WO2010084090A1 - Pyridyl-aza(thio)xanthone sensitizer comprising lanthanide(iii) ion complexing compounds, their luminescent lanthanide (iii) ion complexes and use thereof as fluorescent labels. - Google Patents

Abstract

Lanthanide (III) Ion completing compound comprising: (1) a sensitizer moiety of Formula (I) in which: a is an integer from 1 to 4; b is an integer equal to 1 or 2; c is an integer equal to 1 or 2; (R₁)a, (R₂)b, (R₃)C are the same or different and are chosen from the group consisting of H; alkyl; -COOR₄ where R₄ is H or an alkyl; aryl; heteroaryl; saturated or unsaturated cyclic hydrocarbon; CF3; CN; a halogen atom; L-Rg; L-Sc; or two consecutive R₃, two consecutive R₂ or two consecutive R₁ groups together form an aryl or a heteroaryl group or a saturated or unsaturated cyclic hydrocarbon group; where L is a linker, Rg is a reactive group and Sc is a conjugated substance; X₁ and X₂ are the same or different and are O or S; A is either a direct bond or a divalent group chosen from -CH₂- or -(CH₂)₂-, said moiety being covalentiy attached to (2) a lanthanide (in) ion chelating moiety through A. The above compounds are used in the preparation of luminescent lanthanide (III) ion complexes which are used as fluorescent labels.

Description

Pyridyl-aza(thio)xanthone sensitizer comprising lanthanide (III) ion complexinq compounds, their luminescent lanthanide fill) ion complexes and use thereof as fluorescent labels

Field of the invention

This invention relates to novel compounds that can complex with lanthanide cations. In particular, this invention relates to complexing compounds which contain novel photosensitizers and can produce long-lived fluorescence for use in time-resolved energy transfer fluorescence assays, especially bioassays.

State of the art

Traditional fluorescent labels of organic dyes such as fluoresceins and rhodamines have long been employed as bioanalytical tools in immunoassays. Coordination complexes of the lanthanide (III) ions are more recently developed fluorescence agents and have been found to possess properties which make them very suited as potential labels in the bioassay field. These complexes are capable of giving long-lived and longer wavelength fluorescent emissions upon excitation. Through time- delay measurements, they have demonstrated clear advantages over conventional fluorescent labels in terms of experiencing less quenching and background interference while exhibiting increased detection sensitivity. In addition to these advantages, many lanthanide (III) complexes have improved solubility properties and are able to efficiently transfer energy from their excited states to neighbouring acceptor molecules. As such, they are ideal agents for time-resolved fluorescence use, especially for developing high-throughput automated and miniaturized binding assays with the inclusion of immunoassays, DNA hybridization assays, receptor binding assays, enzyme assays, cell-based assays, immunocytochemical or immunohistochemical assays.

Emissive lanthanide complexes that can be sensitised efficiently have been studied in detail as components of bioassays, spatially localised sensors, or as donors in time-resolved energy transfer systems. They typically comprise a polydentate ligand, often loosely termed a chelating moiety which binds the Lanthanide (III) ion and an organic sensitiser group. The sensitiser group has the function of absorbing light and transferring energy to the lanthanide. It thereby overcomes the inherently low absorbance of the lanthanide ions. There is a developing need to find long-lived emissive probes that are suitable for application in living cells (for recent examples: J. Yu, D. Parker, R.Poole, R. Pal and MJ. Cann, J. Am. Chem. Soc, 2006, 128, 2294; K. Hanoaka, K. Kikuchi, H. Kojima, Y. Urano and T. Nagano, J. Am. Chem. Soc, 2004, 126, 12470; G. Bobba, J-C. Frias and D. Parker, Chem. Commun., 2002, 890; H.C. Manning, S.M. Smith, M. Sexton, S. Haviland, M. F. Bai, K. Cederquist, N. Stella and DJ. Bornhop, Bioconjug. Chem., 2006, 17, 735; D. Parker and R. Pal, Chem. Commun., 2007, 474; H.C. Manning, T. Goebel, R.C. Thompson, R. R. Price, H. Lee and DJ. Bornhop, Bioconjug. Chem., 2004, 15, 1488; J-C. Frias, G. Bobba, MJ. Cann, D. Parker and CJ. Hutchinson, Org. Biomol. Chem., 2003, 1, 905). For such applications, the complexes need to be non-toxic and cell permeable, resistant to photobleaching and photo-fading, exhibit kinetic stability with respect to degradation and preferably should be relatively immune to quenching of the excited state of the lanthanide (III) ion by electron or charge transfer processes.

Several series of cyclic and acyclic ligands have been studied (e.g. R. Ziessel, N. Weibel, LJ. Charbonniere, M. Guardigli and A. Roda, J. Am. Chem. Soc, 2004, 126, 4888; B. Song, E. Wang and J. Yuan, Chem. Commun., 2005, 3553; M. Xiao and P.R. Selvin, J. Am. Chem. Soc, 2001, 123, 7067; D. Parker, R.S. Dickins, C. Crossland, J.A.K. Howard and H. Puschmann, Chem. Rev., 2002, 102, 1977) that present 8 or 9 donor atoms able to bind to the lanthanide ion and also incorporate a heterocyclic sensitising moiety that is able to harvest incident light efficiently (i.e. possess a large molar extinction coefficient, ε) and transfer its excited state energy in an intramolecular process to generate the lanthanide excited state. The ligand is preferably designed to inhibit the vibrational deactivation of the lanthanide (III) excited state, which can be particularly problematic with proximate OH and NH oscillators. (A. Beeby, I. M. Clarkson, R.S. Dickins, S. Faulkner, D. Parker, L. Royle, A.S. de Sousa J.A.G. Williams and M. Woods, J. Chem. Soc, Perkin Trans 2., 1999, 493).

Recently, ligands containing substituted 1-azaxanthone and azathiaxanthones have been introduced (WO 2006/039505 A2; Org. Biomol. Chem., 2006, 4, 1707-1722; WO2006/120444 Al) as effective sensitisers for Eu and Tb emission in aerated aqueous media. The pyrazoyl-1-azaxanthone has also been proposed as sensitiser for Tb luminescence at 355 nm. (Craig P. Montgomery et al. Chem. Commun., 2007, 3841-3843). Definitions

The term "alkyl" is used herein to refer to a branched or unbranched, saturated or unsaturated, monovalent hydrocarbon radical, generally having from about 1-15 carbons, preferably from 1-10 carbons and more preferably from 1-6 carbons. Suitable alkyl radicals include, for example, structures containing one or more methylene, methine and/or methyne groups. Branched structures have a branching motif similar to i-propyl, t-butyl, i-butyl, 2-ethylpropyl, etc. As used herein, the term encompasses "substituted alkyls," and "cyclic alkyls."

"Substituted alkyl" refers to alkyl as just described including one or more substituents such as (C₁-Q) alkyl, aryl, acyl, halogen, hydroxy, amino, alkoxy, alkylamino, acylamino, thioamido, acyloxy, aryloxy, aryloxyalkyl, mercapto, thia, aza, oxo, both saturated and unsaturated cyclic hydrocarbons, heterocydes and the like. These groups may be attached to any carbon or substituent of the alkyl moiety. Additionally, these groups may be pendent from, or integral to, the alkyl chain. "Alkylamino" refers to a secondary amine -NHR where R is an alkyl group as defined above.

"Alkylcarboxyl" refers to a group -RCOOH where R is an alkyl group as defined above.

The term "aryl" is used herein to refer to an aromatic substituent having 5 to 20 carbon atoms, preferably 5 to 10 carbon atoms; said aromatic substituent may be a single aromatic ring or multiple aromatic rings which are fused together, linked covalently, or linked to a common group such as a methylene or ethylene moiety. The common linking group may also be a carbonyl as in benzophenone. The aromatic ring(s) may include phenyl, benzyl, naphthyl, biphenyl, diphenylmethyl and benzophenone among others. The term "aryl" encompasses "arylalkyl" and "substituted aryl."

"Substituted aryl" refers to aryl as just described including one or more groups such as (C₁-C₆) alkyl, acyl, halogen, haloalkyl (e.g. CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, phenoxy, mercapto and both saturated or unsaturated cyclic hydrocarbons which are fused to the aromatic ring(s), linked covalently or linked to a common group such as a methylene or ethylene moiety. The linking group may also be a carbonyl such as in cyclohexyl phenyl ketone. The term "substituted aryl" encompasses "substituted arylalkyl." The term "arylalkyl" is used herein to refer to a subset of "aryl" in which the aryl group is attached to another group by an alkyl group as defined herein.

The term "Substituted arylalkyl" defines a subset of "substituted aryl" wherein the substituted aryl group is attached to another group by an alkyl group as defined herein.

The term "saturated cyclic hydrocarbon" denotes groups having 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 6 carbon atoms. EExamples of these groups are cyclopropyl, cyclobutyl, cyclopentyl, etc., and substituted analogues of these structures. These cyclic hydrocarbons can be single-or multi-ring structures. The term "saturated cyclic hydrocarbon" encompasses "substituted saturated cyclic hydrocarbon".

The term "substituted saturated cyclic hydrocarbon " refers to saturated cyclic hydrocarbon as just described including one or more groups such as lower alkyl, acyl, halogen, haloalkyl (e.g. CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, phenoxy, mercapto, thia, aza, oxo.

The term "unsaturated cyclic hydrocarbon" is used to describe a monovalent non-aromatic group with at least one double bond and having 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms and more preferably 3 to 6 carbon atom, such as cydopentene, cyclohexene, etc. and substituted analogues thereof. These cyclic hydrocarbons can be single-or multi-ring structures. The term "unsaturated cyclic hydrocarbon" encompasses "substituted unsaturated cyclic hydrocarbon"

The term "substituted unsaturated cyclic hydrocarbon " refers to unsaturated cyclic hydrocarbon as just described including one or more groups such as lower alkyl, acyl, halogen, haloalkyl (e.g. CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, phenoxy, mercapto, thia, aza, oxo.

The term "heteroaryl" as used herein refers to aromatic rings having 5 to 20 carbon atoms; preferably 5 to 10 carbon atoms and in which one or more carbon atoms of the aromatic ring(s) are replaced by a heteroatom such as nitrogen, oxygen or sulfur. Heteroaryl refers to structures that may be a single aromatic ring, multiple aromatic ring(s), or one or more aromatic rings coupled to one or more non-aromatic ring(s). In structures having multiple rings, the rings can be fused together, linked covalently, or linked to a common group such as a methylene or ethylene moiety. The common linking group may also be a carbonyl as in phenyl pyridyl ketone. As used herein, rings such as thiophene, pyridine, isoxazole, phthalimide, pyrazole, indole, furan, etc. or benzo-fused analogues of these rings are defined by the term "heteroaryl." The term heteroaryl encompasses "substituted heteroaryl" and

"heteroarylalkyl"

The term "heteroarylalkyl" defines a subset of "heteroaryl" wherein an alkyl group, as defined herein, links the heteroaryl group to another group.

The term "substituted heteroaryl" refers to heteroaryl as described above wherein the heteroaryl nucleus is substituted with one or more groups such as lower alkyl, acyl, halogen, alkylhalos (e.g. CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, mercapto, etc. Thus, substituted analogues of heteroaromatic rings such as thiophene, pyridine, isoxazole, phthalimide, pyrazole, indole, furan, etc. or benzo-fused analogues of these rings are defined by the term "substituted heteroaryl" The term "substituted heteroaryl" encompasses "substituted heteroarylalkyl".

The term "substituted heteroarylalkyl" refers to a subset of "substituted heteroaryl" as described above in which an alkyl group, as defined herein, links the heteroaryl group to another group.

The term "heterocyclic" is used herein to describe a monovalent saturated or unsaturated non-aromatic group having a single ring or multiple condensed rings from

1-12 carbon atoms and from 1-4 heteroatoms selected from nitrogen, sulfur or oxygen within the ring. Such heterocycles are, for example, tetrahydrofuran. morpholine, piperidine, pyrrolidine, etc.

The term "substituted heterocyclic" as used herein describes a subset of "heterocyclic" wherein the heterocyde nucleus is substituted with one or more groups such as lower alkyl, acyl, halogen, alkylhalos (e.g. CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, mercapto, etc. The term "heterocyclicalkyl" defines a subset of "heterocyclic" wherein an alkyl group, as defined herein, links the heterocyclic group to another group.

The term "halogen" is used herein to refer to fluorine, bromine, chlorine and iodine atoms.

The term "alkoxy" is used herein to refer to the -OR group, where R is alkyl, or a substituted analogue thereof. Suitable alkoxy radicals include, for example, methoxy, ethoxy, t-butoxy, etc.

The term "reactive group" is used to mean a first atom or group capable of reacting with a second atom or group forming a covalent bond with it. The term "alkoxycarbonyl" by itself or as part of another substituent refers to a radical -C(O)OR where R represents an alkyl or cycloalkyl group as defined herein. Representative examples include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, cyclohexyloxycarbonyl and the like.

The term "amino acids side chain" refers to the following groups:

Description of the invention

The invention relates to lanthanide (III) ion complexing compounds comprising: (1) a sensitising moiety of formula (I)

(FUb

(R₃)C (R₁Ja

(I) in which: a is an integer from 1 to 4; b is an integer equal to 1 or 2; c is an integer equal to 1 to 3;

(R₁)a, (R2)tv (R3)c are the same or different and are chosen from the group consisting of

H; alkyl; -COOR₄ where R₄ is H or an alkyl; aryl; heteroaryl; saturated or unsaturated cyclic hydrocarbon; CF₃; CN; a halogen atom; L-Rg; L-Sc; or two consecutive R₃, two consecutive R₂ or two consecutive R₁ groups together form an aryl or a heteroaryl group or a saturated or unsaturated cyclic hydrocarbon group; where L is a linker, Rg is a reactive group and Sc is a conjugated substance;

Xi, X₂ are the same or different and are O or S;

A is either a direct bond or a divalent group chosen from: -CH₂- or -(CH₂)₂-, said moiety being covalently attached to

(2) a lanthanide (III) ion chelating moiety through A.

It should be specified that each R₁, R₂ and R₃ in the (R₁)_a groups, (R₂)_b groups and (R₃)_c groups, may be identical or different. For example, if a is 2, the two R₁ groups may be the same or different. Particularly preferred compounds of formula I are: those compounds where Xi=X₂=O; those where a=b=c=l, R₂=R₃=H and R₁ is a (C₁-C₆) alkyl; and those where a=b=c=l, Xi=X₂=O, R₂=R₃=H and R₁ is a (C₁-C₆) alkyl.

Other particularly preferred compounds of formula (I) are those in which: a=b=c=l; R₁=H, (C₁-C₆) alkyl;

R₂=H;

R₃=CF₃; COOR₄, where R₄=H, (C₁-C₆) alkyl, aryl, CN, halo, phenyl;

X₁= X₂=O.

The sensitising moiety of formula (I)

The pyridyl-azaxanthone sensitising moiety of formula (I) is also able to coordinate the lanthanide (III) ion via two nitrogen atoms of the pyridyl and azaxanthone groups. As compared to chelating compounds comprising azaxanthone chromophores disclosed in WO2006/120444 Al and WO 2006/039505 A2, the addition of a pyridyl group extends the conjugation length of the chromophore, shifting the lowest energy absorption band of the lanthanide complex to a longer wavelength and increasing the molar extinction coefficient. The sensitising moiety of formula (I) is obtained by using a sensitising derivative of formula (Ia), which is a further object of the present invention:

in which: (R₁)_a, (R₂)_b and (R₃)_c are as defined hereinabove for moiety of formula (I); A₁ is hydrogen, alkyl, halogen or halogenoalkyl.

Particularly preferred compounds of formula (Ia) are: those in which X₁=X₂=O; those where a=b=c=l, R₂=R₃=H and R₁ is a (C₁-C₆) alkyl; and those where a=b=c=l , X₁=X₂=O , R₂=R₃=H and R₁ is a (C₁-C₆) alkyl.

Other particularly preferred compounds of formula (Ia) are those in which: a=b=c=l; R₁=H, (C₁-C₆) alkyl; R₂=H;

R₃=CF₃; COOR₄, where R₄=H, (C₁-C₆) alkyl, aryl, preferably phenyl, CN, halo; X₁= X₂=O.

The sensitising derivative of formula (Ia) is prepared by reacting a pyridine derivative with a halo (preferably chloro) azaxanthone derivative (6). This reaction is based on carbon-carbon bond formation via a Stille cross coupling reaction that occurs between stannyl pyridine derivative and halo azaxanthone leading to the expected chromophore in a good yield ( around 60%) for this kind of reaction. Another alternative to this carbon-carbon bond formation is the Suzuki cross coupling. Indeed, the bipyridine formation moiety occurs but this time yields are poor (around 16%).

Pyridine derivatives bearing various substituents are commercially available and can be used to prepare compounds of formula (Ia) where R₃ is other that a hydrogen or may be easily prepared according to the substitution processes well-known by the person skilled in the art. Synthesis of the chloroazaxanthone derivative (6) is carried out by using the 2-chloronicotinic acid and the 4-tert-butylphenol as starting materials according to the reaction scheme 1, which comprises the synthesis of 6-ή_yf-butyl-9-oxa-1-aza- anthracen-10-one (2) by a two-step process involving a nucleophilic aromatic substitution reaction between 2-chloronicotinic acid and 4-te/f-butyl phenol in the presence of NaOMe in MeOH, followed by electrophilic cyclisation under acidic conditions. Methylation of azaxanthone (2) with methyl triflate and subsequent anion exchange chromatography yields the water soluble azaxanthone (4) as its chloride salt. Oxidative hydrolysis using a well-known protocol (J. Lewis and T. D. O' Donoghue, J. Chem. Soc, Dalton Trans., 1980, 5, 736) with [K₃Fe(CN)₆] and NaOH affords the N-methylpyridone intermediate (5). The reaction is easily monitored by ¹H NMR, with the loss of pyridine aromaticity accompanied by a proton shift from 7.99 to 6.54 ppm. Finally, chlorination of intermediate (5) with POCI₃ in an appropriate solvent such as C₆H₅N (CH₃)₂ yields the 2-chloro derivative (6) after chromatographic purification on silica. The above steps for preparing the chloroazaxanthone derivative (6) are known by the person skilled in the art.

It would be obvious to the person skilled in the art that the other halo azaxanthone derivatives may be obtained by processes similar to the one of reaction scheme 1 (compounds (1) to (6)) as the halogenation reagents used are commercially available.

The coupling of compound (6) with a pyridine derivative may be made by using either:

1) 6-methyl-2-(tributylstannyl) pyridine (7); or

2) 6-methyl-2-pyridineboronic acid N-phenyldiethanol amine ester which is commercially available.

