GB2467012A

GB2467012A - Lanthanide (III) ion complexing compounds, luminescent lanthanide (III) ion complexes and use thereof as fluorescent labels

Info

Publication number: GB2467012A
Application number: GB0900913A
Authority: GB
Inventors: Laurent Lamarque; Craig Montgomery; David Parker
Original assignee: CIS Bio International SA
Current assignee: CIS Bio International SA
Priority date: 2009-01-20
Filing date: 2009-01-20
Publication date: 2010-07-21
Also published as: GB0900913D0; WO2010084090A1

Abstract

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 2; (R1)a, (R2)b, (R3)c are the same or different and are chosen from the group consisting of H; alkyl; -COOR4where R4is 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 R2or two consecutive R1groups 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; X1and X2are the same or different and are 0 or S; A is either a direct bond or a divalent group chosen from -CH2- or -(CH-2)2-, said moiety being covalently attached to (2) a lanthanide (III) ion chelating moiety through A. These complexing compounds may be used to form lanthanide (III) complexes, particularly with terbium (Tb), europium (Eu), samarium (Sm), dysprosium (Dy) and gadolinium (Gd) (III) ions. These complexes may be useful as fluorescent labels.

Description

Lanthanide (III) ion complexing compounds. luminescent lanthanide (III) 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 potydentate 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, 3-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 D.J. Bornhop, Sioconjug. 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; 3-C. Frias, G. Bobba, M.3.

Cann, D. Parker and CJ. Hutchinson, Org. Bfoniol. 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, U. Charbonniere, M. Guardigli and A. Roda, J. Am. Chem. Soc., 2004, 126, 4888; B. Song, E. Wang and 3. 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, 3.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, E) 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 3.A.G. Williams and M. Woods, J. Chem. Soc., Perk/n Trans 2., 1999, 493).

Recently, ligands containing substituted 1-azaxanthone and azathiaxanthones have been introduced (WO 2006/039505 A2; Org. Bioniol. Chem., 2006, 4, 1707-1722; W02006/120444 Al) as effective sensitisers for Eu and Tb emission in aerated aqueous media. The pyrazoyl-l-azaxanthone has atso been proposed as sensitiser for Tb luminescence at 355 nm. (Craig P. Montgomery et al. Chem. Commun., 2007, 3841-3843).

Definitions The term!alkylu 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-butyi, i-butyt, 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-C6) alkyl, aryl, acyl, halogen, hydroxy, amino, alkoxy, alkytamino, acylamino, thioamido, acyloxy, aryloxy, aryloxyalkyl, mercapto, thia, aza, oxo, both saturated and unsaturated cyclic hydrocarbons, heterocycles 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" refers to aryl as just described including one or more groups such as (C1-C6) alkyt, 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.

Examples 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 cyclopentene, 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 "heteroarylal kyl" 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 heteroaryf 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 heterocycle 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 aiky) 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: Aminoacid Side chain Amino acid Side chain Alanine -CH3 Methionine -CH2CH2SCH3 Cysteine -CH2j Asraragine -Ci-I2CONH2 Aspartic acid -CH2COOH Proline -CH2CH2CH2-Glutamic acid -CH2CH2COOH Giutamine -CH2CH2CONH2 Phenylalanine -CH2C6H5 Arciinine -(CH2)3NH-C(NH)NH2 Aminoacid Side chain Amino acid Side chain Giycine -H Serine -CH2OH Histidine -CH2-j:b Threonine -CH(OH)CH3 Isoleucine -CH(CH3)CH2CH3 Valine -CH(CH3)2 Lysine -(CH2)4NH2 TryDtoDhan -CHfl5N Leucine -CH2CH(CH3)2 Tyrosine -CH2-C6H4OH

Description of the invention

The invention relates to lanthanide (III) ion corn plexing compounds comprising: (1) a sensitising moiety of formula (I) (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; (Ri)a, (R2)b, (R3) 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 Unker, Rg is a reactive group and Sc is a conjugated substance; X1, X2 are the same or different and are 0 or S; A is either a direct bond or a divalent group chosen from: -CH2-or -(Cl-I2)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 (Ri)a groups, (R2)b groups and (R3) 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 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=1, X1=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=1; 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 Ianthanide (III) ion via two nitrogen atoms of the pyridyl and azaxanthone groups. As compared to chelating compounds comprising azaxanthone chromophores disclosed in W02006/120444 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: A1 (Ia) in which: (R1)0, (Rz)b and (R3) 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=1, R2=R3=H and R1 is a (C1-C6) alkyl; and those where a=b=c=1, 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=1; R1=I-1, (C1-C6) alkyl; R2=H; R3=CF3; COOR4, where R4=H, (C1-C5) 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 chioro) 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 (Ta) 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-tertbutyl-9-oxa-1-aza-anthracen-lO-one (2) by a two-step process involving a nucleophilic aromatic substitution reaction between 2-chloronicotinic acid and 4-tert-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 (3. Lewis and T. D. 0' Donoghue, 3.

Chem. Soc., Dalton Trans., 1980, 5, 736) with {K3Fe(CN)6J and NaOH affords the N-methylpyridone intermediate (5). The reaction is easily monitored by H 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 POd3 in an appropriate solvent such as C6H5N (CH3), 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.

SCHEME 1 + NaOMe, MeOH ((LOH PPA

N OH HO 1 2

CF3SO3CH3, Toluene 0 0 [K3Fe(CN)6], NaOH DOWEX 1-X8(Cl) Resin 0°C MeOH, H20 rO Me Me Me SO3CF3 4 3 Pod3, C6H5N(CH3)2 Cl The coupling of compound (6) with a pyridne 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).

