steroid derivative, 14 with the amino chelate 10.

[0056] Compound 14 (6.5 mg, 12 μmol; predisolved in dioxane) was dissolved in MES-buffer (pH 5.5, 1.5 mL). Compound 10 (16.5 mg, 26 μmol) predissolved in MES buffer (550 μL) was added followed by EDAC (5.0 mg, 26 μmol). The reaction was allowed to proceed for 4 h at RT. Purification was performed on HPLC. ESI-TOF MS for C ₆₈ H ₉₀ Eu ₂ N ₁₄ O ₂₂ (M-2H) ^2" : calcd, 879.23; obsd, 879.23.

[0057] Example 15. Stabilities of Amino-Eu-DTPA and the corresponding neutral derivative 10 in DELFIA Enhanchement Solution® and in DELFIA Inducer®.

[0058] The chelates (ca 1 mg) were dissolved either in Inducer or Enhanchement Solution. The dissociation of the europium at 25 ⁰ C were followed using a time-resolved fluorometer. The results are shown below.

[0059] Table 1. Stabilities of DTPA acetate and the corresponding neutral derivative 10 at RT. Approximate times needed for complete dissociation.

Data from PCT WO 03/076939A1

[0060] Example 16. Comparison of the performance of the tracer 13 and the corresponding DTPA derivative to AutoDELFIA Neonatal T ₄ (thyroxine) kit.

[0061] The assay concentrations of the antiserum were optimized for each tracer individually, and the analytical sensitivities of the optimized standard curves were defined. The correlation between the methods were studied with a small sample panel. The on-board stability was tested up to one week in instrument-like conditions. Sensitivity to the interference of EDTA- containing samples was also studied. The results are summarised below. [0062] Table 2. Comparison of the performance of the tracer 13 and the corres ondin DTPA derivative to AutoDELFIA® Neonatal T ₄ kit.

[0063] The shapes of the calibration curves obtained with optimized amounts of tracer and antiserum were slightly different with the three tracers.

All tracers were sensitive enough at clinically important range. Assays with the tested tracers compared well to the AutoDELFIA® Neonatal T ₄ assay and no significant level differences were obtained.

[0064] It will be appreciated that the methods of the present invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent for the expert skilled in the field that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.

EuCIo

[0065] SCHEME 1

Eu ,3+ 6] SCHEME 2

15 Eu ,3+ [0067] SCHEME 3

Eu ³⁺

Amino-Eu-DTPA

[0068] Chart 1

Eu ³⁺

T ₄ -DTPA

[0069] Chart 2