The 6-methyl-2-(tributylstannyl) pyridine (7) may be obtained by the reaction of 2-bromo-6-methyl-pyridine with n-BuLi, followed by the reaction of the 2-lithium-6- methylpyridine with tri-n-butyl-tin chloride according to the reaction scheme 2.

The coupling of compound (6) with a pyridine derivative as hereinabove defined may be carried out according to scheme (3).

6-Methyl-2-(tributylstannyl)pyridine (7), may be used directly in a Stille-coupling with azaxanthone (6), without any purification. Using Pd(PPfIa)₄ as a catalyst, 6-tert- butyl-2-(6-methyl-pyridin-2-yl)-9-oxa-1-aza-anthracen-10-one (8), is obtained preferably following purification by trituration in diethyl ether. Selective bromination of the α-methyl substituent of (8) may be performed using radical bromination with N-bromosuccinimide and benzoyl peroxide in CCI₄. The reaction may be monitored by ¹H NMR to provide ratiometric analysis of the formation of the desired mono-brominated derivative (9), and the competing di-brominated analogue (10). Purification by column chromatography on silica yielded (9) and (10) respectively in a ration of about 4:3. Reaction of compound (10) with diethyl phosphite and DIPEA enabled conversion of the di-brominated material to the corresponding mono-brominated analogue, following chromatographic purification.

The lanthanide fill) ion chelating moietv or liqand. The term "lanthanide (III) chelating moiety" is used to describe a group that is capable of forming a high affinity complex with lanthanide cations such as Tb³⁺, Eu³⁺, Sm³⁺ , Dy³⁺, Gd³⁺ .

A lanthanide chelating moiety typically includes a set of lanthanide coordinating moieties that are heteroatom electron-donating group capable of coordinating a metal cation, such as O^", OPO₃ ^2-, NHR, or OR where R is an aliphatic group. Such a lanthanide chelating moiety should be kinetically stable to exchange the lanthanide ion and preferably have a formation constant (K _f) of greater than 10¹⁰ M^-1.

A variety of useful chelating moieties are known to the person skilled in the art.

Typical examples of lanthanide ion chelating moieties include: EDTA, DTPA, TTHA, DOTA, NTA, HDTA, DTPP, EDTP, HDTP, NTP, DOTP, DO3A, DOTAGA. Organic syntheses of these chelating moieties are known, and they are also available from commercial suppliers.

The following formulae illustrate chelating compounds that can be conjugated to a pyridyl-xanthone sensitizer and lead to the compounds according to the invention.

Most preferably, the sensitising moiety of formula (I) is linked to a lanthanide ion chelating moiety and together form an ion complexing compound of formula (II):

in which:

W is a sensitising moiety of formula (I) as defined above, linked through A,

R₅ to R₁₂, are the same or different and are chosen from the group consisting of H, alkyl, L-Rg, L-Sc;

Yi, Y₂ and Y₃ are the same or different and are chosen from the groups consisting of H,

L-Rg, L-Sc, and groups of the following formulae:

wherein: n is 0, 1 or 2; m is 1 or 2; p is 1 or 2; R₁₃ represents H, alkyl, optionally substituted aryl, preferably optionally subtituted benzyl, L-Rg, L-Sc;

R₁₄, R₁₅ are the same or different and chosen from H, -CHR'R" in which R' and R" being the same or different and being chosen from H, alkyl, optionally substituted aryl, optionally substituted aralkyl, or amino acid side chain, carboxyl group, L-Rg, L-Sc; R₁₆ represents H, alkyl, optionally substituted aryl, preferably optionally substituted benzyl, alkylcarboxyl, alkylamino, L-Rg, L-Sc; provided that when one of Y₁, Y₂, Y₃ is hydrogen, the other two are different from hydrogen.

The compounds of formula II, in which Y₁, Y₂, Y₃ are different from hydrogen, are preferred compounds. Among these preferred compounds, those in which R₅ to R₁₂ are hydrogen, are even more preferred compounds. Among, these compounds, those in which:

- n is 0 or 1 and R₁₃ is a (C₁-C₆) alkyl, preferably tertiobutyl, a benzyl or a phenyl; or

- R₁₄ is hydrogen and R₁₅ is a phenyl or benzyl, are particularly preferred.

In a particular embodiment of the present invention, the lanthanide ion complexing compound is a compound of formula (III):

wherein:

W, R₅ to R₁₂ and m are as defined above,

R₁₇ to R₂₂ are the same or different and are chosen from H, -CHR'R" in which R' and R" being the same or different and being chosen from H, alkyl, optionally substituted aryl, optionally substituted aralkyl, an amino acid side chain , a carboxyl group, L-Rg, L-Sc.

Among this family of compounds, a preferred subfamily comprises compounds of formula (IV):

in which:

W, R₅ to R₁₂ and m are as defined above for a compound of formula (III);

R'i, R'₂, R'₃ identical or different are a (C₁-C₆) alkyl, preferably -CH₃, -C₂H₅;

R"i to R"₃ are the same or different and are an optionally substituted aryl, preferably chosen from optionally substituted benzyl, optionally substituted phenyl, L-Sc or L-Rg.

The compounds of formulae (III) and (IV) in which R₅ to R₁₂ are hydrogen, are preferred compounds.

Among these preferred compounds of formula IV₇ those in which R"i to R"₃ is a benzyl or phenyl, optionally mono-substituted by a carboxy group, a (C₁-C₆) alkoxycarbonyl, L-Rg or L-Sc are particularly preferred compounds.

A particularly preferred subfamily comprises the compounds of formula (V):

in which:

W is as previously defined for a compound of formula (II);

R₂₃ represents H, a carboxyl group, (C₁-C₆) alkoxycarbonyl, L-Sc, L-Rg.

In another embodiment of the present invention, the lanthanide ion chelating complex is a compound of formula (VI):

in which: n is 0, 1 or 2

W, R₅ to R₁₂ are as defined above for a compound of formula (II);

R₂₄ to R₂₆, identical or different, are chosen from the group consisting of H, (C₁-C₆) alkyl, optionally substituted aryl, (preferably optionally substituted benzyl), L-Rg, L-Sc. Among the compounds of formula VI, those in which R₅-R₁₂ are hydrogen, are preferred compounds. Particularly preferred compounds are those in which R₅ to R₁₂ are hydrogen, n is 0 or 1 and R₂₄ to R₂₆ is a (C₁-C₆) alkyl, a phenyl or a benzyl.

In another embodiment, the lanthanide ion chelating complex is a compound of formula (VII):

in which:

W, R₅ to R₁₂, and p are as previously defined for a compound of formula (II);

R₂₇ to R₂₉ are chosen from the group consisiting of H, (C₁-C₆) alkyl, optionally substituted arγl, (preferably optionally substituted benzyl), L-Rg, L-Sc.

In other embodiment, the lanthanide ion chelating complex is a compound of formula (VIII):

in which n is 0, 1 or 2

W, R₅ to R_J2 are as defined above for a compound of formula (II); R₃₀, R_3I and R₃₂ are chosen from the group consisting of H, (C₁-C₆) alkyl, optionally substituted aryl, (preferably optionally substituted benzyl), L-Rg, L-Sc; R₃₃ is a group of formula

in which r is 0, 1 or 2 and R₃₄ is chosen from the group consisting of H, (C₁-QJalkyl, optionally substituted aryl, preferably optionally substituted benzyl.

Among the compounds of formulae (VII) or (VIII) those in which R₅ to R_n are hydrogen are preferred compounds.

Any fluorescent lanthanide metal can be used with the chelating ligands of this invention. Most preferably, the lanthanide metal is europium.

Linker - Reactive group / conjugated substance: (L-Rg, L-Sc)

As mentioned above, compounds of formula (I) to (III) and (V) to (VIII) optionally comprise a linker L that bears a reactive group Rg or a conjugated substance Sc. It is particularly advantageous to use the lanthanide ions complexes of the invention as fluorescent markers, particularly in bioassays where biological molecules have to be labelled with fluorescent compounds.

Some preferred compounds according to the invention comprise at least one group L-Rg or L-Sc, and preferably one or two. The linker L is optionally a single covalent bond, such that either the reactive functional group Rg or the conjugated substance Sc is bound directly to the complexing compound. Alternatively, L may incorporate a series of non-hydrogen atoms that form a stable covalent linkage between the reactive functional group or conjugated substance and the lanthanide (III) ion complexing compound. Typically, L may incorporate 1-20 non-hydrogen atoms in a stable conformation. Stable atom conformations include, without limitation, carbon-carbon bonds, amide linkages, ester linkages, sulfonamide linkages, ether linkages, thioether linkages, and/or other covalent bonds. Preferred covalent linkages may include single bonds, carboxamides, sulfonamides, ethers, and carbon-carbon bonds, or combinations thereof. Particularly preferred linkers are those according to the following formulae:

in which:

- q and r are integers from 1 to 16, preferably 1 to 8;

- s and u are integers from 1 to 16, preferably 1 to 5.

The reactive functional group Rg may include any functional group that exhibits appropriate reactivity to be conjugated with a desired substance. The choice of the reactive group depends on the functional groups present on the substance to be conjugated. Typically, functional groups present on such substances include, but are not limited to, alcohols, aldehydes, amines, carboxylic acids, halogens, ketones, phenols, phosphates, and thiols, or combinations thereof. Suitable Rg groups include activated esters of carboxylic acids, aldehydes, alkyl halides, amines, anhydrides, aryl halides, carboxylic acids, haloacetamides, halotriazines, hydrazines (including hydrazides), isocyanates, isothiocya nates, maleimides, phosphoramidites, sulfonyl halides and thiol groups, or a combination thereof. Typically, Rg is an activated ester of a carboxylic acid, an amino, haloacetamido, a hydrazine, an isothiocyanate, or a maleimide group. In one aspect of the lanthanide complex, Rg is a succinimidyl ester of a carboxylic acid.

Preferred reactive groups Rg are those that are routinely used in conjugation chemistry, and particularly those with following formulae:

in which: p represents 0 to 8 and n represents 0 or 1; Ar is 5 to 6 member aryl, optionally containing 1 to 3 heteroatoms chosen from halo, N, O, S and optionally substituted by a halogen atom.

The lanthanide (III) ion chelating moiety is obtained by using a chelating compound of formula (Ha) which is a 1, 4, 7, 10-tetraazacyclododecane derivative, i.e. a cyclen derivative:

in which:

R₅ to R₁₂ and Yi to Y₃ are as defined above for the lanthanide (III) ion chelating moiety of formulae (II) to (VIII). The chelating compound of formula (Ha) is obtained by conventional substitution by Yi, Y₂, Y₃ of the cyclen or the corresponding cyclen appropriately substituted by R₅-R₁₂. These cyclen compounds used as starting materials are known compounds or may be obtained by appropriate conventional substitution of the cyclen.

The lanthanide (III) ion complexing compounds are obtained by nucleophilic substitution resulting from the reaction of a sensitising derivative of formula (Ia) with a lanthanide (III) ion chelating compound of formula (Ha).

This process is illustrated by the following schemes 4, 5, 6 and 7 in which the lanthanide (III) ion complexing compounds are identified as L compounds (U, L₄, L₉ and U₃) and the lanthanide (III) ion complexes are identified as EuL complexes (EuLi, EuU, EuL₉ and EuLi₃). It would be obvious for the person skilled in the art that these processes may be used starting from the reagents bearing the appropriate substituents.

The lanthanide (III) ion complexing compounds that are substituted with a reactive functional group may be used to prepare a variety of conjugates. The conjugated substance may be a member of a specific binding pair. Alternatively, the conjugated substance may be a molecular carrier. The conjugated substance may include a biomolecule that is an amino acid, a peptide, a protein, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer or a carbohydrate. The conjugated substance may include a polar moiety, or a masked polar moiety, or the conjugated substance may include a solid or semi-solid matrix. The conjugated substance may include one or more additional dyes or luminophores. The conjugated substance Sc also may be a member of a specific binding pair or a molecular carrier. Specific binding pair members typically specifically bind to and are complementary with the complementary member of the specific binding pair. Conjugated members of a specific binding pair can be used to localize compounds of the present teachings to the complementary member of that specific binding pair. Representative specific binding pairs are: antigen/antibody, avidin or streptavidin/Biotin, ligand/receptor, DNA strand /DNA strand.

Lanthanide (III) ion complexes

The invention also encompass those lanthanide (III) ion complexes obtained by contacting the lanthanide (III) ions complexing compounds of the invention and described hereinabove, with a lanthanide (III) ion (such as Tb³⁺, Eu³⁺, Sm³⁺, Dy³⁺). When the resulting complex is a charged compound, it is generally in the form of a salt with a counter ion, such as Cl-, OTf or related common anions.

Examples:

Abbreviations used in the examples:

THF : Tetrahydrofuran

NBS : N-Bromosuccinimide

DO3A : 1, 4, 7-tris(carboxymethyl)-1 ,4,7,10-tetraazacyclododecane

DCM : Dichloromethane

TFA : Trifluoroacetic acid

OTf : Trifluoromethanesulfonate anion (=CF₃SO₃)

HRMS : High resolution mass spectrometry

Vr₂ : Isopropyl *Bu : Tertiobutyl

*Boc : Tertiobutyloxycarbony

HPLC Analysis of the examples were made as follows:

Analytical reverse phase HPLC analysis were performed at 298 K on a Perkin Elmer system comprising of Perkin Elmer Series 200 Pump, Perkin Elmer Series 200

Autosampler, Perkin Elmer Series 200 Diode array detector and Perkin Elmer Series

200 Fluorescence detector.

All analysis were perfomed using a Chromolith performance RP18e 100 x 4.6 nm column, using a flow rate of 1 ml / min and solvents as indicated hereinafter.

Method A:

Analytical and semi-preparative HPLC were performed at 298 K using a Waters HPLC system equipped with a diode array detector.

Analytical HPLC was performed using a Chromolith performance RP18e 100 x 4.6 mm column, using a flow rate of 3 ml / min and one of the selected methods shown below;

Method B:

Method C:

Method D:

Semi-preparative HPLC was performed using a Chromolith performance RP18e 100 x 10 mm column, using a flow rate of 14.1 ml / min and one of the selected methods shown below;

Method E:

Method F:

The invention will now be disclosed in more detail by the following illustrative, but non-limiting, examples 1 to 7 relating to the synthesis of the invention ligands and complexes and the Examples A to C concerning the properties of the invention complexes thus obtained. Example 1: Synthesis of L₁ and [Eu L₁ (See schemes 1 to 4)

2-(4'-tert-Butylphenoxy) nicotinic acid (1)

To a solution of sodium metal (1.02 g, 44.4 mmol) was carefully added to dry MeOH (25 cm³) was added 2-chloronicotinic acid (3.31 g, 21.01 mmol) and 4-tert- butylphenol (15.20 g, 101.18 mmol) to form a thick cream coloured solution. The MeOH was removed under reduced pressure to afford a cream residue which was heated for 20 h at 190 °C with stirring. After cooling, the coloured gum was treated with H₂O (200 cm³) and washed successively with Et₂O (2 x 150 cm³). The aqueous solution was acidified to pH 5 by the addition of acetic acid to afford a fine precipitate. The precipitate was filtered, washed with water and dried under vacuum to yield the title compound as a white fine crystalline solid (4.89 g, 18.02 mmol, 86%). δ_H (CDCI₃, 500 MHz) 1.37 (9H, S, 'Bu), 7.14 (2H, d, J 8.5, H^2'), 7.20 (1H, dd, J 7.5; 5, H²), 7.49 (2H, d, J 9, H^3'), 8.35 (1H, dd, J 4.5; 2, H¹), 8.55 (1H, dd, J 8; 2, H³). δc (CDCI₃, 125 MHz) 31.7 (C^6'), 34.8 (C^5'), 113.5 (C⁴), 119.7 (C²), 121.4 (C^2'), 127.1 (C^3'), 143.8 (C³), 149.3 (C^4'), 149.8 (C^1'), 152.4 (C¹), 161.5 (C⁵), 164.9 (C=O_(aαd)). m/z (ES ) 270.1 (100%, M - H). Found: C, 70.54; H, 6.20; N, 4.91%; C₁₆Hi₇NO₃ requires C, 70.83; H, 6.32; N, 5.16%.

7-tert-Butyl-1-azaxanthone (2)

Polyphosphoric acid (90 g) was added to 2-(4'-tert-butylphenoxy) nicotinic acid (2.15 g, 7.93 mmol) and the mixture heated at 120 °C for 16 h. The light brown mixture was allowed to cool slightly before being poured onto ice water (400 cm³) to afford a pale yellow solution. The pH of the solution was then adjusted to neutral pH 7 by the careful addition of concentrated NaOH _(aq). The solution was extracted with Et₂O (3 x 300 cm³), the organic phases combined, dried over MgSO₄, filtered and the solvent removed under reduced pressure to afford 7-tert-butyl-1-azaxanthone as a cream coloured solid (1.79 g, 7.08 mmol, 89%). 6H (CDCI₃, 500 MHz) 1.42 (9H, s, ⁴Bu), 7.45 (1H, dd, J 7.5; 4.5, H²), 7.58 (1H, d, J 8.5, H¹⁰), 7.86 (1H, dd, J 9; 3, H⁹), 8.30 (1H, d, J 2.5, H⁷), 8.73-8.76 (2H, H7H³). δc (CDCI₃, 125 MHz) 31.6 (C¹⁴), 35.1 (C¹³), 117.0 (C⁴), 118.4 (C¹⁰), 121.1 (C²), 121.1, 122.7 (C⁷), 133.9 (C⁹), 137.6, 148.2 (C⁶), 154.1, 154.3(C¹²), 160.6 (C¹¹), 178.1 (C⁵). m/z (ES⁺) 529.5 (100%, 2M + Na), 782.3 (70%, 3M + Na), 275.8 (25%, M + Na). Found: C, 75.80; H, 5.91; N, 5.61%; C₁₆Hi₅NO₂ requires C, 75.87; H, 5.97; N, 5.53%. 7-tert-Butyl-N-methyl-1-azaxanthonium trifluoromethylsulfonate (3)

7-tert-Butyl-1-azaxanthone (1.00 g, 3.95 mmol) was dissolved in dry toluene (20 cm³) under an atmosphere of argon. The resultant yellow solution was then cooled in an ice bath to approximately 0 °C. An excess of methyl trifluoromethanesulfonate (6 cm³, 8.70 g, 53.02 mmol) was then carefully added to the cooled solution in a dropwise fashion. Almost instantaneously a pale cream precipitate formed in a faint yellow coloured solute. The precipitate was filtered and dried under vacuum to afford the title compound as a white solid (1.49 g, 3.58 mmol, 91%). δ» (CD₃OD, 400 MHz) 1.43 (9H, s, ¹Bu), 4.51 (3H, s, Me), 7.84 (1H, d, J 8.8, H¹⁰), 7.99 (1H, dd, J 8; 6, H²), 8.15 (1H, dd, J 8.8; 2.4, H⁹), 8.33 (1H, d, J 2.4, H⁷), 9.14 (1H, dd, J 6; 2, H¹), 9.30 (1H, dd, J 8; 2, H³). δc (CD₃OD, 100 MHz) 30.3 (C¹⁴), 34.8 (C¹³), 41.7 (CH₃), 118.2 (C¹⁰), 120.4 (C⁴), 120.8 (C⁶), 121.2 (C²), 122.6 (C⁷), 135.4 (C⁹), 145.9 (C³), 149.1 (C¹),

151.1 (C⁸), 152.4 (C¹¹), 156.3 (C¹²), 173.8 (C⁵). δ_F (CD₃OD, 188 MHz) - 80.5 (CF₃). m/z (ES⁺) 268.2 (100%, M).