SCHEME 2 n-BuLl SnBu3CI N Br THF, -78°C [Lil SnBu3 SCHEME 3 (i)or(ii)

CI N

Conditions; Me (I) 6-Methyl-2-(tributylstannyl)pyridine, Pd(PPh3)4, toluene,

NJ

I NBS, (C6H5CO)202 (ii) 6-Methyl-2-pyridinoboronic acid N-phenyldiethanolamine ester, Cs2CO3, Pd2(dba)3, P(teu)3, dioxane, I CCI4 Br Pr2NEt, (C2H50)2P(O)H 6-Methyl-2-(tributylstannyl)pyridine (7), may be used directly in a Stille-coupling with azaxanthone (6), without any purification. Using Pd(PPh3)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 a-methyl substituent of (8) may be performed using radical bromination with ttAbromosuccinimide and benzoyl peroxide in Cd4. 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 (III) ion chelating moiety or ligand.

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 0, 0P032, 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) of greater than 1010 M'.

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.

HOOC-\ ____ // N-CH2 CH2-N

COOH

EDTA HOOC

HOOC HOOC

N\,,/\,t\,,.N\,,,COOH HOOC--\7COOH

COOH

DTPA COOH NTA COOH

HOOC HOOC\

COOH

1THA COOH COOH DOTAHO0 NH2 1" NH2 H2N DOTAM H2N 0 H/N:H3C DTMA NH 0 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): R1N N1R7 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; Y1, 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: O 0 Il -(CH2)---C-0R13 -(CH2)---C-NRR15 0 0 II 1 OH R16O-C-(CH2)-CH-C-OR13 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, alkyamino, 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): R5 R6 NR17R15-\ (CH)m w (III) 30:: 11/\ IN 8 (CH2)m NR19R20 O R10 R9 NR21R22 0 wherein: W, R 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): CR1 R1CH 11 / N 8 R9 (CH2)mNH in which: W, R to R12 and m are as defined above for a compound of formula (III); R'1, R'2, R'3 identical or different are a (C1-C6) alkyl, preferably -CH3, -C2H5; R"1 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 IV, those in which R"1 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): (V) H3NH R23 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): OR24 A5 R6 (CH2)n \ / \flw 25::: (VI) )CH2)n H (CH2)n Rio R9 OR26 0 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 II, (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 R25 is a (C1-c6) alkyl, a phenyl or a benzyl.

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

R R 6

(CH2)p w / (VII) /\IN O\\ ,JCH2)p /\ (C)PR HO-R9 O \

OH R29

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 aryl, (preferably optionally substituted benzyl), L-Rg, L-Sc.

In other embodiment, the lanthanide ion chelating complex is a compound of formula (VIII): OR3 R5 R6 (CH)n 2\W R3C/ H\(CHZn OR3.

(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, (C1-C6) alkyl, optionally substituted aryl, (preferably optionally substituted benzyl), L-Rg, L-Sc; R33 is a group of formula -(CH2)E-C-OR34 in which r is 0, 1 or 2 and R34 is chosen from the group consisting of H, (C1-C6)alkyl, optionally substituted aryl, preferably optionally substituted benzyl.

Among the compounds of formulae (VII) or (VIII) those in which R5 to R12 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: 1) o 0

II II 2)

3) -0-(CH2)---o 0 4) -0-(CH2) -NH--(CH2)-4-5)

II

6) ______ >i S-(CH2)-

-7) o 8)

9) -(CH2)q NH-(CH2)_Nh S-(CH2) -10) 11) 12) (CH2)qNH -(CH2)----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, isothiocyanates, 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: 0 0 /11 \ \ (\ C-NH)-(CH2)NCS ç CNH*(CH2)NCO p p eC_NH) (CH2)NCS C -NH) (CH2) NCO ( C-NH (CH _o_NI1 0 SO0 ( C-NH) (CH2)-C-O-N" ( C-NH) (CH2N ( C-NH) (CH2) -S-S-Ar; ( C_M{+(CH2)fl_NHAr; ( CNH+(CH2)N3; ( NH) (CH7)N3 ( C_NH+CH2)n_NH2 or ( C-NH) (CH2)-SH 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, 0, 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: :2xx:7 11 /N\__N\ 8 R10 R9 ha in which: R5 to R12 and Y1 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 V1. V2, V3 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 (JIa).

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 (L1, L1, L9 and L13) and the lanthanide (III) ion complexes are identified as EuL complexes (EuL1, EuL, EuL9 and EuL13). It would be obvious for the person skilled in the art that these processes may be used starting from the reagents bearing the appropriate SCHEME 4 � ____ (rH N Br N N \ 1) CH2CI2 TFA f-N\ /N \ / C N / _________ 2j Eu(OAc)36H20, K2C03, MeCN N \ MeOH, H20 0 0 0 0 16 [L1] / [EuL1] \ / SCHEME 5 O\ H N N' Br N N \ TFA CH2CI2 _-N N ________ / ________ N N K2C03, MeCN N N H'JH 17 18 19 3+ Ph 0 HN HN _______________ Eu(OTf)36H20, MeCN

H 3 CI

K2C03, MeCN N H 0NH [L4} [EuL4} SCHEME 6 Ph 0 N N PhOCI BnONINM H\/\,H N N Na2HPO4, pH 25 N N Cs2CO3, MeCN F-( \J H H' \J -OBn HNr 0 HN0 OBn N N H2, Pd(OH)2/C N N NN:NN: BnOI \I H' \I °NH 32 33 SCHEME 6 (continued) HN0 HN0 HN ____ fl H

N N + NND

N N MeOH

NH NH NH

NJ

33 37 38 TFA, CH2CI2 SCHEME 7

HN HN0

N N

N N K2CO , MeCN H H' \_J'7 N" \

NH [L

Eu(OTf)36H20, MeCN Eu(OTf)36H20, MOCN 3+ 3+ HN 0 HN H2O 3CV O-r 3C1 °NH 0 H [EuL8] (EuL9J

SCHEMES *

H NH Br N Br NaHCO3 MeCN N N K2C03, MeCN 0 N N N \I \ 55 O ooO 47 d \/ 49 \/ [L13] w

C HO 0

[L13] HBr, CH3CO2H r N N _____________ Eu(OAc)36K20 \

EN H0°

MeOHH2O

OH

HOL

HO [EuL13J 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 S 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, OT or related common anions.