7-tert-Butyl-N-methyl-1-azaxanthonium chloride (4)

Compound (4), having the following properties, was obtained by ion exchange chromatography in water using a DOWEEX 1-X8 (Cl) resin: δ_H (CD₃OD, 500 MHz) 1.46 (9H, s, ¹Bu), 4.55 (3H, S, Me), 7.88 (1H, d, J 9, H¹⁰), 8.03 (1H, t, J 6.5, H²), 8.18 (1H, dd, J 9; 2, H⁹), 8.36 (1H, d, J 2, H⁷), 9.22 (1H, d, J 6.5,

H¹), 9.33 (1H, d, J 7.5, H³). δc (CD₃OD, 125 MHz) 30.4 (C¹⁴), 34.8 (C¹³), 41.8 (CH₃),

118.2 (C¹⁰), 120.5 (C⁴), 120.8 (C⁶), 121.2 (C²), 122.6 (C⁷), 135.4 (C⁹), 145.9 (C³), 149.1 (C¹), 151.1 (C⁸), 152.4 (C¹¹), 156.3 (C¹²), 173.8 (C⁵).

6-te/tf-Butyl-1-methyl-1H-9-oxa-1-aza-anthracene-2,10-dione (5)

7-te/f-Butyl-N-methyl-1-azaxanthonium chloride (0.36 g, 1.18 mmol) dissolved in H₂O (10 cm³) was added in a dropwise fashion to a solution of potassium hexacyanoferrate (III) (1.16 g, 3.54 mmol) in H₂O (6 cm³). The solution was cooled to approximately 0 °C and a solution of NaOH (0.85 g, 21.24 mmol) in H₂O (10 cm³) added to the reaction mixture over a period of 20 min. The solution was stirred at approximately 0 °C for 24 h. The solution was acidified to pH 3 by the addition of sulphuric acid to afford a green precipitate. The material was filtered, dissolved in CHCl₃ (50 cm³) and partitioned with H₂O (2 x 50 cm³). The organic phases were separated, dried over MgSO₄ and the solvent removed under reduced pressure to yield the title compound as a red solid (0.25 g, 0.87 mmol, 74%). 5H (CDCI₃, 500 MHz) 1.41 (9H, s, 'Bu), 3.76 (3H, s, Me), 6.54 (1H, d, J 9.5, H²), 7.47 (1H, d, J 8.5, H¹⁰), 7.79 (1H, dd, J 9; 2, H⁹), 8.21 (1H, d, J 9.5, H³), 8.29 (1H, d, J 2, H⁷). δc (CDCI₃, 125 MHz) 28.5 (CH₃), 31.6 (C¹⁴), 35.2 (C¹³), 102.8 (C⁴), 116.0 (C²), 117.3 (C¹⁰), 121.6 (C⁶), 122.9 (C⁷), 132.3 (C⁹), 135.7 (C³), 149.7 (C⁸), 152.0 (C¹¹), 156.5 (C¹²), 162.3 (C¹), 174.2 (C⁵). m/z (ES⁺) 284.3 (100%, M + H). HRMS (ES⁺) 284.12809; Cj₇Hi₈O₃N₁ requires 284.12812, [M + H]⁺. Found: C, 71.82; H, 5.91; N, 4.90%; Ci₇Hi₇NO₃ requires C, 72.07; H, 6.05; N, 4.94%.

6-te/tf-Butyl-2-chloro-9-oxa-anthracen-10-one (6)

N,N-Dimethylaniline (0.3 cm³) was added to a solution of 6-te^f-butyl-1-methyl- 1H-9-oxa-1-aza-anthracene-2,10-dione (0.18 g, 0.63 mmol) in POCI₃ (10 cm³) and the solution heated at reflux for 24 h. The solvent was removed under reduced pressure to yield a dark green residual solid. The residue was treated with H₂O (100 cm³) and the aqueous phase extracted with CH₂CI₂ (2 x 50 cm³). The combined organic phases were washed with aqueous K₂CO₃ (0.1 M, 100 cm³), dried over K₂CO₃, filtered and the filtrate concentrated under reduced pressure. The residue purified by chromatography on silica (gradient elution: Hexane to 10% EtOAc/Hexane, R_F = 0.33, 10% EtOAc/Hexane) to yield the title compound as a pink solid (0.09g, 0.31 mmol, 49%). δ_H (CDCI₃, 500 MHz) 1.41 (9H, S, ¹Bu), 7.43 (1H, d, J 8, H²), 7.54 (1H, d, J 9, H¹⁰), 7.86 (1H, dd, J 9; 2.5, H⁹), 8.27 (1H, d, J 2.5, H⁷), 8.65 (1H, d, J 8, H³). δc (CDCI₃, 125 MHz) 31.5 (C¹⁴), 35.1 (C¹³), 115.6 (C⁴), 118.4 (C¹⁰), 121.1 (C⁶), 121.9 (C²), 122.7 (C⁷), 134.1 (C⁹), 139.9 (C³), 148.8 (C⁸), 153.8 (C¹¹), 155.6 (C¹²), 159.7 (C¹), 177.2 (C⁵).

2-Methyl-6-tributylstannanyl-pyridiπe (7)

Following the procedure disclosed by U. S. Schubert et al. in Org. Lett., 2000, 2, 3373, /hButyllithium (1.94 ml, 3.13 mmol, 1.6 M in hexane) was added dropwise to a stirred solution of 2-bromo-6-methylpyridine (0.50 g, 0.33 ml, 2.90 mmol) in anhydrous THF (15 ml), at -78 °C. After stirring the solution at -78 °C for 2 h, tributyltinchloride (0.94 ml, 3.49 mmol) was added dropwise, and the mixture stirred while allowed to warm to room temperature. H₂O (20 ml) was poured into the reaction mixture and the phases separated. The aqueous phase was extracted with diethyl ether (2 x 20 ml). The combined organic extracts were dried over MgSO₄, filtered, and the solvent removed under reduced pressure to yield the crude title compound 7. The material was used directly without any further purification.

6-te/*-Butγl-2-(6-methyl-pyridin-2-yl)-9-oxa-1-aza-anthracen-10-one (8)

Procedure A: StHIe Cross-Coupling

6-te/?-Butyl-2-chloro-9-oxa-anthracen-10-one 6 (0.201 g, 0.696 mmol) and 6-methyl- 2-(tributylstannyl)pyridine 7 (0.293 g, 0.766 mmol) were added to a Schlenk tube which was evacuated and back filled with argon three times. Degassed toluene (5 ml) was added to the vessel which was then evacuated and back filled with argon five times. Tetrakis(triphenylphosphine)palladium (0) (0.040 g, 0.034 mmol) was added to the solution under an atmosphere of argon. The reaction mixture was stirred and heated at reflux, under argon, for 16 h. The reaction mixture was allowed to cool to room temperature, filtered, and the solute concentrated under reduced pressure to afford a residual brown oil. The crude material was triturated with diethyl ether (10 ml) to yield a fine precipitate in red solute. The solvent was decanted and the solid dried under vacuum to yield the title compound % as a colourless solid (0.145 g, 0.422 mmol, 61 %); m.p. 191-192 °C; ¹H NMR (CDCI₃, 700 MHz) δ 1.37 (9H, s, ¹Bu CH₃), 2.60 (3H, s, CH₃), 7.18 (1H, d, J = 8.0 Hz, H^5'), 7.53 (1H, d, J = 8.0 Hz, H¹⁰), 7.69 (1H, t, J= 8.0 Hz, H^4'), 7.79 (1H, dd, J= 8.0; 2.0 Hz, H⁹), 8.26 (1H, d, J= 2.0 Hz, H⁷), 8.29 (1H, U₁ J = 8.0 Hz, H^3'), 8.54 (1H, d, J = 8.5 Hz, H²), 8.74 (1H, d, J = 8.5 Hz, H³); ¹³C NMR (CDCI₃, 176 MHz, ¹H decoupled 700 MHz) δ 24.8 (1C, CH₃), 31.5 (3C, C¹⁴), 35.0 (1C, C¹³), 116.4 (1C, C⁴), 118.3 (1C, C¹⁰), 118.5 (1C, C²), 119.7 (1C, C^5'), 121.3 (1C, C⁶), 122.7 (1C, C⁷), 124.9 (1C, C^2'), 133.6 (1C, C⁹), 137.4 (1C, C)₁ 138.2 (1C, C³), 148.0 (1C, C⁸), 153.6 (1C, C^1'), 154.2 (1C, C¹¹), 158.6 (1C, C^6'), 160.2 (1C, C¹), 160.6 (1C, C¹²), 177.8 (1C, C⁵); MS (ES⁺) m/z 345.2 (100 %, [M + H]⁺); HRMS (ES⁺) m/z found 345.1596, C₂₂H_2IU₂N₂ requires 345.1597, [M + H]⁺. Procedure B: Suzuki-Miyaura Cross-Coupling

6-ήs^-Butyl-2-chloro-9-oxa-anthracen-10-one 6 (0.040 g, 0.14 mmol), 6- methyl-2-pyridineboronic acid N-phenyldiethanolamine ester (0.047 g, 0.17 mmol) and Cs₂CO₃ (0.054 g, 0.17 mmol) were added to a Schlenk tube which was evacuated and back filled with argon three times. Degassed 1,4-dioxane (4 ml) was added to the vessel, which was evacuated and back filled with argon three times. Pd₂(dba)₃ (2 mg, 1.5 % mol) and P(¹Bu)₃ (1 mg, 3.6 % mol) were added to the reaction mixture, which was stirred and heated at 85 °C, under argon, for 18 h. After 18 h a further addition of Pd₂(dba)₃ (2 mg, 1.5 % mol) and P(¹Bu)₃ (1 mg, 3.6 % mol) were introduced to the vessel, with heating continued at 85 °C for a further 24 h. The reaction mixture was allowed cool to room temperature and the solvent removed under reduced pressure. The residue was dissolved in diethyl ether (50 ml) and washed with H₂O (50 ml), followed by brine (50 ml). The organic phase was dried over K₂CO₃, filtered and the solvent removed under reduced pressure. The crude material was purified by column chromatography on silica (gradient elution: CH₂CI₂ to 8 % CH₃OH:CH₂CI₂, utilising 0.1 % CH₃OH increments). The product was recrystallised from warm EtOH and dried under vacuum to yield the title compound 8 as a colourless solid (0.008 g, 0.015 mmol, 16 %); R_F = 0.38 (Silica, CH₃OH - CH₂CI₂, 9 : 1 v/v). Characterisation as reported in procedure A.

2-(6-Bromomethyl-pyridin-2-yl)-6-te/*-butyl-9-oxa-1-aza-anthracen-10-one (2)

Procedure A:

A stirred mixture of 6-te/f-butyl-2-(6-methyl-pyridin-2-yl)-9-oxa-1-aza- anthracen-10-one 8 (0.200 g, 0.581 mmol), N-bromosuccinimide (0.129 g, 0.725 mmol) and benzoyl peroxide (0.010 g, 0.041 mmol) in CCI₄ (5 ml) was heated at reflux, for 16 h. The reaction progress was monitored by ¹H NMR analysis. After 8 h, N- bromosuccinimide (0.100 g, 0.562 mmol) and dibenzoyl peroxide (0.010 g, 0.041 mmol) were added to the reaction mixture, which was heated at reflux for a further 16 h. The reaction mixture was allowed to cool to room temperature, filtered and the solvent removed under reduced pressure to yield a yellow residue. The crude material was purified by column chromatography on silica (using 100 % CH₂CI₂ elution) to yield the title compound 9 as a colourless solid (0.101 g, 0.406 mmol, 41 %); R_? = 0.35 (Silica, CH₂CI₂, 100 v); m.p. 186-187 °C; ¹H NMR (CDCI₃, 500 MHz) δ 1.42 (9H, s, ¹Bu CH₃), 4.66 (2H, s, CH₂Br), 7.56 (1H, d, J = 7.5 Hz, H^5'), 7.60 (1H, d, J = 9.0 Hz, H¹⁰), 7.85 (1H, dd, J = 8.5; 2.5 Hz, H⁹), 7.90 (1H, t, J = 8.0 Hz, H^4'), 8.32 (1H, d, J = 2.5 Hz, H⁷), 8.48 (1H, d, J= 8.0 Hz, H^3'), 8.63 (1H, d, J= 8.0 Hz, H²), 8.82 (1H, d, J= 8.0 Hz, H³); ¹³C NMR (CDCI₃, 126 MHz, ¹H decoupled 500 MHz) δ 31.6 (3C, C¹⁴), 34.0 (1C, CH₂Br), 35.1 (1C, C¹³), 116.8 (1C, C⁴), 118.3 (1C, C¹⁰), 118.7 (1C, C²), 121.3 (1C, C⁶), 121.8 (1C, C^5'), 122.8 (1C, C⁷), 125.1 (1C, C^3'), 133.8 (1C, C⁹), 138.4 (1C, C^4'), 138.6 (1C, C³), 148.2 (1C, C⁸), 154.0 (1C, C^1'), 154.2 (1C, C¹¹), 157.0 (1C, C^6'), 160.0 (1C, C¹), 160.2 (1C, C¹²), 177.9 (1C, C⁵); MS (ES⁺) m/z 423.2 (100 %, [M + H]⁺); HRMS (ES⁺) /77/zfound 423.0700, C₂₂H₂₀O₂N₂Br₁ requires 423.0702, [M + H]⁺.

Procedure B:

A solution of 6-te/t-butyl-2-(6-dibromomethyl-pyridin-2-yl)-9-oxa-1-aza- anthracen-10-one 10 (0.060 g, 0.120 mmol), Pr₂NEt (0.062 g, 0.084 ml, 0.480 mmol) and diethyl phosphite (0.066 g, 0.062 ml, 0.478 mmol) in anhydrous 'THF (5 ml), was stirred at room temperature, for 16 h. The solvent was removed under reduced pressure and the residue partitioned between CHCl₃ (10 ml) and H₂O (10 ml). The organic phase was separated and concentrated under reduced pressure to afford a residual oil. The crude material was purified by column chromatography on silica (using 100 % CH₂CI₂ elution), to yield the title compound 9 as a colourless solid (0.042 g, 0.095 mmol, 79 %). Characterisation as reported in procedure A.

6-te/^Butyl-2-(6-dibromomethyl-pyridin-2-yl)-9-oxa-1-aza-anthracen-10- one (10)

β-te/f-Butyl-Z-Cβ-dibromomethyl-pyridin-Z-yO-Q-oxa-1-aza-anthracen-lO-one 10 was isolated using an identical procedure to that described for 2-(6-bromomethyl-pyridin-2- yl)-6-te/f-butyl-9-oxa-1-aza-anthracen-10-one 9. The procedure yielded the title compound 10 as a colourless solid (0.061 g, 0.119 mmol, 33 %); /?_F = 0.71 (Silica, CH₂CI₂, 100 v); ¹H NMR (CDCI₃, 500 MHz) δ 1.41 (9H, s, ¹Bu CH₃), 6.78 (1H, S, CHBr₂), 7.58 (1H, ά, J = 8.5 Hz, H¹⁰), 7.84 (1H, dd, J = 9.0; 2.5 Hz, H⁹), 7.92 (1H, U₁ J = 8.0 Hz, H^5'), 7.98 (1H, t, J= 8.0 Hz, H^4'), 8.30 (1H, d, J= 2.5 Hz, H⁷), 8.50 (1H, U₁ J= 7.5 Hz, H^3'), 8.59 (1H, d, J= 8.0 Hz, H²), 8.81 (1H, d, J= 8.0 Hz, H³); ¹³C NMR (CDCI₃, 126 MHz, ¹H decoupled 500 MHz) δ 31.6 (3C, C¹⁴), 35.1 (1C, C¹³), 41.6 (1C, CHBr₂), 116.9 (1C, C⁴), 118.3 (1C, C¹⁰), 118.7 (1C, C²), 121.3 (1C, C⁶), 122.8 (1C, C⁷), 122.9 (1C, C^3'), 123.6 (1C, C^5'), 133.9 (1C, C⁹), 138.7 (1C, C³), 139.1 (1C, C^4'), 148.3 (1C, C⁸), 152.7 (1C, C²O, 154.2 (1C, C¹¹), 159.0 (1C, C^6'), 159.1 (1C, C¹), 160.2 (1C, C¹²), 177.8 (1C, C⁵); MS (ES⁺) m/z 503.2 (100 %, [M + H]⁺).

(4,7-bis-fert-Butoxycarbonylmethyl-1,4,7,10-tetraaza-cyclododec-1-yl)- acetic acid tert butyl ester (16)

This cyclen derivative was prepared according to the procedure described by O. Reany et al., J. Chem. Soc. Perkin. Trans 2., 2000, 1819. A mixture of cyclen (2.54 g, 14.7 mmol), te/t-butγl bromoacetate (8.67 g, 44.2 mmol) and NaHCO₃ (3.72 g, 44.2 mmol) in anhydrous XH₃CN (75 ml) was stirred at it, under argon, for 24 h. The solution was filtered and the filtrate concentrated under reduced pressure to afford a residual orange oil, which crystallised upon standing. The crude material was purified by column chromatography on silica (gradient elution: CH₂CI₂ to 5 % CH₃OH : CH₂CI₂, utilising 0.1 % CH₃OH increments) to yield the title compound 16 as a colourless crystalline solid (2.41 g, 4.68 mmol, 32 %); m.p. 179-181 °C; ¹H NMR (CDCI₃, 500 MHz) δ 1.47 (27H, s, *Boc CH₃), 2.88 (12H, br s, cyclen CH₂), 3.11 (4H, br s, cyclen CH₂), 3.30 (2H, s, CH₂CO₂^Bu), 3.39 (4H, s, CH₂CO₂^Bu), 10.04 (1H, br s, NH); ¹³C NMR (CDCI₃, 125 MHz, ¹H decoupled 500 MHz) δ 28.4 (9C, feoc CH₃), 30.6- 31.2 (6C, cyclen CH₂), 47.8 (1C, cyclen CH₂), 49.4 (2C, CH₂CO), 51.4 (1C, cyclen CH₂), 58.5 (1C, CH₂CO), 81.9 (2C, feoqq)), 82.1(1C, teoc_(q)), 169.9 (2C, C = O), 170.8 (1C, C = O); MS (ES⁺) m/z 515.6 (100 %, [M + H]⁺).