ExamDles: 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 r2: Isopropyl tBu: Tertiobutyl tBoc: 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 Fluorescence detector.

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

Method A: Time (mm) H20 + 0.1 % HCO2H CH3CN + 0.1 % HCO2H 0 95 5 2 95 5 0 100 17 0 100 22 95 5 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 / mm and one of the selected methods shown below; Method B: Time (mm) : H2Ô + 0.1 % HCOH CH3CN + 0.1 % HCO2H 0 95 5 1 95 5 8 35 65 0 100 11 85 15 13 95 5 Method C: Time (mm) H20 + 0.1 % HCO2H CH3CN + 0.1 % HCO2H 0 85 15 1 85 15 0 100 11 85 15 13 85 15 Method 0: Time (mm) H20 + 0.1 % HCO2H CFI3CN + 0.1 % HCO2H 0 90 10 1 90 10 11 30 70 12 0 100 14 0 100 90 10 17 90 10 Semi-preparative HPLC was performed using a Chromolith performance RP18e 100 x mm column, using a flow rate of 14.1 ml / mm and one of the selected methods shown below; Method E: Time (mm) H20 + 0.1 % HCO2H CH3CN + 0.1 % HCO2H 0 80 20 1 80 20 8 45 55 0 100 11 0 100 12 80 20 13 80 20 Method F: Time (mm) H20 + 0 1 % HCO2H CH3CN + 0 1 % HCO2H 15 3 85. 15 0 100 33 0 100 36 85 15 85 15 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 IEuL1l(See schemes 1 to 4) 2-(4'-tert'Butylphenoxy) nicotinic acid (fl 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 H20 (200 cm3) and washed successively with Et20 (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%). SH (CDCI3, 500 MHz) 1.37 (9H, s, tBU), 7.14 (2H, d, 3 8.5, H2'), 7.20 (1H, dd, 3 7.5; 5, H2), 7.49 (2H, d, 3 9, H3'), 8.35 (1H, dd, 3 4.5; 2, H'), 8.55 (1H, dd, 3 8; 2, H3). & (CDCI3, 125 MHz) 31.7 (C6'), 34.8 (C5'), 113.5 (cr), 119.7 (C2), 121.4 (C2'), 127.1 (C3'), 143.8 (C3), 149.3 (c'), 149.8 (C1'), 152.4 (C'), 161.5 (C5), 164.9 (C0(acid)). m/z (ES) 270.1 (100%, M -H). Found: C, 70.54; H, 6.20; N, 4.91%; C,6H17N03 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 Et20 (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%). (CDCI3, 500 MHz) 1.42 (9H, s, 7.45 (1H, dd, 3 7.5; 4.5, H2), 7.58 (1H, d, 3 8.5, H'°), 7.86 (1H, dd, 3 9; 3, H9), 8.30 (1H, d, 3 2.5, H7), 8.73-8.76 (2H, H1/H3). & (CDCI3, 125 MHz) 31.6 (C'4), 35.1 (C13), 117.0 (C4), 118.4 (C'°), 121.1 (C2), 121.1, 122.7 (C7), 133.9 (C9), 137.6, 148.2 (C6), 154.1, 154.3(C'2), 160.6 (C'1), 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%; C16H15N02 requires C, 75.87; H, 5.97; N, 5.53%.

7-tett-Butyl-N-methyl-1-azaxanthonium trifluoromethylsulfonate Q) 7-tert-Butyl-1-azaxanthone (1.00 g, 3.95 mmol) was dissolved in thy 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 conipoundas a white solid (1.49 g, 3.58 mmol, 91%). (CD3OD, 400 MHz) 1.43 (9H, s, tBu), 4.51 (3H, s, Me), 7.84 (1H, d, 3 8.8, H'°), 7.99 (1H, dd, 3 8; 6, H2), 8.15 (1H, dd, J 8.8; 2.4, H9), 8.33 (1H, d, 3 2.4, H7), 9.14 (1H, dd, 3 6; 2, H'), 9.30 (1H, dd, 3 8; 2, H3). &, (CD3OD, 100 MHz) 30.3 (C'4), 34.8 (C'3), 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 (C'), 151.1 (C8), 152.4 (C"), 156.3 (C12), 173.8 (C5). 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 DOWEX 1-X8 (Cl) resin: H (CD3OD, 500 MHz) 1.46 (9H, s, tBu), 4.55 (3H, s, Me), 7.88 (1H, d, 3 9, H'°), 8.03 (1H, t, 3 6.5, H2), 8.18 (1H, dd, 3 9; 2, H9), 8.36 (111, d, 3 2, H7), 9.22 (1H, d, 3 6.5, H'), 9.33 (1H, d, 3 7.5, H3). & (CD3OD, 125 MHz) 30.4 (C'4), 34.8 (C'3), 41.8 (CH3), 118.2 (C'°), 120.5 (C4), 120.8 (C6), 121.2 (C2), 122.6 (C7), 135.4 (C9), 145.9 (C3), 149.1 (C'), 151.1 (C8), 152.4 (C"), 156.3 (C'2), 173.8 (C5).

6-te*Butyl-1-methyl-1H-9-oxa-1-aza-anthracene-2, lO-dione () 7-tert-Butyl-N-methyl-1-azaxanthonium chloride (0.36 g, 1.18 mmol) dissolved in H20 (10 cm3) was added in a dropwise fashion to a solution of potassium hexacyanoferrate (III) (1.16 g, 3.54 mmol) in HzO (6 cm3). The solution was cooled to approximately 0 °C and a solution of NaOH (0.85 g, 21.24 mmol) in H20 (10 cm3) added to the reaction mixture over a period of 20 mm. 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 CHCI3 (50 cm3) and partitioned with H20 (2 x 50 cm3). The organic phases were separated, dried over MgSO4 and the solvent removed under reduced pressure to yield the title cornpoundas a red solid (0.25 g, 0.87 mmol, 74%). (CDCI3, 500 MHz) 1.41 (9H, s, tBu), 3.76 (3H, s, Me), 6.54 (1H, d, 3 9.5, H2), 7.47 (1H, d, 3 8.5, H10), 7.79 (1H, dd, 3 9; 2, H9), 8.21 (1H, d, 3 9.5, H3), 8.29 (1H, d, 3 2, H7). & (CDCI3, 125 MHz) 28.5 (CH3), 31.6 (C'4), 35.2 (C13), 102.8 (C4), 116.0 (C2), 117.3 (C'°), 121.6 (C6), 122.9 (C7), 132.3 (C9), 135.7 (C3), 149.7 (C8), 152.0 (C'1), 156.5 (C'2), 162.3 (C'), 174.2 (C5).

m/z (ES) 284.3 (100%, M + H). HRMS (ES) 284.12809; C17H,803N, requires 284.12812, [M + HJ. Found: C, 71.82; H, 5.91; N, 4.90%; C,7H17N03 requires C, 72.07; H, 6.05; N, 4.94%.

6-tert'Butyl-2-chloro-9-oxa-anthracen-10-one () N,N-Dimethylaniline (0.3 cm3) was added to a solution of 6-tertbutyl-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 H20 (100 cm3) and the aqueous phase extracted with CH2CI2 (2 x 50 cm3). The combined organic phases were washed with aqueous K2C03 (0.1 M, 100 cm3), dried over K2C03, filtered and the filtrate concentrated under reduced pressure. The residue purified by chromatography on silica (gradient elution: Hexane to 10% EtOAc/Hexane, RF = 0.33, 10% EtOAc/Hexane) to yield the title compound as a pink solid (0.09g, 0.31 mmol, 49%). H (CDCI3, 500 MHz) 1.41 (9H, 5, tBU), 7.43 (1H, d, 3 8, H2), 7.54 (1H, d, 3 9, H'°), 7.86 (1H, dd, 3 9; 2.5, H9), 8.27 (1H, d, 3 2.5, H7), 8.65 (1H, d, 3 8, H3). &2(CDCI3, 125 MHz) 31.5 (C14), 35.1 (C'3), 115.6 (C4), 118.4 (C'°), 121.1 (C6), 121.9 (C2), 122.7 (C7), 134.1 (C9), 139.9 (C3), 148.8 (C8), 153.8 (C"), 155.6 (C'2), 159.7 (C'), 177.2 (C5).

2-Methyl-6-tributylstannanyl-pyridine (73 Following the procedure disclosed by U. S. Schubert et al. in Org. Lett., 2000, 2, 3373, frButyllithium (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 an/iydrous 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. H20 (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-tert'Butyl-2-(6-methyl-pyridin-2-yJ)-9-oxa-1-aza-anthracen-lO-one () Procedure A: St/lie Cross-Coupling 6-tertButyl-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 wfth 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 8 as a colourless solid (0.145 g, 0.422 mmol, 61 %); m.p. 191-192 °C; H NMR (CDCI3, 700 MHz) 6 1.37 (9H, s, Bu CH3), 2.60 (3H, 5, CH3), 7.18 (1H, d, 3= 8.0 Hz, H5'), 7.53 (1H, d, 3= 8.0 Hz, H'°), 7.69 (1H, t, 3= 8.0 Hz, H4'), 7.79 (1H, dd, 3= 8.0; 2.0 Hz, H9), 8.26 (1H, d, 3= 2.0 Hz, H7), 8.29 (1H, d, 3= 8.0 Hz, H3'), 8.54 (1H, d, 3= 8.5 Hz, H2), 8.74 (1H, d, 3= 8.5 Hz, H3); 3C NMR (CDCI3, 176 MHz, H decoupled 700 MHz) 6 24.8 (1C, CH3), 31.5 (3C, C'4), 35.0 (1C, C13), 116.4 (1C, C4), 118.3 (1C, C'°), 118.5 (1C, C2), 119.7 (1C, C5'), 121.3 (1C, C6), 122.7 (1C, C7), 124.9 (1C, C2'), 133.6 (1C, C9), 137.4 (1C, C4'), 138.2 (1C, C3), 148.0 (1C, C8), 153.6 (1C, C"), 154.2 (1C, C"), 158.6 (1C, C6'), 160.2 (1C, C'), 160.6 (1C, C'2), 177.8 (1C, C5); MS (ES) m/z 345.2 (100 %, [M + HJ); HRMS (ES) m/z found 345.1596, C22H2102N2 requires 345.1597, [M + HJ.