{4,7-bis-te^Butoxycarbonylmethyl-10-[6-(6-te/^butyl-10-oxo-10/^9-oxa- l-aza-anthracen-2-yl)-pyridin-2-ylmethyl]-1,4,7,10-tetraaza-cγclododec-1- yl}-acetic acid fe/t-butyl ester ([L¹])

A stirred mixture of (4,7-bis-te/f-butoxycarbonylmethyl-1,4,7,10-tetraaza-cyclododec-1- yl)-acetic acid tert butyl ester 16 (0.032 g, 0.062 mmol), 2-(6-bromomethyl-pyridin-2- yl)-6-te/?-butyl-9-oxa-1-aza-anthracen-10-one 9 (0.024 g, 0.057 mmol) and Cs₂CO₃

(0.026 g, 0.080 mmol) in anhydrous CH₃CN (5 ml), was heated at reflux, under argon, for 16 h. The mixture was allowed to cool to room temperature, syringe filtered and the filtrate concentrated under reduced pressure to afford a residual yellow oil. The crude material was purified by column chromatography on alumina (gradient elution; CH₂CI₂ to 2 % CH₃OH : CH₂CI₂, utilising 0.1 % CH₃OH increments) to yield the title compound [L¹] as a pale yellow crystalline solid (0.035 g, 0.042 mmol, 74 %); Rp = 0.22 (Alumina, CH₂CI₂ - CH₃OH, 19 : 1 v/v); ¹H NMR (CDCI₃, 700 MHz) δ 1.29 (9H, s, feu CH₃), 1.32 (18H, br s, *Boc CH₃), 1.35 (9H, br s, *Boc CH₃), 2.71 (8H, br s, cyclen CH₂), 3.24 (8H, br s, cyclen CH₂), 3.50 (4H, br s, CH₂CO₂^Bu), 3.55 (2H, br s, CH₂CO₂^Bu), 3.69 (2H, s, CH₂ pyridine), 7.25 (1H, d, J = 8.0 Hz, H^5'), 7.45 (1H, d, J = 8.5 Hz, H¹⁰), 7.69 (1H, t, J= 8.0 Hz, H^4'), 7.71 (1H, dd, J= 9.0; 3.0 Hz, H⁹), 8.17 (1H, d, J= 3.0 Hz, H⁷), 8.32 (1H, 6, J= 8.0 Hz, H^3'), 8.36 (1H, d, J= 7.0 Hz, H²), 8.64 (1H, U₁ J = 8.0 Hz, H³); ¹³C NMR (CDCI₃, 176 MHz, ¹H decoupled 700 MHz) δ 28.3 (3C, C¹⁴), 31.5 (6C, 'Boc CH₃), 35.1 (3C, *Boc CH₃), 50.6 (2C, cyclen CH₂), 51.8 (2C, cyclen CH₂), 56.1 (2C, cyclen CH₂), 56.7 (2C, cyclen CH₂), 57.1 (1C, CH₂ pyridine), 82.4 (4C, CBu), 116.4 (1C, C⁴), 118.2 (1C, C¹⁰), 118.3 (1C, C²), 121.0 (1C, C^3'), 121.2 (1C, C⁶), 122.6 (1C, C⁷), 126.2 (1C, C^5'), 133.6 (1C, C⁹), 137.4 (1C, C^4'), 138.2 (1C, C³), 148.0 (1C, C⁸), 153.6 (1C, C^1'), 154.2 (1C, C¹¹), 158.6 (1C, C^6'), 160.2 (1C, C¹), 160.6 (1C, C¹²), 177.8 (1C, C⁵); MS (ES⁺) m/z 857.5 (100 %, [M + H]⁺); HRMS (ES⁺) m/z found 857.5176 [M + H]⁺ C₄₈H₆₉O₈N₆ requires 857.5171.

[EuL¹]

A solution of {4,7-bis-te/f-butoxycarbonylmethyl-10-[6-(6-ή9/t-butyl-10-oxo-10/f 9-oxa- l-aza-anthracen-2-yl)-pyridin-2-ylmethyl]-1,4,7,10-tetraaza-cyclododec-1-yl}-acetic acid tert-butyl ester [L¹] (0.035 g, 0.0419 mmol) in CH₂CI₂ - TFA (1:1 v/v, 2 ml) was stirred at room temperature, in a sealed flask, for 16 h to afford an orange solution. The solvent was removed under reduced pressure to yield a glassy solid. The crude material was repeatedly (x 3) dissolved in CH₂CI₂ (5 ml) and the solvent removed under reduced pressure to facilitate elimination of excess acid and te/f-butyl alcohol. The desired ligand, as its TFA salt, was examined by ¹H NMR to ensure complete ester hydrolysis, with the material used immediately for complexation.

The hydrolysed ligand was dissolved in CH₃OH - H₂O (1:1 v/v, 4 ml) and

Eu(OAc)₃.6H₂O (0.017 g, 0.039 mmol) added to the mixture. The pH of the solution was raised to 5.5 by the addition of 1 M KOH _(aq), then stirred and heated at 80 °C, for

15 h. The reaction mixture was allowed to cool to rt before raising the pH of the solution to 10.0 using dilute KOH _(aq). The reaction mixture was stirred for 1 h to allow precipitation of excess Eu metal as its hydroxide salt, Eu(OH)₃. The solid precipitate was removed by syringe filtration and the pH of the colourless aqueous filtrate lowered to pH 5.5 using a solution of 1 M HCl _(aq). The solvent was removed under reduced pressure using a freeze-drier to yield a colourless solid. The material was purified by column chromatography on neutral alumina (elution; CH₂CI₂ : CH₃OH, 8 : 2 v/v) to yield the title complex [EuL¹] as a colourless solid (0.024 g, 0.028 mmol, 68 %); /?_F = 0.41 (Alumina, CH₂CI₂ - CH₃OH, 7:3 v/v); λ_max (H₂O) = 356 nm; T (H₂O) = 1.00 ms; T (D₂O) = 1.34 ms; φ_Eu (H₂O; pH 7.4; A₆^ 365 nm) = 14 %.

Example 2: Synthesis of L4 and TEuL41 (see schemes 1 to 3 and 5)

1,4,7-Tetraaza-cyclododecane-1,4,7-tricarboxylic acid tri-tert-butyl ester (17)

This cyclen derivative was prepared by using the procedure described by S. Brandes et al., Bull.. Soc. CMm. Fn₁ 1996, 133, 65.

A solution of di-te/t-butyl dicarbonate (6.08 g, 27.8 mmol) in anhydrous CH₂CI₂ (100 ml) was added dropwise to a stirred solution of cyclen (2.00 g, 11.61 mmol) in anhydrous CH₂CI₂ (300 ml). The reaction mixture was stirred at room temperature, for 18 h. The solvent was removed under reduced pressure to afford a transparent oil. The crude material was purified by column chromatography on silica (gradient elution: CH₂CI₂ to 5 % CH₃OH : CH₂CI₂, utilising 0.1 % CH₃OH increments) to yield the title compound 17 as a colourless crystalline solid (3.08 g, 6.51 mmol, 56 %); R_F = 0.29 (Silica, CH₂CI₂ - CH₃OH, 9 : 1, v/v); ¹H NMR (CDCI₃, 500 MHz) δ 1.42 (18H, s, feoc CH₃), 1.44 (9H, s, *Boc CH₃), 2.81 (4H, br s, cyclen CH₂), 3.28 (8H, br s, cyclen CH₂), 3.60 (4H, br s, cyclen CH₂); ¹³C NMR (CDCI₃, 125 MHz, ¹H decoupled 500 MHz) δ 28.9 (6C, 'Boc CH₃), 29.0 (3C, 'Boc CH₃), 46.1 (2C, cyclen CH₂), 49.9 (2C, cyclen CH₂), 51.2 (4C, cyclen CH₂), 79.4 (2C, fex*,)), 79.6 (1C, ^oq_q)), 155.8 (2C, feoc C = O), 156.0 (1C, 'Boc C = O); MS (ES⁺) m/z 473.3 (100 %, [M + H]⁺); HRMS (ES⁺) m/z found 473.3330 [M + H]⁺ C₂₃H₄₅O₆N₄ requires 473.3333.

10-[6-(6-te^Butyl-10-oxo-10//-9-oxa-1-aza-anthracen-2-yl)-pyridin-2- ylmethyl]-lA7_/10-tetraaza-cydododecane-1,4,7-tricarboxylic acid tri-tett- butyl ester (18)

A stirred mixture of 1,4,7-tetraaza-cyclododecane-1,4,7-tricarboxylic acid tri-te/f-butyl ester 17 (0.117 g, 0.248 mmol), 2-(6-bromomethyl-pyridin-2-yl)-6-ή_γf-butyl-9-oxa-1- aza-anthracen-10-one 9 (0.100 g, 0.236 mmol) and K₂CO₃ (0.049 g, 0.354 mmol) in anhydrous CH₃CN (4 ml), was heated at reflux, under argon, for 16 h. The reaction mixture was allowed to cool to room temperature, syringe filtered and the filtrate concentrated under reduced pressure to afford a residual yellow oil. The crude material was purified by column chromatography on silica (gradient elution: CH₂CI₂ to 3 %

CH₃OH : CH₂CI₂, utilising 0.1 % CH₃OH increments) to yield the title compound IS as a pale yellow coloured solid (0.173 g, 0.212 mmol, 90 %); R_f = 0.70 (Silica, CH₂CI₂ -

CH₃OH, 49 : 1, v/v); ¹H NMR (CDCI₃, 700 MHz) δ 1.29 (9H, s, *Bu CH₃), 1.32 (9H, s,

'Boc CH₃), 1.35 (18H, br s, 'Boc CH₃), 2.71 (4H, br s, cyclen CH₂), 3.24 (8H, br s, cyclen CH₂), 3.50 (4H, br s, cyclen CH₂), 3.87 (2H, s, CH₂ pyridine), 7.25 (1H, d, J =

8.0 Hz, H^5'), 7.45 (1H, d, J = 8.5 Hz, H¹⁰), 7.69 (1H, t, J = 8.0 Hz, H^4'), 7.71 (1H, dd, J = 9.0; 3.0 Hz, H⁹), 8.17 (1H, d, J = 3.0 Hz, H⁷), 8.32 (1H, d, J = 8.0 Hz, H^3'), 8.36 (1H, ύ, J = 7.0 Hz, H²), 8.64 (1H, d, J = 8.0 Hz, H³); ¹³C NMR (CDCI₃, 176 MHz, ¹H decoupled 700 MHz) δ 28.6 (9C, 'Boc CH₃), 31.5 (3C, C¹⁴), 35.0 (1C, C¹³), 47.3 (1C, cyclen CH₂), 47.7 (1C, cyclen CH₂), 48.0 (1C, cyclen CH₂), 48.4 (1C, cyclen CH₂), 50.1 (1C, cyclen CH₂), 51.1 (1C, cyclen CH₂), 54.4 (1C, cyclen CH₂), 55.2 (1C, cyclen CH₂), 57.1 (1C, CH₂ pyridine), 79.5 (4C, teoc_(q); C¹⁴), 116.4 (1C, C⁴), 118.2 (1C, C¹⁰), 118.3 (1C, C²), 121.0 (1C, C^3'), 121.2 (1C, C⁶), 122.6 (1C, C⁷), 126.2 (1C, C^5'), 133.6 (1C, C⁹), 137.4 (1C, C^4'), 138.2 (1C, C³), 148.0 (1C, C⁸), 153.6 (1C, C^1'), 154.2 (1C, C¹¹), 155.9 (3C, Boc C = O), 158.6 (1C, C^6'), 160.2 (1C, C¹), 160.6 (1C, C¹²), 177.8 (1C, C⁵); MS (ES⁺) m/z 815.4 (100 %, [M + H]⁺); HRMS (ES⁺) m/z found 815.4710 [M + H]⁺ C₄₅H₆₃O₈N₆ requires 815.4710.

6-te/^Butyl-2-[6-(1,4,7,10-tetraaza-cγclododec-1-ylmethyl)-pyridin-2-yl]- 9-oxa-1-aza-anthracen-10-one (19)

A solution of 10-[6-(6-fø/?-butyl-10-oxo-10/f9-oxa-1-aza-anthracen-2-yl)-pyridin-2- ylmethyl]-1,4,7,10-tetraaza-cyclododecane-1,4,7-tricarboxylic acid tri-tert-butyl ester 18 (0.173 g, 0.212 mmol) in CH₂CI₂ - TFA (2 : 1 v/v, 3 ml) was stirred at room temperature, in a sealed flask, for 6 h, to afford an orange solution. The solvent was removed under reduced pressure to yield a glassy orange solid. The crude material was repeatedly (x 3) dissolved in CH₂CI₂ (5 ml) and the solvent removed under reduced pressure to facilitate elimination of excess acid and tert-butyl alcohol. The residue was finally taken into KOH_(aq) (1 M, 10 ml) and extracted with CH₂CI₂ (3 x 5 ml). The organic extracts were combined, dried over K₂CO₃, filtered and the filtrate concentrated under reduced pressure to yield the title compound 19 as an orange coloured crystalline solid (0.065 g, 0.129 mmol, 94 %); ¹H NMR (CDCI₃, 500 MHz) δ 1.36 (9H, s, tøu CH₃), 2.54 (4H, s, cyclen CH₂), 2.68 (8H, s, cyclen CH₂), 2.77 (4H, s, cyclen CH₂), 3.85 (2H, s, CH₂ pyridine), 7.44 (1H, d, J = 7.5 Hz, H^5'), 7.54 (1H, d, J = 9.0 Hz, H¹⁰), 7.79 (1H, dd, J = 8.5; 2.5 Hz, H^4'), 7.81 (1H, t, J = 7.5 Hz, H⁹), 8.26 (1H, d, J = 2.5 Hz, H⁷), 8.38 (1H, d, J = 7.5 Hz, H^3'), 8.62 (1H, d, J = 8.0 Hz, H²), 8.73 (1H, d, J= 8.0 Hz, H³); ¹³C NMR (CDCI₃, 126 MHz, ¹H decoupled 500 MHz) δ 31.5 (3C, C^H), 35.0 (1C, C¹³), 45.3 (2C, cyclen CH₂), 46.6 (2C, cyclen CH₂), 47.4 (2C, cyclen CH₂), 51.8 (2C, cyclen CH₂), 60.8 (1C, CH₂ pyridine), 116.4 (1C, C)₁ 118.2 (1C, C¹⁰), 118.6 (1C, C²), 121.0 (1C, C^5'), 121.3 (1C, C⁶), 122.6 (1C, C⁷), 124.6 (1C, C^2'), 133.7 (1C, C⁹), 137.8 (1C, C)₁ 138.1 (1C, C³), 148.1 (1C, C⁸), 153.5 (1C, C^1'), 154.2 (1C, C¹¹), 159.7 (1C, C^6'), 160.2 (1C, C¹), 160.6 (1C, C¹²), 178.0 (1C, C⁵); MS (ES⁺) m/z 289.4 (100 %, [M + Zn]²⁺); HRMS (ES⁺) /77/zfound 515.3130 [M + H]⁺; C₃₀H₃₉O₂N₆ requires 515.3129.

(S)-N-2-Chloroethanoyl-2-phenylethylamine

This compound was prepared according to the procedure described by R. S. Dickins et al., Chem. Eur. J₁ 1999, 5, 1095.

Chloroacetyl chloride (10.5 g, 7.40 ml, 93.0 mmol) was added dropwise to a stirring solution of (5)-1-phenylethylamine (9.40 g, 10.0 ml, 77.50 mmol) and anhydrous NEt₃ (13.0 ml, 93.00 mmol) in anhydrous diethyl ether (70 ml) at - 10 °C, under argon The solution was allowed to warm to room temperature then stirred for a further 3 h. The reaction mixture was washed with H₂O (150 ml) followed by HCl_(aq) (0.1 M, 150 ml). The organic phase was separated, dried over Na₂SO₄, filtered and the filtrate concentrated under reduced pressure to afford a colourless solid. Recrystallisation from warm diethyl ether yielded the title compound as colourless needle-like crystals (10.46 g, 53.09 mmmol, 69 %); m.p. 95-96 °C; ¹H NMR (CDCI₃, 500 MHz) δ 1.56 (3H, d, J = 6.5 Hz, CH₃), 4.05 (2H, dd, J = 15.5; 8.0 Hz, CH₂), 5.15 (1H, q, J = 7.5 Hz, CH), 6.83 (1H, br s, NH), 7.34-7.39 (5H, m, Ph); ¹³C NMR (CDCI₃, 125 MHz, ¹H decoupled 500 MHz) δ 21.9 (1C, CH₃), 42.9 (1C, CH₂), 49.5 (1C, CH), 126.4 (2C, Ph₍₀₎), 127.9 (1C, Ph_(p)), 129.1 (2C, Ph_(m)), 142.6 (1C, Ph_(q)), 165.2 (1C, C = O); C₁₀H₁₂CINO (%): calcd C 60.80, H 6.12, N 7.08; found C 60.06, H 6.15, N 6.94. (/7)-N-2-Chloroethanoyl-2-phenylethylamine

An analogous procedure to that described for 2-chloro-N-[(5)- methylbenzyl]ethanamide was followed using chloroacetyl chloride (5.25 g, 3.70 ml, 46.5 mmol) and a stirring solution of (/^-1-phenylethylamine (4.70 g, 5.00 ml, 38.8 mmol) and anhydrous NEt₃ (6.51 ml, 46.52 mmol) in anhydrous diethyl ether (100 ml) at - 10 °C. The procedure yielded the title compound as colourless needle-like crystals (3.71 g, 18.83 mmol, 49 %). Characterisation was identical to that reported for 2- chloro-N-[(S)-methylbenzyl]ethanamide.

2-{4-[6-(6-fe/^Butyl-10-oxo-10/^-9-oxa-1-aza-anthracen-2-yl)-pyridin-2- ylmethyl]-7,10-bis-[((S)-1-phenyl-ethylcarbamoyl)-methyl]-1,4,7,10- tetraaza-cyclododec-1-yl}-N-((S)-1-phenyl-ethyl)-acetamide ([L⁴])

A stirred mixture of 6-ήe/f-butyl-2-[6-(1,4,7,10-tetraaza-cyclododec-1-ylmethyl)-pyridin- 2-yl]-9-oxa-1-aza-anthracen-10-one 19 (0.060 g, 0.116 mmol), (S)- N-2- chloroethanoyl-2-phenylethylamine (0.080 g, 0.408 mmol) and K₂CO₃ (0.064 g, 0.466 mmol) in anhydrous CH₃CN (5 ml), was heated at reflux, under argon, for 16 h. The resultant mixture was allowed to cool to room temperature, syringe filtered and the filtrate concentrated under reduced pressure to afford a residual yellow oil. The crude material was purified by column chromatography on neutral alumina (gradient elution: CH₂CI₂ to 2 % CH₃OH : CH₂CI₂, utilising 0.1 % CH₃OH increments) to yield the title compound [L⁴] as a free flowing pale yellow coloured solid (0.074 g, 0.0741 mmol, 64 %); R_F = 0.21 (Alumina, CH₂CI₂ - CH₃OH, 19 : 1, v/v); ¹H NMR (CDCI₃, 700 MHz) δ 1.38 (9H, s, feu CH₃), 1.45 (9H, br s, CH₃), 2.60 (8H, br s, cyclen CH₂), 2.92 (8H, br s, cyclen CH₂), 3.06 (2H, br s, CH₂CO), 3.54 (2H, br s, CH₂CO), 3.67 (2H, br s, CH₂CO), 3.96 (2H, br s, CH₂ pyridine), 4.96 (1H, q, CH), 5.11 (2H, q, CH), 7.11-7.30 (15H, m, Ph), 7.45 (1H, br s, H^5'), 7.58 (1H, d, J = 9.0 Hz, H¹⁰), 7.70 (1H, br s, H^4'), 7.82 (1H, dd, J = 9.0; 2.0 Hz, H⁹), 8.28 (1H, d, J = 3.0 Hz, H⁷), 8.43 (1H, br s, H^3'), 8.62 (1H, d, J = 8.5 Hz, H²), 8.77 (1H, d, J = 8.5 Hz, H³); MS (ES⁺) m/z 998.6 (100 %, [M + H]⁺); HRMS (ES⁺) /77/zfound 998.5667 [M + H]⁺; C₆₀H₇₂O₅N₉ requires 998.5651.

[EuL⁴]

3 +

A solution of 2-{4-[6-(6-te/f-butyl-10-oxo-10/f9-oxa-1-aza-anthracen-2-yl)-pyridin-2- ylmethyl]-7,10-bis-[((S)-1-phenyl-ethylcarbamoyl)-methyl]-1,4,7,10-tetraaza- cyclododec -1-yl}-N-((S)-1-phenyl-ethyl)-acetamide [L⁴] (0.040 g, 0.040 mmol) and Eu(OTf)₃.6H₂O (0.034 g, 0.041 mmol) in anhydrous CH₃CN (1 ml) was heated at reflux, under argon, for 18 h. The resultant solution was allowed to cool to room temperature, followed by the removal of solvent under reduced pressure to afford a glassy orange solid. CH₂CI₂ (10 ml) was added to the solid, and the mixture sonicated for 10 min. The solvent was then decanted and the solid material dissolved in a minimum volume of CH₃CN (0.5 ml). The solution was added dropwise onto diethyl ether (25 ml) to induce precipitation. The solid material was isolated by centrifugation, and the process of induced precipitation repeated twice more to yield the complex as its triflate salt. The off white solid was made water soluble by the exchange of triflate anions for chloride anions using OOWEX 1 x 8 200-400 mesh Cl' resin. The solid material was dissolved in a mixture of H₂O - CH₃OH (1:1 v/v, 16 ml) and 0.8 g of prepared resin added to the solution, which was stirred at room temperature, for 3 h. The resin was removed by filtration, and the filtrate concentrated under reduced pressure to yield the title complex [EuL⁴] (0.034 g, 0.027 mmol, 68 %); λ_max (H₂O) = 356 nm; T (H₂O) = 1.00 ms; T (D₂O) = 1.34 ms; φ (H₂O; pH 7.4; λeχ_C 365 nm) = 24 %; HPLC (Method A) & = 10.9 min.

Example 3: Synthesis of cvclen derivatives (31) to (37) (see scheme 6)

The following intermediate compounds (24), (25), (26), (27), (29) and (30) were prepared according to the procedures disclosed by B.B. Shankar et al. in Bioorg. Med. Chem. Lett., 2005, 15, 4417. Compound (28) was prepared by the procedure disclosed by L-O. Pllsson et al. in Da/ton Trans., 2007, 5726.

(5)-N-Ethanoyl-1-(4-bromophenyl)ethylamine (24)

Acetyl chloride (2.40 ml, 30.0 mmol) was added dropwise to a stirring solution of (S)- l-(4-bromophenyl)ethylamine (5.00 g, 24.99 mmol) and anhydrous NEt₃ (4.40 ml, 31.0 mmol) in anhydrous diethyl ether (300 ml) at - 10 °C. The solution was allowed to warm to rt then stir for a further 3 h. The reaction mixture was washed with H₂O (150 ml) and then 0.1 M HCl_(aq₎ (150 ml). The organic phase was separated, dried over Na₂SO₄, filtered and the filtrate concentrated under reduced pressure to afford a colourless solid. Recrystallisation from warm diethyl ether yielded the t/t/e compound 24 as colourless needle-like crystals (4.56 g, 18.8 mmol, 76 %). m.p. 127-129 °C; ¹H NMR (CDCI₃, 500 MHz) δ 1.47 (3H, d, J = 7.0 Hz, CH₃), 2.00 (3H, s, C(O)CH₃), 5.08 (1H, q, J = 7.0 Hz, CH), 5.71 (1H, br s, NH), 7.20 (2H, d, J = 8.5 Hz, H²), 7.46 (2H, d, J = 8.5 Hz, H³); ¹³C NMR (CDCI₃, 125 MHz, 1H decoupled 500 MHz) δ 21.9 (1C, CH₃), 23.7 (1C, C(O)CH₃), 48.5 (1C, CH), 121.4 (1C, C¹), 128.2 (2C, C³), 132.0 (2C, C²), 142.5 (1C, C⁴), 169.3 (1C, C = O); MS (ES⁺) m/z 263.9 (100 %, [M + Na]⁺); HRMS (ES⁺) m/z found 263.9994 [M + Na]⁺ C₁₀Hi₂ON⁷⁹Br²³Na requires 263.9995; C₁₀H₁₂BrNO (%): calcd C 49.61, H 5.00, N 5.79; found C 49.48, H 4.98, N 5.52. (S)-N-Ethanoyl-1-(4-cyanophenyl)ethylamine (25)

A stirred solution of (5)-N-ethanoyl-1-(4-bromophenyl)ethylamine 24 (4.00 g, 16.5 mmol) and CuCN (1.55 g, 17.3 mmol) in anhydrous, degassed DMF (40 ml) was heated at 180 °C, for 78 h. The resultant dark green solution was allowed to cool to rt followed by the removal of solvent under reduced pressure. The residue was taken up into 6 M HCl₍a_q) (50 ml) in a well ventilated fumehood and the resulting aqueous solution extracted with CH₂CI₂ (3 x 50 ml). The organic extracts were combined, washed with H₂O (100 ml), separated and the solvent removed under reduced pressure to yield the title compound 25 as a yellow crystalline solid (2.20 g, 11.7 mmol, 71 %); m.p. 188-190 °C; ¹H NMR (CDCl₃, 500 MHz) δ 1.47 (3H, U₁ J = 7.0 Hz, CH₃), 2.00 (3H, s, C(O)CH₃), 5.12 (1H, q, J = 7.0 Hz, CH), 6.02 (1H, d, J = 6.5 Hz, NH), 7.43 (2H, d, J = 8.0 Hz, H²), 7.63 (2H, ά, J = 8.0 Hz, H³); ¹³C NMR (CDCI₃, 125 MHz, ¹H decoupled 500 MHz) δ 22.0 (1C, CH₃), 23.5 (1C, C(O)CH₃), 48.9 (1C, CH), 111.2 (1C, CN), 119.0 (1C, C⁴), 127.1 (2C, C³), 132.7 (2C, C²), 149.2 (1C, C¹), 169.7 (1C, C = O); MS (ES⁺) m/z 189.1 (100 %, [M + H]⁺); HRMS (ES⁺) /77/zfound 189.1023 [M + H]⁺ CnH₁₃ON₂ requires 189.1022; C₁₁Hj₂N₂O (%): calcd C 70.19, H 6.43, N 14.88; found C 70.05, H 6.49, N 15.00.

N-[(5)-1-(4-Bromo-phenyl)-ethyl]-2,2,2-trifluoro-acetamide (27)

A solution of (S)-(-)-α-methylbenzylamine (7.81 g, 39.1 mmol) in anhydrous CH₂CI₂ (20 ml) was added dropwise to a stirring solution of trifluoroacetic anhydride (14.0 g. 9.27 ml, 66.7 mmol) in anhydrous CH₂CI₂ (35 ml), under argon, at 0 °C. The solution was allowed to warm to rt then stir for a further 3 h. The solution was cooled to -10 °C for the addition of 70 % methanesulfonic acid (16.3 g, 11.0 ml, 169 mmol) followed by 1,3-dibromo-5,5-dimethylhydantoin (9.00 g, 31.6 mmol). The suspension was allowed to warm to rt then stir for 16 h. 1 M NaHSO_3(aq) (100 ml) was introduced into the reaction mixture and the organic phase washed with H₂O (200 ml). The organic phase was separated, dried over MgSO₄, filtered and the filtrate concentrated under reduced pressure to afford a crude colourless solid. The crude material was recrystallised from diethyl ether to yield the title compound 27 as colourless needle-like crystals (5.80 g, 19.7 mmol, 51 %); m.p. 154-156 °C; ¹H NMR (CDCI₃, 500 MHz) δ 1.57 (3H, d, J = 7.0 Hz, CH₃), 5.10 (1H, q, J = 7.0 Hz, CH), 6.57 (1H, br s, NH), 7.20 (2H, d, J = 8.0 Hz, H³), 7.51 (2H, d, J= 8.0 Hz, H²); ¹³C NMR (CDCI₃, 125 MHz, ¹H decoupled 500 MHz) δ 21.2 (1C, CH₃), 49.5 (1C, CH), 122.3 (1C, C¹), 128.1 (2C, C³), 132.4 (2C, C²), 140.2 (1C, C⁴), 156.7 (1C, C = O); ¹⁹F NMR (CDCI₃, 470 MHz, ¹H decoupled 500 MHz) δ -76.2 (3F, s, CF₃); MS (ES⁺) m/z 318.1 (100 %, [M + Na]⁺); HRMS (ES⁺) m/z found 317.9713 [M + Na]⁺ C₁₀H₉ON⁷⁹BrF₃ ²³Na requires 317.9712; C₁₀H₉BrF₃NO (%): calcd C 40.57, H 3.06, N 4.73; found C 40.47, H 3.03, N 4.67.

N-[(5)-1-(4-Cyano-phenyl)-ethyl]-2,2,2-trifIuoro-acetamide (28)

A stirring solution of /\A[(S)-1-(4-bromo-phenyl)-ethyl]-2,2,2-trifluoro-acetamide 27 (5.80 g, 19.7 mmol) and CuCN (2.12 g, 23.7 mmol) in anhydrous, degassed DMF (30 ml) was heated at 180 °C, under argon, for 48 h. The resultant dark green solution was allowed to cool to rt followed by the removal of the solvent under reduced pressure. The residue was taken up into 6 M HCl(_aq) (50 ml) in a well ventilated fumehood and the resulting aqueous solution extracted with CH₂CI₂ (3 x 50 ml). The combined organic extracts were combined and concentrated under reduced pressure to afford a brown solid. The crude material was purified by column chromatography on silica (using 100 % CH₂CI₂ elution) to yield the title compound 1Α as a colourless solid (2.86 g, 11.8 mmol, 60 %); R_? = 0.34 (Silica, CH₂CI₂, 100 v); m.p. 98-99 °C; ¹H NMR (CDCI₃, 500 MHz) δ 1.53 (3H, d, J = 7.5 Hz, CH₃), 5.10 (1H, q, J = 7.0 Hz, CH), 7.36 (1H, ύ, J= 7.5 Hz, NH), 7.41 (2H, d, J= 8.0 Hz, H²), 7.60 (2H, d, J = 8.0 Hz, H³); ¹³C NMR (CDCI₃, 125 MHz, ¹H decoupled 500 MHz) δ 21.3 (1C, CH₃), 49.9 (1C, CH), 111.7 (1C, C¹), 118.7 (1C, CN), 127.1 (2C, C³), 132.9 (2C, C²), 147.1 (1C, C⁴), 156.9 (1C, C = O); ¹⁹F NMR (CDCI₃, 470 MHz, ¹H decoupled 500 MHz) δ -75.7 (3F, s, CF₃); MS (ES-) m/z 241.2 (100 %, [M - H] ); HRMS (ES ) m/z found 241.0591 [M - H]- C₁₁H₈ON₂F₃ requires 241.0594; CnH₉F₃N₂O (%): calcd C 54.55, H 3.75, N 11.57; found C 54.48, H 3.85, N 11.65.

(5)-4-(l-Aminoethyl)benzoic acid (26)

Procedure A:

A solution of (S)-N-ethanoyl-1-(4-cyanophenyl)ethylamine 25 (2.00 g, 10.6 mmol) in 6 M HCl_(aq₎ (35 ml) was heated at reflux, for 72 h. The solution was allowed to cool to it followed by the removal of solvent under reduced pressure to yield the hydrochloride salt of the title compound 26, as a colourless crystalline solid, in quantitative yield. ¹H NMR (D₂O, 500 MHz) δ 1.52 (3H, d, J = 7.0 Hz, CH₃), 4.48 (1H, q, J = 7.0 Hz, CH), 7.37 (2H, dd, J = 6.5; 1.5 Hz, H³), 7.86 (2H, dd, J = 7.0; 2.0 Hz, H²); ¹³C NMR (D₂O, 125 MHz, ¹H decoupled 500 MHz) δ 19.4 (1C, CH₃), 50.8 (1C, CH), 126.9 (2C, C³), 130.6 (2C, C²), 132.3 (1C, C¹), 143.1 (1C, C)₁ 170.2 (1C, C = O); MS (ES-) /77/2- 164.4 (100 %, [M - H] ); HRMS (ES ) m/z found 164.0715 [M - H]- C₉H₁₀O₂N requires 164.0717.

Procedure B: An analogous procedure to that described in procedure A was followed using

N-[(S)-1-(4-cyano-phenyl)-ethyl]-2,2,2-trifluoro-acetamide 28 (2.67 g, 11.0 mmol) in 6 M HCl_(aq) (50 ml) for 72 h. The procedure yielded the hydrochloride salt of (S)-A-(I- aminoethyl)benzoic acid 26, as a colourless crystalline solid, in quantitative yield. Character/sat/on was identical to that reported in procedure A.

(5)-Methyl-4-(l-aminoethyl)benzoate (29)

Concentrated HCl_(aq) (12 M, 2.00 ml) was added to a stirring solution of (5)-4-(l- aminoethyl)benzoic acid 26 (2.60 g, 11.9 mmol) in anhydrous CH₃OH (30 ml) and the solution heated at reflux, under argon, for 48 h. The solution was allowed to cool to rt followed by the removal of the solvent under reduced pressure to yield the hydrochloride salt of the title compound 29, as a bright yellow crystalline solid, in quantitative yield. ¹H NMR (CH₃OD, 500 MHz) δ 1.66 (3H, d, J = 7.0 Hz, CH₃), 3.93 (3H, s, CO₂CH₃), 4.58 (1H, q, J = 7.0 Hz, CH), 7.61 (2H, d, J = 8.5 Hz, H³), 8.10 (2H, 6, J = 8.5 Hz, H²); ¹³C NMR (CH₃OD, 125 MHz, ¹H decoupled 500 MHz) δ 19.5 (1C, CH₃), 50.8 (1C, CH), 51.7 (1C, CO₂CH₃), 126.9 (2C, C³), 130.2 (2C, C²), 133.0 (1C, C¹), 143.5 (1C, C⁴), 166.7 (1C, C = O); MS (ES⁺) m/z 180.0 (100 %, [M + H]⁺); HRMS (ES⁺) /77/zfound 180.1019 [M + H]⁺ Ci₀H₁₄NO₂ requires 180.1019.

4-[(5)-1-(2-Chloro-acetylamino)-ethyl]-benzoic acid methyl ester (30)

Chloroacetyl chloride (0.540 g, 0.380 ml, 4.78 mmol) was added dropwise to a stirring solution of (5)-methyl-4-(l-aminoethyl)benzoate 29 (0.791 g, 3.68 mmol) and anhydrous NEt₃ (1.43 ml, 10.1 mmol) in anhydrous diethyl ether (100 ml) at - 10 °C. The solution was allowed to warm to rt then stir for a further 4 h. The reaction mixture was washed with H₂O (150 ml) and then 0.1 M HCl _(aq) (150 ml). The organic phase was separated, dried over K₂CO₃, filtered and the filtrate concentrated under reduced pressure to afford a crude solid. Recrystallisation from warm diethyl ether yielded the title compouπd30 as colourless solid (0.704 g, 2.76 mmol, 75 %); ¹H NMR (CDCI₃, 500 MHz) δ 1.55 (3H, d, J = 7.5 Hz, CH₃), 3.92 (3H, s, CO₂CH₃), 4.08 (2H, dd, J = 15; 6.5 Hz, CH₂), 5.18 (1H, q, J = 7.0 Hz, CH), 6.85 (1H, d, J= 6.0 Hz, NH), 7.39 (2H, d, J = 8.5 Hz, H³), 8.03 (2H, d, J = 8.0 Hz, H²); ¹³C NMR (CDCI₃, 125 MHz, ¹H decoupled 500 MHz) δ 22.0 (1C, CH₃), 42.8 (1C, CH₂), 49.3 (1C, CH), 52.4 (1C, CO₂CH₃), 126.3 (2C, C³), 129.7 (1C, C⁴)_/ 130.4 (2C, C²), 147.7 (1C, C¹), 165.4 (1C, C(O)CH₂), 167.0 (1C, C(O)CH₃); MS (ES⁺) m/z 256.0 (100 %, [M + H]⁺); HRMS (ES⁺) /77/zfound 256.0735 [M + H]⁺ Ci₂Hi₅O₃Ni³⁵CI₁ requires 256.0735. 1,4,7,10-Tetraaza-cyclododecane-l,7-dicarboxylic acid dibenzyl ester (31)

This cyclen derivative was prepared according to the procedure described by Z. Kovacs et al., J. Chem. Soc, Chem. Commun., 1995, 2, 185.