Procedure B: Suzuki-Miyaura Cross-Coupling 6-tertButyl-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 Schienk 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(tBu)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(tBU)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 H20 (50 ml), followed by brine (50 ml). The organic phase was dried over K2C03, 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 = 0.38 (Silica, CH3OH -CH2CI2, 9: 1 v/v). Character/sat/on as reported in procedure A. 2-(6-Bromomethyl-pyridin-2-yI)-6-teii'butyl-9-oxa-1-aza-anthracen-10-one () Procedure A: A stirred mixture of 6-tertbutyl-2-(6-methyl-pyridi n-2-yl)-9-oxa-1-aza-anthracen-lO-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 Cd4 (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 %); RF = 0.35 (Silica, CH2CI2, 100 v); m.p. 186-187 °C; H NMR (CDCI3, 500 MHz) 5 1.42 (9H, s, tBU CH3), 4.66 (2H, s, CH2Br), 7.56 (1H, d, J = 7.5 Hz, H5), 7.60 (HI, d, 3= 9.0 Hz, H'°), 7.85 (1H, dd, J-8.5; 2.5 Hz, H9), 7.90 (1H, t, 3= 8.0 Hz, H4'), 8.32 (1H, d, 3= 2.5 Hz, H7), 8.48 (1H, d, 3= 8.0 Hz, H3), 8.63 (1H, d, 3= 8.0 Hz, H2), 8.82 (1H, d, 3= 8.0 Hz, H3); 3C NMR (CDCI3, 126 MHz, H decoupled 500 MHz) 6 31.6 (3C, C'4), 34.0 (1C, CH2Br), 35.1 (1C, C'3), 116.8 (1C, C4), 118.3 (1C, C'°), 118.7 (1C, C2), 121.3 (1C, C6), 121.8 (1C, C5'), 122.8 (1C, C7), 125.1 (1C, C3'), 133.8 (1C, C9), 138.4 (1C, C4'), 138.6 (1C, C3), 148.2 (1C, C8), 154.0 (1C, C1'), 154.2 (1C, C"), 157.0 (1C, C6'), 160.0 (1C, C'), 160.2 (1C, C'2), 177.9 (1C, C5); MS (ES) m/z423.2 (100 %, [M + H]); HRMS (ES) m/zfound 423.0700, C22H20O2N2Br1 requires 423.0702, [M + H].

Procedure B: A solution of 6-tertbutyl-2-(6-dibromomethyl-pyridin-2-yl)-9-oxa-1-aza-anthracen-lO-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 CHCI3 (10 ml) and H20 (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, 0.09 5 mmol, 79 %). Character,ation as repotted in procedure A. 6-tert-Butyl-2-(6-dibromomethyl-pyridin-2-yI)-9-oxa-1-aza-anthracen-10-one (jQ) 6-teiButyl-2-(6-dibromomethyl-pyridin-2-yl)-9-oxa-1-aza-anth racen-10-one 10 was isolated using an identical procedure to that described for 2-(6-bromomethyl-pyridin-2-yl)-6-tert-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 %); R1 0.71 (Silica, CH2CI2, 100 v); H NMR (CDCI3, 500 MHz) 6 1.41 (9H, 5, tBu CH3), 6.78 (1H, S, CHBr2), 7.58 (1H, d, 3= 8.5 Hz, H'°), 7.84 (1H, dd, 3= 9.0; 2.5 Hz, H9), 7.92 (1H, d, 3= 8.0 Hz, H5'), 7.98 (1H, t, J= 8.0 Hz, H4'), 8.30 (1H, d, J= 2.5 Hz, H7), 8.50 (1H, d, 3= 7.5 Hz, H3'), 8.59 (1H, d, J= 8.0 Hz, H2), 8.81 (1H, d, 3= 8.0 Hz, H3); 3C NMR(CDCI3, 126 MHz, H decoupled 500 MHz) 6 31.6 (3C, C14), 35.1 (1C, C'3), 41.6 (1C, CHBr2), 116.9 (1C, C4), 118.3 (1C, C'°), 118.7 (1C, C2), 121.3 (1C, C6), 122.8 (1C, C7), 122.9 (1C, C3'), 123.6 (1C, C5'), 133.9 (1C, C9), 138.7 (1C, C3), 139.1 (1C, C'), 148.3 (1C, C8), 152.7 (1C, C2'), 154.2 (1C, C"), 159.0 (1C, C6'), 159.1 (1C, C'), 160.2 (1C, C'2), 177.8 (1C, C5); MS (ES) m/z503.2 (100 %, [M + H]).

(4,7-bis-tert Butoxycarbonylmethyl-1,4,7, 1O-tetraaza-cyclododec-1-yl)-acetic acid tertbutyl ester (16) 0 0 This cyclen derivative was prepared according to the procedure described by 0. Reany et al., 3. Chem. Soc. Perk/n. Trans 2., 2000, 1819.

A mixture of cyclen (2.54 g, 14.7 mmol), tert-butyl bromoacetate (8.67 g, 44.2 mmol) and NaHCO3 (3.72 g, 44.2 mmol) in anhydrousCH3CN (75 ml) was stirred at rt, 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: CH2C12, utilising 0.1 % CH3OH increments) to yield the title compound 16 as a colourless crystalline solid (2.41 g, 4.68 mmot, 32 %); m.p. 179-181 °C; H NMR (CDCI3, 500 MHz) a 1.47 (27H, S, CH3), 2.88 (12H, br s, cyclen CH2), 3.11 (4H, br s, cyclen CH2), 3.30 (2H, s, CH2CO21Bu), 3.39 (4H, s, CH2CO21Bu), 10.04 (1H, br s, NH); 3C NMR (CDCI3, 125 MHz, H decoupled 500 MHz) 6 28.4 (9C, tBOC 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, tBoc(q)), 82.1(1C, tBoc(q)), 169.9 (2C, C = 0), 170.8 (1C, C = 0); MS (ES) m/z515.6 (100 %, [M + H]).