Disodium hydrogen phosphate (14.0 g, 98.6 mmol) was added to a solution of cyclen (5.00 g, 29.0 mmol) in H₂O - 1,4-dioxane (50 : 20 v/v, 70 ml) and the pH adjusted to pH 2.5 by the addition of cone. HCl_(aq) (12 M). Benzyl chloroformate (10.0 ml, 70.1 mmol) in dioxane (20 ml) was added dropwise to the stirred solution at room temperature, over 2 h, followed by stirring for a further 18 h to afford a colourless solution containing a white precipitate. The solvent was removed under reduced pressure and the residue dissolved in H₂O (100 ml). The pH of the aqueous phase was then raised to pH 7 by the addition of 1 M KOH_(aq). The aqueous phase was then extracted with diethyl ether (2 x 100 ml), followed by CH₂CI₂ (2 x 100 ml). The CH₂CI₂ extracts were combined, dried over MgSO₄, filtered and the filtrate concentrated under reduced pressure to afford a colourless oil. The material was repeatedly washed with diethyl ether and concentrated under reduced pressure (3 x 50 ml) to yield the title compound 31 as a colourless crystalline solid (9.47 g, 21.5 mmol, 74 %); m.p. 113- 116 °C; ¹H NMR (CDCI₃, 500 MHz) δ 2.05 (2H, br s, NH), 2.86-3.12 (8H, br m, cyclen CH₂), 3.47-3.79 (8H, br m, cyclen CH₂), 5.18 (4H, s, Cbz CH₂), 7.33-7.40 (1OH, m, Ph); ¹³C NMR (CDCI₃, 125 MHz, ¹H decoupled 500 MHz) δ 49.2 (1C, cyclen CH₂), 49.4 (1C, cyclen CH₂), 49.9 (1C, cyclen CH₂), 50.1 (1C, cyclen CH₂), 50.5 (1C, cyclen CH₂), 50.7 (1C, cyclen CH₂), 50.9 (1C, cyclen CH₂), 68.1 (2C, Cbz CH₂), 68.2 (1C, cyclen CH₂), 128.3 (2C, Ph), 128.4 (2C, Ph), 128.7 (2C, Ph), 128.8 (2C, Ph), 129.0 (2C, Ph), 129.1 (2C, Ph), 136.1 (2C, Ph_(q)), 136.2, 156.4 (1C, C = O), 156.5 (1C, C = O); MS (ES⁺) m/z 441.4 (100 %, [M + H]⁺). 4_/10-bis-[((5)-1-Phenyl-ethylcarbamoyl)-methyl]-1,4,7_/10-tetraaza- cydododecane-l,7-dicarboxylic acid di benzyl ester (32)

A stirred mixture of 1,4,7,10-tetraaza-cγdododecane-l,7-dicarboxylic acid dibenzyl ester 31 (1.34 g, 3.05 mmol), (S)-N-2-chloroethanoyl-2-phenylethylamine (1.31 g, 6.65 mmol), Cs₂CO₃ (1.97 g, 6.06 mmol) and KI (0.010 g) in anhydrous CH₃CN (50 ml) was heated at reflux, under argon, for 24 h. The resulting orange solution was allowed to cool to room temperature, followed by the removal of the solvent under reduced pressure. The residual oil was dissolved in CH₂CI₂ (20 ml) and washed with H₂O (2 x 20 ml). The organic layer was concentrated under reduced pressure to afford a residual oil which was then sonicated in diethyl ether (2 x 50 ml). The solid precipitate was collected by centrifugation, dissolved in CH₂CI₂ and then dried under reduced pressure to yield the title compound '32 as a colourless crystalline solid (1.72 g, 2.26 mmol, 74 %); m.p. 64-66 °C; ¹H NMR (CDCI₃, 500 MHz) δ 1.45 (6H, br s, CH₃), 2.75 (8H, br s, cyclen CH₂), 3.16 (4H, br s, CH₂CO), 3.43 (8H, br s, cyclen CH₂), 4.96 (4H, br s, Cbz CH₂), 5.11 (2H, m, CH), 7.22-7.39 (2OH, m, Ph), 7.59 (2H, br s, NH); ¹³C NMR (CDCI₃, 125 MHz, ¹H decoupled 500 MHz) δ 22.0 (2C, CH₃), 48.1 (1C, CH), 48.7 (1C, CH), 55.0 - 56.4 (4C, br s, cyclen CH₂), 59.3 (2C, CH₂CO), 67.6 (2C, Cbz CH₂), 126.3 (2C, Ph), 126.6 (2C, Ph), 127.4 (2C, Ph), 128.5 (2C, Ph), 128.6 (2C, Ph), 136.5 (2C, Cbz Ph_(q)), 143.8 (2C, amide arm Ph_(q)), 157.1 (2C, C(O)OBn), 170.2 (2C, NHCO); MS (ES⁺) m/z 763.0 (100 %, [M + H]⁺); HRMS (ES⁺) m/z found 763.4187 [M + H]⁺ C₄₄H₅₅N₆O₆ requires 763.4178. N-((5)-1-Phenyl-ethyl)-2-(7-[((5)-1-phenyl-ethylcarbamoyl)-methyl]- 1,4,7,10-tetraaza-cyclododec-1-yl)-acetamide (33)

4, 10-bis-[((5)-1-Phenyl-ethylcarbamoyl)-methyl]-1,4,7, lO-tetraaza-cydododecane-1,7- dicarboxylic acid dibenzyl ester 32 (3.14 g, 4.12 mmol) in CH₃OH - H₂O (40 : 20 v/v, 60 ml) was shaken in a Parr hydrogenation flask at 40 psi H₂ over Pd(OH)₂ZC (0.35 g) for 48 h. The resulting mixture was filtered through celite to afford a colourless solution which was concentrated under reduced pressure to yield the title compound 33 as a colourless crystalline solid (1.77 g, 3.58 mmol, 87 %); m.p. 140-143 °C; ¹H NMR (CD₃OD, 500 MHz) δ 1.46 (6H, U₁ J = 7.0 Hz, CH₃), 2.88-3.18 (16H, br m, cyclen CH₂), 3.41 (2H, d, J = 16.5 Hz, CHCO), 3.54 (2H, d, J = 17.0 Hz, CHCO), 5.04 (2H, m, CH), 7.21-7.28 (1OH, m, Ph); ¹³C NMR (CDCI₃, 125 MHz, ¹H decoupled 500 MHz) δ 23.5 (2C, CH₃), 45.2 (4C, cyclen CH₂), 50.7 (2C, cyclen CH₂), 51.1 (2C, cyclen CH₂), 52.4 (2C, CH), 57.2 (2C, CH₂CO), 128.0 (4C, Ph_(o/m)), 129.0 (4C, Ph_(o/m)), 130.5 (2C, Ph_(p)), 145.8 (2C, Ph_(q)), 172.7 (2C, C = O); MS (ES⁺) m/z 495.3 (100 %, [M + H]⁺); HRMS (ES⁺) /77/zfound 495.3447 [M + H]⁺ C₂₈H₄₃N₆O₂ requires 495.3442.

4,10-bis-[((S)-1-Phenyl-ethylcarbamoyl)-methyl]-1,4,7,10-tetraaza- cyclododecane-1-carboxylic acid te/f-butyl ester (37)

This cyclen derivative was prepared following the procedure described by L-O. PSIsson et al., Da/ton Trans., 2007, 5726.

A solution of di-te/f-butyl dicarbonate (0.205 g, 0.940 mmol) in anhydrous CH₃OH (25 ml) was added dropwise over 3 h, at room temperature, to a stirring solution of N-((S)- l-phenyl-ethyl)-2-(7-[((5)-1-phenyl-ethylcarbamoyl)-methyl]-1,4,7,10-tetraaza- cyclododec -1-yl)-acetamide 33 (0.664 g, 1.34 mmol) in anhydrous CH₃OH (200 ml). The reaction mixture was left to stir for 16 h, then concentrated under reduced pressure to afford a residual yellow viscous oil. The crude material was purified by column chromatography on neutral alumina (gradient elution; CH₂CI₂ to 2 % CH₃OH : CH₂CI₂, utilising 0.1 % CH₃OH increments) to yield the title compound ' 37 as a glassy colourless solid (0.354 g, 0.596 mmol, 63 %); R_F = 0.46 (Alumina, CH₂CI₂ - CH₃OH, 49:1 v/v); m.p. 98-101 °C; ¹H NMR (CDCI₃, 500 MHz) δ 1.39 (9H, s, feoc CH₃), 1.51 (6H, d, J = 7.0 Hz, CH₃), 2.61 (8H, br s, cyclen CH₂), 2.86 (4H, br s, cyclen CH₂), 2.94 (4H, br s, cyclen CH₂), 3.09 (4H, br s, CH₂CO), 4.95 (2H, q, CH), 7.17-7.23 (1OH, m, Ph), 8.34 (1H, d, J = 5.5 Hz, NH); ¹³C NMR (CDCI₃, 125 MHz, ¹H decoupled 500 MHz) δ 22.0 (2C, CH₃), 28.8 (3C, feoc CH₃), 48.7 (2C, CH), 49.0-53.8 (8C, br m, cyclen CH₂), 59.6 (2C, CH₂CO), 80.3 (1C, CBu), 126.8 (4C, Ph_(o/m)), 127.5 (4C, Ph_(0/m)), 128.8 (2C, Ph(_P)), 143.8 (2C, Ph_(q)), 156.7 (1C, 'Boc C=O), 170.5 (2C, amide C=O); MS (ES⁺) m/z 595.4 (100 %, [M + H]⁺); HRMS (ES⁺) m/z found 595.3972 [M + H]⁺ C₃₃H₅₁O₄N₆ requires 595.3966. 4,10-bis-[(($)-1-Phenyl-ethylcarbamoyl)-methyl]-1,4,7,10-tetraaza- cydododecane-l,7-dicarboxylic acid di-tert-butyl ester (38)

The cyclen derivative (38) was isolated using an identical procedure to that described for cyclen derivative (37). The derivative (33) may be obtained from this derivative (38) by removing the BOC residues.

Example 4: Synthesis of EuLg (see scheme 7)

7-[6-(6-te/*-Butyl-10-oxo-10//-9-oxa-1-aza-anthraceπ-2-yl)-pyridin- 2ylmethyl] -4,10-bis-[((5)-1-phenyl-ethylcarbamoyl)-methyl]-1 ,4,7_/10- tetraaza-cyclo dodecane-1-carboxylic acid te/f-butyl ester (39)

A stirring mixture of 4,10-bis-[((5)-1-phenyl-ethylcarbamoyl)-methyl]-1,4,7,10- tetraaza-cydododecane-1-carboxylic acid tert-buty\ ester 33 (0.325 g, 0.547 mmol), 2- (6-bromomethyl-pyridin-2-yl)-6-te/f-butyl-9-oxa-1-aza-anthracen-10-one 9 (0.254 g, 0.601 mmol) and K₂CO₃ (0.113 g, 0.821 mmol) in anhydrous CH₃CN (10 ml) was heated at reflux, under argon, for 16 h. The mixture was allowed to cool to room temperature, syringe filtered and the filtrate concentrated under reduced pressure to afford a residual yellow oil. The crude material was purified by column chromatography on neutral alumina (gradient elution; CH₂CI₂ to 0.5 % CH₃OH : CH₂CI₂, utilising 0.1 % CH₃OH increments) to yield the title compound 2S as an orange crystalline solid (0.379 g, 0.404 mmol, 74 %); R* = 0.74 (Alumina, CH₂CI₂ - CH₃OH, 49 : 1 v/v); m.p. 87-89 °C; ¹H NMR (CDCI₃, 500 MHz) δ 1.38 (18H, br s, feu CH₃; feoc CH₃), 1.46 (6H, d, J = 4.0 Hz, CH₃), 2.61 (8H, br s, cyclen CH₂), 2.82 (4H, br s, cyclen CH₂), 3.07 (4H, br s, cyclen CH₂), 3.33 (2H, br s, CH₂CO), 3.40 (2H, br s, CH₂CO), 3.61 (2H, br s, CH₂ pyridine), 5.12 (2H, br s, CH), 7.19 (1H, d, J = 8.0 Hz, H^5'), 7.26 (1OH, br s, Ph), 7.46 (1H, br s, NH), 7.58 (1H, d, J = 8.5 Hz, H¹⁰), 7.64 (1H, t, J= 8.0 Hz, H^4'), 7.74 (1H, br s, NH), 7.82 (1H, dd, J = 9.0; 2.5 Hz, H⁹), 8.29 (1H, d, J = 2.5 Hz, H⁷), 8.41 (1H, d, J = 8.0 Hz, H^3'), 8.46 (1H, d, J = 8.0 Hz, H²), 8.78 (1H, d, J = 8.0 Hz, H³); ¹³C NMR (CDCI₃, 125 MHz, ¹H decoupled 500 MHz) δ 21.3 (1C, CH₃), 22.0 (1C, CH₃), 28.7 (3C, tSoc CH₃), 31.5 (3C, C¹⁴), 35.1 (1C, C¹³), 48.0 (1C, CH), 48.4 (1C, CH), 52.5 (2C, cyclen CH₂), 53.2 (2C, cyclen CH₂), 53.8 (2C, cyclen CH₂), 54.6 (2C, cyclen CH₂), 59.4 (1C, CH₂ pyridine), 59.8 (1C, CH₂CO), 60.6 (1C, CH₂CO), 80.2 (1C, W_(q)), 116.6 (1C, C⁴), 118.3 (1C, C¹⁰), 121.1 (1C, C^3'), 121.3 (1C, C⁶), 122.7 (1C, C⁷), 124.8 (1C, C^5'), 126.5 (1C, Pco/_rr,)), 126.8 (1C, P_(0/m)), 127.3 (2C, P_(o/m)), 127.8 (2C, P₍o/_m)), 128.7 (1C, P_(o/m)), 128.9 (1C, P₍o/_m)), 133.8 (1C, C⁹), 137.6 (1C, C^4'), 138.5 (1C, C³), 143.0 (1C, Ph_(q)), 143.8 (1C, Ph_(q)), 148.2 (1C, C⁸), 153.8 (1C, C^1'), 154.2 (1C, C^u), 156.1 (1C, feoc C = O), 158.0 (1C, C^6'), 160.1 (1C, C¹), 160.3 (1C, C¹²), 170.1 (2C, amide C = O), 177.8 (1C, C⁵); MS (ES⁺) /77/z937.5 (100 %, [M + H]⁺); HRMS (ES⁺) /77/zfound 937.5331 [M + H]⁺ C₅₅H₆₉O₆N₈ requires 937.5334.

2-{4-[6-(6-fe/*-Butyl-10-oxo-10//-9-oxa-1-aza-anthracen-2-yl)-pyridin- 2ylmethyl]-7-[((S)-1-phenyl-ethylcarbamoyl)-methyl]-1,4,7_/10-tetraaza- cyclododec-1-yl}-N-((S)-1-phenyl-ethyl)-acetamide (40) (also named L₈)

A solution of 7-[6-(6-te/f-butyl-10-oxo-10/f9-oxa-1-aza-anthracen-2-yl)-pyridin- 2ylmethyl]-4,10-bis-[((5)-1-phenyl-ethylcarbamoyl)-methyl]-1 ,4,7,10-tetraaza-cyclo dodecane-1-carboxylic acid te/t-butyl ester 39 (0.320 g, 0.341 mmol) in CH₂CI₂ - TFA (2 : 1 v/v, 6 ml) was stirred at room temperature, in a sealed flask, for 12 h, to afford a yellow solution. The solvent was removed under reduced pressure to yield a glassy solid. The crude material was repeatedly (x 3) dissolved in CH₂CI₂ (5 ml) and the solvent removed under reduced pressure to facilitate elimination of excess acid and tert-butyl alcohol. The residue was finally taken into 0.2 M KOH _(aq) (10 ml) and extracted with CH₂CI₂ (3 x 15 ml). The organic layers were combined, dried over K₂CO₃, filtered and the filtrate concentrated under reduced pressure to yield the title compound AQ as an orange coloured crystalline solid (0.254 g, 0.304 mmol, 89 %); m.p. °C; ¹H NMR (CDCI₃, 500 MHz) δ 1.38 (9H, s, feu CH₃), 1.47 (6H, d, J = 7.0 Hz, CH₃), 2.58 (8H, br s, cyclen CH₂), 2.64 (8H, br s, cyclen CH₂), 3.09 (4H, q, J = 17.0 Hz, CH₂CO), 3.60 (1H, d, J = 15.0 Hz, CH₂ pyridine), 3.68 (1H, d, J = 15.0 Hz, CH₂ pyridine), 5.14 (2H, q, J= 7.5 Hz, CH), 7.16 (3H, q, J= 8.0 Hz, H^5': Ph₀₀), 7.24 (4H, t, J = 7.5 Hz, Ph_(m)), 7.30 (4H, d, J = 7.5 Hz, Ph₍₀₎), 7.56 (1H, U₁ J = 9.0 Hz, H¹⁰), 7.62 (1H, t, J = 7.5 Hz, H^4'), 7.82 (1H, dd, J = 8.5; 2.0 Hz, H⁹), 8.06 (2H, d, J = 8.5 Hz, NH), 8.28 (1H, ύ, J = 2.5 Hz, H⁷), 8.37 (1H, d, J = 7.5 Hz, H^3'), 8.44 (1H, d, J = 8.5 Hz, H²), 8.77 (1H, d, J = 8.5 Hz, H³); ¹³C (CDCI₃, 125 MHz, ¹H decoupled 500 MHz) δ 21.7 (2C, CH₃), 31.5 (3C, C¹⁴), 35.0 (1C, C¹³), 47.4 (2C, cyclen CH₂), 48.6 (2C, CH), 52.5 (2C, cyclen CH₂), 53.4 (2C, cyclen CH₂), 53.7 (1C, cyclen CH₂), 54.8 (1C, cyclen CH₂), 58.7 (1C, CH₂ pyridine), 60.2 (2C, CH₂CO), 116.6 (1C, C⁴), 118.3 (2C, C²; C¹⁰), 121.0 (1C, C^3'). 121.3 (1C, C⁶), 122.7 (1C, C⁷), 125.0 (1C, C^5'), 126.7 (4C, Ph₍₀₎), 127.5 (2C, Ph_(p)), 128.8 (4C, Ph_(m)), 133.8 (1C, C⁹), 137.6 (1C, C^4'), 138.5 (1C, C³), 143.5 (2C, Ph_(q)), 148.2 (1C, C⁸), 153.6 (1C, C^1'), 154.2 (1C, C¹¹), 158.0 (1C, C^6'), 160.2 (1C, C¹), 160.3 (1C, C¹²), 170.8 (2C, amide C=O), 177.8 (1C, C⁵); MS (ES⁺) m/z 837.5 (100 %, [M + H]⁺); HRMS (ES⁺) /tf/zfound 837.4817 [M + H]⁺ C₅₀H_6IO₄N₈ requires 837.4810.