{4,7-bis-ten' Butoxycarbonylmethyl-1O-[6-(6-teit-butyl-1O-oxo-1OH-9-oxa- 1-aza-anthracen-2-yI)-pyridin-2-ylmethyl]-1,4,7, 1O-tetraaza-cyclododec-1-yI}-acetic acid tert-butyl ester ((L1]) *oD / N\ o-J 00 \/ A stirred mixture of (4, 7-bis-terbutoxycarbonylmethyl-1,4,7, 1O-tetraaza-cyclododec-1- yl)-acetic acid tert butyl ester 16 (0.032 g, 0.062 mmot), 2-(6-bromomethyl-pyridin-2-yl)-6-tertbutyl-9-oxa-1-aza-anthracen-10-one 9 (0.024 g, 0.057 mmol) and Cs2CO3 (0.026 g, 0.080 mmol) in an/iydrous 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 EL'] as a pale yellow crystalline solid (0.035 g, 0.042 mmol, 74 %); RF = 0.22 (Alumina, CH2CI2 -CH3OH, 19: 1 v/v); H NMR (CDCI3, 700 MHz) 6 1.29 (9H, s, tBu CH3), 1.32 (18Ff, br s, 60 CH3), 1.35 (9H, br tBOC CH3), 2.71 (8H, br s, cycien Cl-i2), 3.24 (8Ff, br s, cyclen CH2), 3.50 (4H, br s, CH2CO2tBU), 3.55 (2H, br 5, CH2CO2tBu), 3.69 (2Ff, s, CH2 pyridine), 7.25 (1H, d, 3= 8.0 Hz, H5'), 7.45 (1H, d, 3= 8.5 Hz, H10), 7.69 (1Ff, t, 3= 8.0 Hz, H4'), 7.71 (1Ff, dd, 3= 9.0; 3.0 Hz, H9), 8.17 (1Ff, d, 3= 3.0 Hz, H7), 8.32 (1H, d, 3= 8.0 Hz, H3'), 8.36 (1H, d, 3= 7.0 Hz, H2), 8.64 (1H, d, 3= 8.0 Hz, H3); 13C NMR (CDCI3, 176 MHz, H decoupled 700 MHz) 6 28.3 (3C, C'4), 31.5 (6C, tBoc CH3), 35.1 (3C, tBoc CH3), 50.6 (2C, cyclen CH2), 51.8 (2C, cyclen CH2), 56.1 (2C, cycten CH2), 56.7 (2C, cyclen CH2), 57.1 (1C, CH2 pyridine), 82.4 (4C, C1Bu), 116.4 (1C, C4), 118.2 (1C, C'°), 118.3 (1C, C2), 121.0 (1C, C3'), 121.2 (1C, C6), 122.6 (1C, C7), 126.2 (1C, C5'), 133.6 (1C, C9), 137.4 (1C, C'), 138.2 (1C, C3), 148.0 (1C, C8), 153.6 (1C, C"), 154.2 (1C, C"), 158.6 (1C, C6'), 160.2 (1C, C'), 160.6 (1C, C'2), 177.8 (1C, C5); MS (ES) m/z857.5 (100 %, [M + H]); HRMS (ES) m/zfound 857.5176 [M + H] C48H6908N6 requires 857.5171.

(EuL1] N/N /

U

O'' J) Nfl o-\ \/ A so'ution of {4,7-bis-te,Dbutoxycarbonyf methyl-10-[6-(6-terbutyf-lO-oxo-lOfl-9-oxa- 1-aza-anthracen-2-yl)-pyridin-2-ylmethyl]-1,4,7, 10-tetraaza-cyclododec-1-yl}-acetic acid tertbutyl 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 tertbutyl alcohol.

The desired ligand, as its TFA salt, was examined by H NMR to ensure complete ester hydrolysis, with the material used immediately for complexation.

The hydrolysed ligand was dissolved in CH3OH -H20 (1:1 v/v, 4 ml) and Eu(OAc)3.6H20 (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, then stirred and heated at 80 °C, for h. The reaction mixture was allowed to cool to rt before raising the pH of the solution to 10.0 using dilute KOH. 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 HCI. 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: CH3O1-I, 8: 2 v/v) to yield the title complex (EuL1] as a colourless solid (0.024 g, 0.028 mmol, 68 %); RF = 0.41 (Alumina, CH2CI2 -CH3OH, 7:3 v/v); A1. (H20) = 356 nm; i (H20) = 1.00 ms; T (D20) = 1.34 ms; 4Eu (H20; pH 7.4; 365 nm) = 14 %.

Examnle 2: Synthesis of L4 and FEuL41 (see schemes 1 to 3 and 5) 1,4,7-Tetraaza-cyclododecane-1,4,7-tricarboxylic acid tn-te,t-butyt ester (17) This cyclen derivative was prepared by using the procedure described by S. Brandes et al., BulL Soc. Chim. Fr., 1996, 133, 65.

A solution of di-tebutyl dicarbonate (6.08 g, 27.8 mmol) in anhydmus 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 %); RF = 0.29 (Silica, CH2CI2 -CH3OH, 9: 1, v/v); 1H NMR (CDCI3, 500 MHz) 6 1.42 (18H, s, tBOC CH3), 1.44 (9H, s, tBoc CH3), 2.81 (4H, br s, cyclen CH2), 3.28 (8H, br s, cyclen CH2), 3.60 (4H, br s, cyclen CH2); 3C NMR (CDCI3, 125 MHz, H decoupled 500 MHz) 5 28.9 (6C, tBoc CH3), 29.0 (3C, tBoc CH3), 46.1 (2C, cyclen CH2), 49.9 (2C, cyclen CH2), 51.2 (4C, cyclen CH2), 79.4 (2C, tBOC(q)), 79.6 (1C, tBOC(q)), 155.8 (2C, tBoc C = 0), 156.0 (1C, tBoc C = 0); MS (ES) m/z 473.3 (100 %, [M + H]); HRMS (ES) in/z found 473.3330 {M + H] C23H4506N4 requires 473.3333.