[EuL⁸]

A stirring solution of 2-{4-[6-(6-te/t-butyl-10-oxo-10A£9-oxa-1-aza-anthracen-2-yl)- pyridin-2ylmethyl]-7-[((5)-1-phenyl-ethylcarbamoyl)-methyl]-1,4,7,10-tetraaza- cydododec-1-yl}-N-((^-1-phenyl-ethyl)-acetamide 40 (0.020 g, 0.024 mmol) and Eu(OTf)₃.6H₂O (0.017 g, 0.024 mmol) in anhydrous CH₃CN (1 ml) was heated at reflux, under argon, for 14 h. The solution was allowed to cool to room temperature followed by the removal of solvent under reduced pressure to afford a glassy orange solid. CH₂CI₂ (10 ml) was added to the solid, and the mixture sonicated for 10 min. The solvent was then decanted and the solid material dissolved in a minimum volume of CH₃CN (0.5 ml). The solution was added dropwise onto diethyl ether (25 ml) to induce precipitation. The solid material was isolated by centrifugation, and the process of induced precipitation repeated twice more to yield the complex as its triflate salt. The pale yellow coloured solid was made water soluble by the exchange of triflate anions for chloride anions using OOWEX 1 x 8 200-400 mesh Cl' resin. The solid material was dissolved in a mixture of H₂O - CH₃OH (1:1 v/v, 10 ml) and 0.5 g of prepared resin added to the solution, which was stirred at room temperature, for 3 h. The resin was removed by filtration, and the filtrate concentrated under reduced pressure to yield the title complex [EuL⁸] as a colourless solid (0.009 g, 0.008 mmol, 34 %); λ_max (H₂O) = 355 nm; T (H₂O) = 0.36 ms; HPLC (Method A) & = 11.0 min. Example 5: Synthesis of U and EuU (see scheme 7)

4-[(5)-1-(2-{7-[6-(6-te/*Butyl-10-oxo-10/*-9-oxa-1-aza-anthracen-2-yl)- pyridin-2-ylmethyl]-4,10-bis-[((5)-1-phenyl-ethylcarbamoyl)-methyl]- 1,4,7,10-tetraaza-cyclododec-1-yl}-acetylamino)-ethyl]-benzoic acid methyl ester ([L⁹])

A stirring mixture of 2-{4-[6-(6-te/f-butyl-10-oxo-10/f9-oxa-1-aza-anthracen-2-yl)- pyridin-2ylmethyl]-7-[((S)-1-phenyl-ethylcarbamoyl)-methyl]-1,4,7,10-tetraaza- cydododec-1-yl}-N-((^-1-phenyl-ethyl)-acetamide 40 (0.131 g, 0.157 mmol), 4-[(S)- l-(2-chloro-acetylamino)-ethyl]-benzoic acid methyl ester 30 (0.050 g, 0.196 mmol) and K₂CO₃ (0.043 g, 0.313 mmol) in anhydrous CH₃CN (7 ml) was heated at reflux, under argon, for 18 h. The resultant mixture was allowed to cool to room temperature, syringe filtered and the filtrate concentrated under reduced pressure to afford a residual orange oil. The crude material was purified by column chromatography on neutral alumina (gradient elution; CH₂CI₂ to 1.0 % CH₃OH : CH₂CI₂, utilising 0.1 % CH₃OH increments) to yield the title compound as an orange crystalline solid (0.105 g, 0.102 mmol, 64 %); R* = 0.27 (Alumina, CH₂CI₂ - CH₃OH, 49 : 1 v/v); m.p. 87-89 °C; ¹H NMR (CDCI₃, 500 MHz) δ 1.40 (9H, S, feu CH₃), 1.44 (9H, d, J = 7.0 Hz, CH₃), 2.71 (16 H, br s, cyclen CH₂), 3.25 (6H, br s, CH₂CO), 3.75 (2H, s, CH₂ pyridine), 3.84 (3H, s, C(O)OCH₃), 5.05 (3H, q, CH), 7.16 (2H, d, J = 8.0 Hz, H^5'; Ph_(p)), 7.28 (1OH, m, Ph), 7.33 (2H, d, J = 8.0 Hz, Ph_(o/m)), 7.60 (1H, U₁ J= 8.5 Hz, H¹⁰), 7.73 (1H, t, J = 8.0 Hz, H^4'), 7.85 (1H, dd, J= 8.5; 2.5 Hz, H⁹), 7.89 (2H, d, J = 8.0 Hz, Ph_(0/m)), 8.30 (1H, d, J = 2.5 Hz, H⁷), 8.43 (2H, d, J = 8.0 Hz, H²; H^3'), 8.77 (1H, d, J = 8.0 Hz, H³); ¹³C (CDCI₃, 125 MHz, ¹H decoupled 500 MHz) δ 21.9 (3C, CH₃), 31.5 (3C, C¹⁴), 35.0 (1C, C¹³), 48.9 (3C, CH), 51.5 (2C, cyclen CH₂), 52.3 (1C, C(O)OCH₃), 52.5 (2C, cyclen CH₂), 53.0 (2C, cyclen CH₂), 53.7 (2C, cyclen CH₂), 58.5 (3C, CH₂CO), 60.2 (1C, CH₂ pyridine), 116.6 (1C, C⁴), 118.3 (2C, C²; C¹⁰), 121.3 (1C, C^3'). 121.4 (1C, C⁶), 122.7 (1C, C⁷), 125.4 (2C, Ph), 126.2 (2C, Ph), 126.5 (4C, Ph), 127.5 (1C, C^5'), 128.8 (4C, Ph), 129.2 (1C, Ph), 130.1 (2C, Ph), 133.8 (1C, C⁹), 137.8 (1C, C)₁ 138.6 (1C, C³), 143.5 (2C, Ph_(q)), 148.2 (1C, C⁸), 153.6 (1C, C^1'), 154.2 (1C, C¹¹), 159.9 (1C, C^6'), 160.2 (1C, C¹), 160.3 (1C, C¹²), 166.9 (1C, C(O)OCH₃), 170.8 (3C, amide C = O), 177.8 (1C, C⁵); MS (ES⁺) m/z 1056.6 (100 %, [M + H]⁺); HRMS (ES⁺) /77/zfound 1056.5733 [M + H]⁺ C₆₂H₇₄O₇N₉ requires 1056.5706.

[EuL⁹]

A stirred solution of 4-[(5)-1-(2-{7-[6-(6-te/t-butyl-10-oxo-10/^t/- 9-oxa-1-aza-anthracen- 2-yl)-pyridin-2-ylmethyl]-4,10-bis-[((5)-1-phenyl-ethylcarbamoyl)-methyl]-1,4,7,10- tetraaza-cyclododec-1-yl}-acetylamino)-ethyl]-benzoic acid methyl ester [L⁹] (0.061 g, 0.057 mmol) and Eu(OTf)₃.6H₂O (0.045 g, 0.063 mmol) in anhydrous CH₃CN (2ml) was heated at reflux, under argon, for 17 h. The resultant solution was allowed to cool to room temperature followed by the removal of solvent under reduced pressure to afford a glassy orange solid. CH₂CI₂ (10 ml) was added to the solid, and the mixture sonicated for 10 min. The solvent was then decanted and the solid material dissolved in a minimum volume of CH₃CN (1.0 ml). The solution was added dropwise onto diethyl ether (50 ml) to induce precipitation. The solid material was isolated by centrifugation, and the process of induced precipitation repeated twice more to yield the complex as its triflate salt. The yellow solid was made water soluble by the exchange of triflate anions for chloride anions using 'DOWEEX 1 x 8 200-400 mesh Cl' resin. The solid material was dissolved in a mixture of H₂O - CH₃OH (1:1 v/v, 20 ml) and 0.8 g of prepared resin added to the solution, which was stirred at rt, for 6 h. The resin was removed by filtration, and the filtrate concentrated under reduced pressure to yield the title complex [EuL⁹] as a pale yellow coloured solid (0.044 g, 0.036 mmol, 64 %); λ_max (H₂O) = 348 nm; T (H₂O) = 1.02 ms; T (D₂O) = 1.34 ms; HPLC (Method A) ft = 10.9 min.

Example 6: Activation and conjugation of EuU with BG-NH?

[EuL⁹_CO₂H]

A solution of [EuL⁹] (1.5 μmol) in CH₃OH - 0.02 M KOH_(aq) (1:1 v/v, 2 ml) was stirred at rt. The progress of hydrolysis was monitored by reverse phase HPLC using a

Chromolith performance RP18e 100 x 4.6 mm column. (Method D; t_R (ester) = 6.40 mins, t_R (add) = 6.24 mins).

The reaction ran to completion in approximately 2 h and was neutralised using a dilute

HCl₍aq) solution. The solvent was removed under reduced pressure and the crude residue used directly in the next step without further purification. [EuL⁹_CONHBG]

To a stirred solution of [EuL⁹_CO₂H] in anhydrous DMF (2 ml) was added incremential equivalents of TSTU and DIPEEA (50 μl of a 100 mM solution in DMF in each case, 5 μmol). The progress of the reaction was monitored by reverse phase HPLC using a Chromolith performance RP18e 100 x 4.6 mm column. BG-NH₂ was added to the stirred solution of in situ [EuL⁹_CO₂H] (1 μmol) in anhydrous DMF (400 μl). The mixture was stirred at rt and the progress of the reaction was monitored by reverse phase HPLC using a Chromolith performance RP18e 100 x 4.6 mm column. (Method D; t_R (NHS) = 6.28 mins, t_R(BG) = 5.97 mins). The reaction ran to completion in approximately 1 h. The product was purified by reverse phase semi-preparative HPLC using a Chromolith performance RP18e 100 x 10 mm column. (Method E; t_R (BG) = 6.67 mins). The product was obtained as a colourless solid (320 nmol, 34 %).

Example 7 : Synthesis of L₁₃ and EuL₁₃ (see schema 8)

(5)-2-Bromo-pentanedioic acid 5-benzyl ester

This compound was prepared following the procedure disclosed by K-P. Eisenwiener and al., Bioorg. Med. Chem. Lett., 2000, 10, 2133.

A solution of NaNO₂ (5.50 g, 80.1 mmol) in H₂O (50 ml) was added dropwise over 30 min to a stirred solution of (5)-glutamic acid 5-benzyl ester (10.0 g, 42.1 mmol) and NaBr (16.0 g, 116 mmol) in 1 M HBr (250 ml), cooled at -5 °C. After 10 h, concentrated H₂SO_4(aq) (5 ml) was slowly added to the reaction mixture, which was then extracted with diethyl ether (3 x 300 ml). The combined organic extracts were washed with brine (200 ml), dried over Na₂SO₄, filtered and the filtrate concentrated under reduced pressure. The crude material was purified by column chromatography on silica (gradient elution: Hexane - 20 % EtOAc : Hexane, utilising 1 % EtOAc increments) to yield the title compound '44 as a yellow oil (7.40 g, 24.6 mmol, 58 %); R_F = 0.25 (Silica, Hexane - EtOAc 17 : 3 v/v); ¹H NMR (CDCI₃, 700 MHz) δ 2.30 (1H, m, CH₂CHBr), 2.42 (1H, m, CH₂CHBr), 2.60 (2H, m, CH₂CH₂CHBr), 4.41 (1H, dd, J = 9.0; 6.0 Hz, CH), 5.14 (2H, s, CH₂Ph), 7.33-7.39 (5H, m, Ph); ¹³C NMR (CDCI₃, 176 MHz, ¹H decoupled 700 MHz) δ 29.7 (1C, CH₂CH₂CHBr), 31.6 (1C, CH₂CHBr), 44.2 (1C, CH), 66.9 (1C, CH₂Ph), 128.4 (1C, Ph_(0/m)), 128.5 (1C, Ph_(0/m)), 128.6 (1C, Ph_(0/m)), 128.7 (1C, Ph_(0/m)), 128.8 (1C, Ph_(p)), 135.8 (1C, Ph_(q)), 172.1 (1C, Cbz C = O), 173.4 (1C, Acid C = O); MS (ES⁺) m/z 323.2 (100 %, [M + H]⁺).

(S)-2-Bromo-pentanedioic acid 5-benzyl ester 1-tert-butyl ester

A solution of (5)-2-bromo-pentanedioic acid 5-benzyl ester 44 (2.00 g, 6.85 mmol) in te/f-butyl acetate (25 ml) and HClO₄ in H₂O (70 %, 0.34 mmol) was stirred, at rt, for 16 h. H₂O (35 ml) was added to the reaction mixture, and the organic phase separated. The organic phase was washed with H₂O (25 ml), followed by 5 % Na₂CO_{3 (aq)} (25 ml). The solvent was removed under reduced pressure to yield the title compound AS as a yellow coloured oil (2.15 g, 6.03 mmol, 88 %); ¹H NMR (CDCI₃, 700 MHz) δ 1.46 (9H, s, *Boc CH₃), 2.25 (1H, m, CH₂CHBr), 2.35 (1H, m, CH₂CHBr), 2.55 (2H, m, CH₂,CH₂,CHBr), 4.23 (1H, q, J = 5.5; 2.0 Hz, CH), 5.12 (2H, s, CH₂Ph), 7.31- 7.36 (5H, m, Ph); ¹³C NMR (CDCI₃, 176 MHz, ¹H decoupled 700 MHz) δ 27.9 (3C, *Boc CH₃), 29.9 (1C, CH₂CH), 31.8 (1C, CH₂CH₂), 46.9 (1C, CH), 66.7 (1C, CH₂Ph), 82.8 (1C, <Boc_(q)), 128.4 (1C, Ph_(0/m)), 128.5 (1C, Ph_(o/m)), 128.6 (1C, Ph_(0/m)), 128.7 (1C, Ph_(0/m)), 128.8 (1C, Phφ₎), 136.0 (1C, Ph_(q)), 168.5 (1C, fcoc C = O), 172.2 (1C, Cbz C = O); MS (ES⁺) m/z 379.0 (100 %, [M + Na]⁺).

4_/10-bis-/e/^Butoxycarbonylmethyl-l_f4_/7_/10-tetraaza-cyclododecane-l_/7- dicarboxylic acid dibenzyl ester

This cyclen derivative was prepared according to the procedure described by Z. Kovacs and al., Synthesis., 1997, 7, 759. A stirred mixture of 1,4,7, 10-tetraaza-cyclododecane-l,7-dicarboxylic acid dibenzyl ester 31 (2.65 g, 6.02 mmol), te/f-butyl bromoacetate (2.64 g, 2.00 ml, 13.5 mmol) and Cs₂CO₃ (5.88 g, 18.1 mmol) was heated at reflux in anhydrous CH₃CN (25 ml), under argon, for 18 h. The reaction mixture was allowed to cool to room temperature, syringe filtered and the filtrate concentrated under reduced pressure to yield a residual yellow oil. The crude material was purified by column chromatography on silica (gradient elution: CH₂CI₂ to 1.5 % CH₃OH : CH₂CI₂, utilising 0.1 % CH₃OH increments) to yield the title compound 4$ as a yellow coloured oil (2.46 g, 3.68 mmol, 61 %); R_F = 0.53 (Silica, CH₂CI₂ - CH₃OH, 39 : 1 v/v); ¹H NMR (CDCI₃, 700 MHz) δ 1.46 (18H, s, teoc CH₃), 2.86 (8H, br s, cyclen CH₂), 3.31 (4H, br s, CH₂CO), 3.42 (8H, br s, cyclen CH₂), 5.12 (4H, s, CH₂Ph), 7.25-7.33 (1OH, br s, Ph); ¹³C NMR (CDCI₃, 176 MHz, ¹H decoupled 700 MHz) δ 28.0 (6C, 'Boc CH₃), 46.7 (2C, cyclen CH₂), 46.8 (2C, cyclen CH₂), 54.2 (2C, cyclen CH₂), 54.5 (2C, cyclen CH₂), 55.9 (2C, CH₂CO), 66.8 (2C, CH₂Ph), 80.7 (2C, *Boc_(q)), 127.7 (2C, Ph), 127.8 (2C. Ph), 128.3 (2C, Ph), 128.5 (2C, Ph), 136.8 (2C, Ph), 156.3 (2C, Cbz C = O), 170.4 (2C, feoc C = O), MS (ES⁺) m/z 669.4 (100 %, [M + H]⁺); HRMS (ES⁺) m/z found 669.3860 [M + H]⁺ C₃₆H₅₃O₈N₄ requires 669.3858. (7-te/*-Butoxycarbonylmethyl-1,4,7,10-tetraaza-cyclododec-1-yl)-acetic acid tert-but/l ester (47)

^lO-Bis-teAf-butoxycarbonylmethyl-l^^_/lO-tetraaza-cyclododecane-l^-dicarboxylic acid dibenzyl ester (2.46 g, 3.68 mmol) in CH₃OH - H₂O (3:1 v/v, 20 ml) was shaken in a Parr hydrogenation flask at 40 psi H₂ over Pd(OH)₂ZC (0.25 g) for 48 h. The resulting mixture was filtered through Celite to afford a colourless solution which was concentrated under reduced pressure to yield the title compound 47 as a colourless crystalline solid (1.46 g, 0.365 mmol, 99 %); ¹H NMR (CDCI₃, 500 MHz) δ 1.46 (18H, s, teoc CH₃), 2.65 (8H, br s, cyclen CH₂), 2.84 (8H, br s, cyclen CH₂), 3.33 (4H, s, CH₂CO); ¹³C (CDCI₃, 125 MHz, ¹H decoupled 500 MHz) δ 27.3 (6C, tøoc CH₃), 51.8-53.4 (8C, cyclen CH₂), 82.7 (2C, i3oc_(q)), 171.9 (2C, C = O); MS (ES⁺) m/z 401.4 (100 %, [M + H]⁺); HRMS (ES⁺) /π/zfound 401.3121 [M + H]⁺ C₂₀H_4JO₄N₄ requires 401.3122.

2-(4,10-bis-te/*-Butoxycarbonylmethyl-1,4,7,10-tetraaza-cyclododec-1-yl)- pentanedioic acid 5-benzyl ester 1-tert-butyl ester (49)

A stirred mixture of (7-te/f-butoxycarbonylmethyl-1,4,7,10-tetraaza-cydododec-1-yl)- acetic acid te/f-butyl ester 47 (0.369 g, 0.925 mmol), (5)-2-bromo-pentanedioic acid 5-benzyl ester 1-tetf-butyl ester (0.325 g, 0.923 mmol) and NaHCO₃ (0.074 g, 0.925 mmol) was heated at 55 °C in anhydrous CH₃CN (15 ml), under argon, for 18 h. The reaction mixture was allowed to cool to room temperature, syringe filtered and the filtrate concentrated under reduced pressure to afford an orange residual oil. The crude material was purified by column chromatography on neutral alumina (gradient elution: CH₂CI₂ - 1.0 % CH₃OH : CH₂CI₂, utilising 0.1 % CH₃OH increments) to yield the title compound '49 as a yellow coloured solid (0.373 g, 0.552 mmol, 60 %); R_? = 0.28 (Alumina, CH₂CI₂ - CH₃OH, 49 : 1 v/v); ¹H NMR (CDCI₃, 700 MHz) δ 1.34 (27H, s, tøoc CH₃), 1.80 (2H, m, arm CH₂), 2.41 (4H, m, cyclen CH₂; arm CH₂), 2.70 (6H, m, cyclen CH₂), 3.05 (8H, m, cyclen CH₂), 3.24 (4H, q, CH₂CO), 3.31 (1H, t, J = 7.5 Hz, CH), 5.03 (2H, q, J = 12.5; 5.5 Hz, Cbz CH₂), 7.19-7.27 (5H, m, Ph); ¹³C NMR (CDCI₃, 176 MHz, ¹H decoupled 700 MHz) δ 25.0 (1C, CH₂), 28.3 (9C, fcoc CH₃), 30.9 (1C, CH₂), 46.6 (2C, cyclen CH₂), 49.8 (2C, cyclen CH₂), 50.8 (2C, cyclen CH₂), 51.4 (2C, cyclen CH₂), 56.5 (2C, CH₂CO), 60.8 (1C, CH), 66.6 (1C, Cbz CH₂), 81.7 (2C, teoq_q)), 82.2 (1C, teoc_(q)), 128.4 (2C, Ph_(0/m)), 128.5 (1C, Ph₀₀), 128.7(2C, Ph_(QΛn)), 136.0 (1C, Ph_(q)), 170.4 (2C, teoc C = O), 171.4 (1C, feoc C = O), 172.9 (1C, Cbz C = O); MS (ES⁺) m/z 677 A (100 %, [M + H]⁺); HRMS (ES⁺) m/z found 677.4484 [M + H]⁺ C₃₆H₆₁O₈N₄ requires 677.4484.

2-C4,10-bis-fø^Butoxycarbonylmethyl-7-[6-(6-te^butyl-10-oxo-10/^-9- oxa-1-aza-anthracen-2-yl)-pyridin-2-ylmethyl]-1,4,7,10-tetraaza- cyclododec-1-yl}-pentanedioic acid 5-benzyl ester 1-te/t-butyl ester ([L¹³])

A stirred mixture of 2-(6-bromomethyl-pyridin-2-yl)-6-te/f-butyl-9-oxa-1-aza- anthracen-10-one 9 (0.110 g, 0.318 mmol), 2-(4,10-bis-tert-butoxycarbonylmethyl- 1,4,7,10-tetraaza-cyclododec-1-yl)-pentanedioic acid 5-benzyl ester 1-tetf-butyl ester 49 (0.195 g, 0.289 mmol) and K₂CO₃ (0.086 g, 0.636 mmol) in anhydrous CH₃CN (10 ml) was heated at reflux, under argon, for 40 h. The reaction mixture was allowed to cool to room temperature, filtered and the filtrate concentrated under reduced pressure to afford an orange residual oil. The crude material was purified by column chromatography on neutral alumina (gradient elution: CH₂CI₂ - 1.0 % CH₃OH : CH₂CI₂, utilising 0.1 % CH₃OH increments) to yield the title compound ' [L¹²] as a yellow oil (0.188 g, 0.116 mmol, 40 %); R_? = 0.37 (Alumina, CH₂CI₂ - CH₃OH, 19 : 1 v/v); ¹H NMR (CDCI₃, 700 MHz) δ 1.34 (27H, s, ttoc CH₃), 1.38 (9H, s feu CH₃), 2.37 (4H, m, cyclen CH₂; arm CH₂), 2.73 (6H, m, cyclen CH₂), 3.07 (8H, m, cyclen CH₂), 3.26 (4H, q, CH₂CO), 3.31 (1H, t, J = 7.5 Hz, CH), 5.04 (2H, q, J = 12.5; 5.5 Hz, Cbz CH₂), 7.19- 7.27 (6H, m, Ph; H^5'), 7.54 (1H₇ d, J = 8.5 Hz, H¹⁰), 7.72-7.78 (2H, br s, H^4'; H⁹), 8.24 (1H, U₁ J = 3.0 Hz, H⁷), 8.32 (1H, U₁ J = 8.0 Hz, H^3'), 8.40 (1H, br s, cyclen NH), 8.48 (1H, ύ, J = 7.0 Hz, H²), 8.75 (1H, d, J = 8.0 Hz, H³); ¹³C NMR (CDCI₃, 176 MHz, ¹H decoupled 700 MHz) δ 25.0 (1C, CH₂), 28.3 (9C, feoc CH₃), 30.9 (1C, CH₂), 31.5 (3C, C¹⁴), 34.9 (1C, C¹³), 46.6 (2C, cyclen CH₂), 49.8 (2C, cyclen CH₂), 50.8 (2C, cyclen CH₂), 51.4 (2C, cyclen CH₂), 56.5 (2C, CH₂CO), 63.3 (1C, CH), 66.6 (1C, Cbz CH₂), 81.7 (2C, feoctø), 82.2 (1C, feoc_(q)), 116.4 (1C, C⁴), 118.3 (1C, C¹⁰), 118.5 (1C, C²), 119.7 (1C, C^5'), 121.3 (1C, C⁶), 122.7 (1C, C⁷), 123.8 (1C, C^2'), 128.4 (2C, Ph_(0/m)), 128.5 (1C, Phjp)), 128.7(2C, Ph_(0/m)), 133.6 (1C, C⁹), 136.1 (1C, Ph_(q)), 137.9 (1C, C^4'), 138.4 (1C, C³), 148.1 (1C, C⁸), 153.6 (1C, C^1'), 154.2 (1C, C¹¹), 158.6 (1C, C^6'), 160.2 (1C, C¹), 160.6 (1C, C¹²), 170.4 (2C, feoc C = O), 171.4 (1C, feoc C = O), 172.9 (1C, Cbz C = O), 177.8 (1C, C⁵); MS (ES⁺) m/z 1019.6 (100 %, [M + H]⁺); HRMS (ES⁺) m/z found 1019.5866 [M + H]⁺ C₅₈H₇₉O₁₀N₆ requires 1019.5852; HPLC (Method A) fc = 12.0 min.

[EuL¹³]

A stirred mixture of 2-{4,10-bis-te^-butoxycarbonylmethyl-7-[6-(6-te/t-butyl-10-oxo- 10/f9-oxa-1-aza-anthracen-2-yl)-pyridin-2-ylmethyl]-1,4,7,10-tetraaza-cyclododec-1- yl}-pentanedioic acid 5-benzyl ester l-tert-butyl ester [L¹³] (0.021 g, 0.021 mmol) in hydrobromic acid (5 ml) was heated at 40 °C, for 4 h. The solvent was removed under reduced pressure to yield a glassy solid. The crude material was analysed by 1H NMR to ensure complete deprotection, with the material used immediately for complexation. HPLC (Method A) & = 10.83 min.

The deprotected ligand was dissolved in CH₃OH - H₂O (2: 2 v/v, 4 ml) and Eu(OAc)₃.6H₂O (0.008 g, 0.023 mmol) added to the solution. The pH of the solution was raised to 5.4 by the addition of 1 M KOH _(aq), then stirred and heated at 90 °C, for 14 h. The reaction mixture was allowed to cool to room temperature before raising the pH of the solution to 10.0 using dilute KOH _(aq). The reaction mixture was stirred for 1 h to allow precipitation of excess Eu metal as its hydroxide salt, Eu(OH)₃. The solid precipitate was removed by syringe filtration and the pH of the colourless aqueous filtrate reduced to pH 5.5 using a solution of 1 M HCl _(aq). The solvent was removed under reduced pressure using a freeze-drier to yield the title complex [EuL¹²] as a colourless solid (0.008 g, 0.009 mmol, 45 %); λ_max (H₂O) = 355 nm.

Example A: Absorption spectra

The absorption spectrum of the complex EuLl prepared in Example 1 was recorded in water at 295 K. For purpose of comparison, the absorption spectrum for the complex TbLl having the following formula was also recorded.

Said complex TbL¹ which has a pyrazoyl-azaxanthone moiety instead of a pyridyl-azaxanthone is described in Chem. Commun. 2007, 3841-3843. The absorption spectra are contained in figure 1. It is observed that with the pyridyl- azaxanthone complex EuL¹ of the invention, complexation was accompanied by a change in spectra form. A red shift in λ_max was observed to a wavelength > 350 nm, while a gradual decrease in absorption was observed above 350 nm. The pyridyl-azaxanthone complex EuL¹ is more suitable than the pyrazoyl- azaxanthone complex TbL¹ to excitation at the wavelength of the common lasers (especially 337 nm and 355 nm).

Similar results were obtained with the complexes of Examples 2, 4, 5, 6 and 7 of the present invention.

Example B: Emission spectra

Emission from complex [EuL¹] (figure 2) reveals the expected Eu spectral fingerprint from the ⁵D₀ emissive state. The spectrum also shows azaxanthone fluorescence centred at 445 nm. This ligand-based emission (Φ^f _em = ~ 20 %), whilst limiting the metal-based quantum yield, provides an observable band for luminescence microscopy and facilitates flow cytometric studies.

The emission spectra of the other invention complexes EuL⁸, EuL⁹, EuL⁹-BG and EuL¹³ are represented respectively on figures 3 to 7.

Example C: PH sensitivity

Luminescent titrations of [EuL⁴] revealed no apparent pH sensitivity of the pyridyl azaxanthone, with luminescent behaviour remaining constant over the pH range 3 to 9.

Claims

1. Lanthanide (III) ion complexing compound comprising: (1) a sensitizer moiety of formula (I)

in which: a is an integer from 1 to 4; b is an integer equal to 1 or 2; c is an integer equal to 1 or 3;

(R₁)a, (R₂)b, (R₃)C are the same or different and are chosen from the group consisting of: H; alkyl; -COOR₄ where R₄ is H or an alkyl; aryl; heteroaryl; saturated or unsaturated cyclic hydrocarbon; CF₃; CN; a halogen atom; L-Rg; L-Sc; or two consecutive R₃, two^* consecutive R₂ or two consecutive R₁ groups together form with the carbon atoms to which they are linked, an aryl or a heteroaryl group or a saturated or unsaturated cyclic hydrocarbon group; where L is a linker, Rg is a reactive group and Sc is a conjugated substance;

Xi and X₂ are the same or different and are O or S;

A is either a direct bond or a divalent group chosen from -CH₂- or -(CH₂)₂-, said moiety being covalently attached to

(2) a lanthanide (III) ion chelating moiety through A,

2. Lanthanide (III) ion complexing compound according to claim 1 wherein X₁=X₂=O.

3. Lanthanide (III) ion complexing compound according to claim 1 wherein a=b=c=1, R₂=R₃=H and R₁ is a (C₁-C₆) alkyl group.

4. Lanthanide (III) ion complexing compound according to claim 1 wherein X1=X2=O, a=b=c=l, R₂=R₃=H and R₁ is a (C₁-C₆) aikyl group.

5. Lanthanide (III) ion complexing compound according to claim 1, wherein: a=b=c=l; R₁=H or a (C₁-C₆) alkyl group; R₂=H;

R₃=CF₃; COOR₄, where R₄=H, (C₁-C₆) alkyl, aryl, CN, halo, phenyl; X₁= X₂=O.

6. Lanthanide (III) ion complexing compound according to any one of claims 1 to 5, wherein the chelating moiety comprises a set of heteroatom-containing electron- donating groups, such as carboxyl, amino, amido, oxo, alkylphosphinate or phosphonate.

7. Lanthanide (III) ion complexing compound according to claim 6, wherein it comprises 3, 4, 5, 6, 7, 8 or 9 heteroatom electron-donating groups.

8. Lanthanide (III) ion complexing compound according to any one of claims 1 to 7, wherein the formation constant (K _f) of the lanthanide (III) ion complex is greater than 10¹⁰ M^'1.

9. Lanthanide (III) ion complexing compound according to any one of claims 1 to

8, wherein the chelating moiety is chosen from the group consisting of: NTA, EDTA, DTPA, TTHA, a tetraazacyclododecane derivative such as DOTA, DOTAM or DTMA.

10. Lanthanide (III) ion complexing compound according to any one of claim 1 to

9, which is a compound of formula (II):

in which:

W is a sensitising moiety of formula (I) as defined in claim 1, linked through A, R₅ to R₁₂ are the same or different and are chosen from the group consisting of H, an alkyl group, L-Rg and L-Sc; Yi, Y₂ and Y₃ are the same or different and are chosen from the groups consisting of L-Rg, L-Sc and groups of the following formulae:

wherein: n is O, 1 or 2; m is 1 or 2; p is 1 or 2; R₁₃ is H, alkyl, optionally substituted aryl, preferably optionally substituted benzyl, L-Rg, L-Sc ; R₁₄, R₁₅ are the same or different and chosen from H, -CHR'R" in which R' and

R" being the same or different and being chosen from H, alkyl, optionally substituted aryl, optionally substituted aralkyl, or amino acid side chain, carboxyl group, L-Rg, L-Sc; R₁₆ represents H, alkyl, optionally substituted aryl, preferably optionally substituted benzyl, alkylcarboxyl, alkylamino, L-Rg, L-Sc; provided that when one of Y₁, Y₂, Y₃ is hydrogen, the other two are different from hydrogen. Ii. Lanthanide (III) ion complexing compound according to claim 10, which is a compound of formula (III):

wherein:

W, R₅ to R₁₂, m are as defined in claim 10, R₁₇ to R₂₂ are the same or different and are chosen from H, -CHR'R" in which R' and R" being the same or different and are chosen from H, (C₁-C₆) alkyl, optionally substituted aryl, optionally substituted aralkyl, amino acid side chain, carboxyl, L-Rg, L-Sc.

12. Lanthanide (III) ion complexing compound according to claim 11 which is a compound of formula (IV):

in which:

W, FVR₁₂, are as defined in claim 10,

R*l# R'2/ R^?3 are identical and represent a (C _!-C₆) alkyl, preferably -CH₃, -C₂ H₅; W₁ to R"₃ are the same or different and are an optionally substituted aryl, preferably optionally substituted benzyl or optionally substituted phenyl.

13. Lanthanide (III) ion complexing compound according to claim 12, which is a compound of formula (V):

W is as defined in claim 10;

R₂₃ is H, a carboxyl group, a (C_rC₆)alkoxycarbonyl, L-Sc, L-Rg.

14. Lanthanide (III) ion complexing compound according to claim 10, which is a compound of formula (VI);

in which: n is 0, 1 or 2,

W, R₅-R₁₂, are as defined in claim 10,

R₂₄ to R₂6 are chosen from the group consisiting of H, (C₁-C₆) alkyl, optionally substituted aryl, (preferably optionally substituted benzyl), L-Rg, L-Sc.

15. Lanthanide (III) ion complexing compound according to claim 10, which is a compound of formula (VII)

in which:

W, R₅-R₁₂, are as defined in claim 10,

R₂₇ to R₂9 are chosen from the group consisting of: H, (CrC₆) alkyl, optionally substituted aryl, (preferably optionally substituted benzyl), L-Rg, L-Sc.

16. Lanthanide (III) ion complexing compound according to claim 10, which is a compound of formula (VIII):

in which n is 0, 1 or 2

W, R₅ to R₁₂ are as defined above for a compound of formula (II); R30, R31 and R₃₂ are chosen from the group consisting of H, (CrQ) alkyl, optionally substituted aryl, (preferably optionally substituted benzyl), L-Rg, L-Sc; R₃₃ is a group of formula

in which r is 0, 1 or 2 and R₃₄ is chosen from the group consisting of H, (d-Cs)alkyl, optionally substituted aryl, preferably optionally substituted benzyl.

17. Lanthanide (III) ion complexing compound according to any of the claims 1 to 16, wherein the linker L is a single covalent bond or L comprises 1-20 non-hydrogen atoms in a stable conformation such as carbon-carbon bonds, amide linkages, ester linkages, sulfonamide linkages, ether linkages, thioether linkages.

18. Lanthanide (III) ion complexing compound according to claim 17, wherein L is chosen from the group consisting of;

in which:

- q and r are integers from 1 to 16, preferably 1 to 8;

- s and u are integers from 1 to 16, preferably 1 to 5.

19. Lanthanide (III) ion complexing compound according to any one of claims 1 to 18, wherein the reactive group Rg is chosen from the group consisting of : carboxylic acids esters, activated carboxylic acid ester, aldehydes, alkyl halides, amines, anhydrides, aryl halides, carboxylic acids, haloacetamides, halotriazines, hydrazines (including hydrazides), isocyanates, isothiocyanates, maleimides, phosphoramidites, sulfonyl halides, and thiol groups, succinimidyl ester of a carboxylic acid, or a combination thereof

20. Lanthanide (III) ion complexing compound according to any one of claims 1 to 19, wherein the reactive group Rg is chosen from the group consisting of:

in which: p represents 0 to 8 and n represents 0 or 1;

Ar is 5 to 6 member aryl, optionally containing 1 to 3 heteroatoms chosen from halo,

N, O, S and optionally substituted by a halogen atom.

21. Lanthanide (III) ion complexing compound according to any one of claims 1 to 20, wherein the conjugated substance Sc is biomolecule chosen from an amino acid, a peptide, a protein, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, or a carbohydrate.

22. Lanthanide (III) ion complexing compound according to claim 21 wherein the conjugated substance Sc is a member of a specific binding pair chosen from: antigen/antibody, avidin or streptavidin/biotin, ligand/receptor, DNA strand / complementary DNA strand.

23. Luminescent lanthanide (III) ion complex comprising a (III) ion complexing compound according to any one of claims 1 to 22 and a lanthanide ion chosen from the group consisting of Tb³⁺, Eu³⁺, Sm³⁺ , Dy³⁺, Gd³⁺.

24. Use of a luminescent lanthanide (III) ion complex according to claim 23 as a fluorescent label.

25. Sensitising derivative of formula (Ia):

in which:

(R₁)a, (R₂Jb and (R₃)_care as defined for moiety of formula (I) in claim 1; Ai is hydrogen, alkyl, halogen or halogenoalkyl.

26. Sensitising derivative of formula (Ia) according to claim 25, wherein Xi=X₂=O.

27. Sensitising derivative of formula (Ia) according to claim 25, wherein a=b=c=l, R₂=R₃=H and R₁ is a (C₁-C₆) alkyl.

28. Sensitising derivative of formula (Ia) according to claim 25, wherein X_x=X₂=O, a=b=c=l, R₂=R₃=H and R₁ is a (C₁-C₆) alkyl.

29. Sensitising derivative of formula (Ia) according to claim 25, wherein a=b=c=l;

R₁=H, (C₁-C₆) alkyl;

R₂=H;

R₃=CF₃; COOR₄, where R₄=H, (C_rC₆) alkyl, aryl, CN, halo, phenyl;

X₁= X₂=O.