1O-(6-(6-tert-Butyl-1O-oxo-1OH-9-oxa-1-aza-anthracen-2-yI)-pyridifl-2- ylmethyl] -1,4,7, iO-tetraaza-cyclododecane-1,4,7-tricarboxylic acid tn-tert-butyl ester (18) CN ND\N< çOJO N \/ A stirred mixture of 1,4,7-tetraaza-cyclododecane-1,4,7-tricarboxyl ic acid tn-teibutyl ester 17 (0.117 g, 0.248 mmol), 2-(6-bromomethyl-pyridin-2-yl)-6-tertbutyl-9-oxa-1-aza-anthracen-lO-one 9 (0.100 g, 0.236 mmol) and K2C03 (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 18 as a pale yellow coloured solid (0.173 g, 0.212 mmol, 90 %); RF = 0.70 (Silica, CH2CI2 -CH3OH, 49 1, v/v); H NMR (CDCI3, 700 MHz) 6 1.29 (9H, s, tBu CH3), 1.32 (9H, s, tBoc CH3), 1.35 (18H, br s, tBOC CH3), 2.71 (4H, br 5, 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, 3 = 8.0 Hz, H5'), 7.45 (1H, d, 3= 8.5 Hz, H'°), 7.69 (1H, t, .1= 8.0 Hz, H4'), 7.71 (1H, dd, 3 = 9.0; 3.0 Hz, H9), 8.17 (lii, d, 3= 3.0 Hz, H7), 8.32 (1H, d, J= 8.0 Hz, H3'), 8.36 (1H, d, 3 7.0 Hz, H2), 8.64 (1H, d, 3 = 8.0 Hz, H3); 3C NMR (CDCI3, 176 MHz, 1H decoupled 700 MHz) ô 28.6 (9C, tBoc CH3), 31.5 (3C, C'4), 35.0 (1C, C'3), 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, tBoc(q); C'4), 116.4 (1C, C4), 118.2 (1C, C'°), 118.3 (1C, C2), 121.0 (1C, C3'), 121.2 (1C, C6), 122.6 (1C, C7), 126.2 (1C, C5'), 133.6 (1C, C9), 137.4 (1C, C4'), 138.2 (1C, C3), 148.0 (1C, C8), 153.6 (1C, C"), 154.2 (1C, C"), 155.9 (3C, Boc C = 0), 158.6 (1C, C6'), 160.2 (1C, C1), 160.6 (1C, C'2), 177.8 (1C, C5); MS (ES) m/z 815.4 (100 %, [M + HJ); HRMS (ES) m/z found 815.4710 [M + HJ C45H5308N6 requires 815.4710.

6-te*Butyl-2-(6-( 1,4,7,1O-tetraaza-cyclododec-1-ylmethyl)-pyricjin-2-yI]- 9-oxa-1-aza-anthracen-1O-one (19) c:D< H' \I "H oo A solution of 10-[6-(6-teiDbutyl-10-oxo-1OH-9-oxa-1-aza-anthracen-2-yl) -pyridin-2-ylmethyl]-1,4,7, 10-tetraaza-cyclododeca ne-1,4, 7-tricarboxylic acid tn-tertbutyl 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 tetbutyl 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 K2C03, 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 %); H NMR (CDCI3, 500 MHz) 6 1.36 (9H, s, tBU 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, H5'), 7.54 (1H, d, 3= 9.0 Hz, H'°), 7.79 (1H, dd, 3 = 8.5; 2.5 Hz, H4'), 7.81 (1H, t, 3= 7.5 Hz, H9), 8.26 (1H, d, J= 2.5 Hz, H7), 8.38 (1H, d, 3= 7.5 Hz, H3'), 8.62 (1H, d, J= 8.0 Hz, H2), 8.73 (1H, d, 3= 8.0 Hz, H3); 3C NMR (CDCI3, 126 MHz, H decoupled 500 MHz) 6 31.5 (3C, C'4), 35.0 (1C, C'3), 45.3 (2C, cyclen CH2), 46.6 (2C, cyclen CH2), 47.4 (2C, cyclen CH2), 51.8 (2C, cyden Cl-I2), 60.8 (1C, CH2 pyridine), 116.4 (1C, C4), 118.2 (1C, C'°), 118.6 (1C, C2), 121.0 (1C, C5'), 121.3 (1C, Cb), 122.6 (1C, C7), 124.6 (1C, C2), 133.7 (1C, C9), 137.8 (1C, C4'), 138.1 (1C, C3), 148.1 (1C, C8), 153.5 (1C, C"), 154.2 (1C, C"), 159.7 (1C, C6'), 160.2 (1C, C'), 160.6 (1C, C'2), 178.0 (1C, C5); MS (ES) m/z289.4 (100 %, [M + Zn]2); HRMS (ES) m/zfound 515.3130 [M + H]; C30H3902N6 requires 515.3129.

(S)-N-2-ChoroethanoyI-2-phenyIethyamine This compound was prepared according to the procedure described by R. S. Dickins et at., Cheni. Eur. J., 1999, 5, 1095.

Chioroacetyl chloride (10.5 g, 7.40 ml, 93.0 mmol) was added dropwise to a stirring solution of (S)-1-phenylethylamine (9.40 g, 10.0 ml, 77.50 mmol) and anhydrous NEt3 (13.0 ml, 93.00 mmol) in anhydrousdiethyl 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 H20 (150 ml) followed by HCI(aq) (0.1 M, 150 ml).

The organic phase was separated, dried over Na2504, 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; H NMR (CDCI3, 500 MHz) 6 1.56 (3H, d, 3= 6.5 Hz, CH3), 4.05 (2H, dd, 3= 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); 3C NMR (CDCI3, 125 MHz, 1H decoupled 500 MHz) 6 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 = 0); C,0H,2CINO (%): calcd C 60.80, H 6.12, N 7.08; found C 60.06, H 6.15, N 6.94.

(R-N-2-ChIoroethanoyI-2-phenyJethyIamine An analogous procedure to that described for 2-chloro-N-[(S)-methylbenzyljethanamide was followed using chloroacetyl chloride (5.25 g, 3.70 ml, 46.5 mmol) and a stirring solution of (R)-1-phenylethylamine (4.70 g, 5.00 ml, 38.8 mmol) and anhydrous NEt3 (6.51 ml, 46.52 mmol) in an/iydrous 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)-methy/benzyl]ethanamide.

2-{4-(6-(6-tert-Butyl-lO-oxo-1OH-9-oxa-1-aza-anthracen-2-yI)-pyridin-2- ylmethyl]-7,1O-bis-[((S)-1-phenyl-ethylcarbamoyl)-methyl]-1,4, 740-tetraaza-cyclododec-1-yI}-N-((S)-1-phenyl-ethy!)-acetamide ([La])

H NH

A stirred mixture of 6-tertbutyl-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 K2C03 (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 % Cl-l3OH increments) to yield the title compound(L4] as a free flowing pale yellow coloured solid (0.074 g, 0.0741 mmol, 64 %); RF = 0.21 (Alumina, CH2CI2 -CH3OH, 19: 1, v/v); H NMR (CDCI3, 700 MHz) 6 1.38 (9H, S. tBu CH3), 1.45 (9H, br s, CH3), 2.60 (8F-l, 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, H5'), 7.58 (1H, d, 3 9.0 Hz, H10), 7.70 (1H, br s, H4'), 7.82 (1H, dd, 3= 9.0; 2.0 Hz, H9), 8.28 (11-4, d, 3= 3.0 Hz, H7), 8.43 (1H, br s, H3'), 8.62 (1H, d, 3= 8.5 Hz, H2), 8.77 (1H, d, 3= 8.5 Hz, H3); MS (ES) m/z998.6 (100%, [M + HJ); HRMS (ES) m/zfound 998.5667 {M + H]; C60H7205N9 requires 998.5651.

[EuL4] \/ 3C

NHO

A solution of 2-{4-[6-(6-teiD-butyl-lO-oxo-1OH-9-oxa-1-aza-anthracen-2-yl)-pyridin-2- ylmethylj-7, 10-bis-[((S)-1-phenyl-ethylcarbamoyl)-methyl]-1,4,7, 10-tetraaza-cyclododec -1-yl}-N-((S)-1-phenyl-ethyl)-acetamide (ii] (0.040 g, 0.040 mmol) and Eu(OTf)3.6H20 (0.034 g, 0.041 mmol) in anhydrousCH3CN (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 mm. 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 DOWEX 1 x 8 200-400 mesh Cl' resin. The solid material was dissolved in a mixture of H20 -CH3OH (1:1 v/v1 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 %); Amax (1120) = 356 nm; i (H20) = 1.00 ms; i (D20) = 1.34 ms; (1120; pH 7.4; Aexc 365 nm) = 24 %; HPLC (Method A) = 10.9 mm.

Examvle 3: Synthesis of cvcten 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 aI. in Siocrg. Med. Cliem. Lett., 2005, 15, 4417. Compound (28) was prepared by the procedure disclosed by L-0. Pâlsson et al. in Dalton Trans., 2007, 5726.

(.S)-N-Ethanoyl-1-(4-bromophenyl)ethylamine (24) Br Acetyl chloride (2.40 ml, 30.0 mmol) was added dropwise to a stirring solution of (5)- 1-(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 1120 (150 ml) and then 0.1 M HCI(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 title compound 24 as colourless needle-like crystals (4.56 g, 18.8 mmol, 76 %). m.p. 127-129 °C; H NMR (CDCI3, 500 MHz) 6 1.47 (311, d, J = 7.0 Hz, CH3), 2.00 (3H, s, C(0)CH3), 5.08 (1H, q, J= 7.0 Hz, CH), 5.71 (1H, br S. NH), 7.20 (2H, d, J= 8.5 Hz, 112), 7.46 (2H, d, 3= 8.5 Hz, H3); 3C NMR (CDCI3, 125 MHz, 1H decoupled 500 MHz) 6 21.9 (1C, CH3), 23.7 (1C, C(0)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 = 0); MS (ES) m/z 263.9 (100 %, [M + NaJ); HRMS (ES) m/zfound 263.9994 [M + NaJ C,0H,20N79Br23Na requires 263.9995; C10H12BrNO (%): calcd C 49.61, H 5.00, N 5.79; found C 49.48, H 4.98, N 5.52.

($)-N-Ethanoyl-1-(4-cyanophenyl)ethylamine (25) NC' A stirred solution of (S)-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 it followed by the removal of solvent under reduced pressure. The residue was taken up into 6 M HCI(aq) (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 H20 (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; H NMR (CDCI3, 500 MHz) 6 1.47 (3H, d, 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, .1 = 6.5 Hz, NH), 7.43 (2H, d, J = 8.0 Hz, H2), 7.63 (2H, d, 3= 8.0 Hz, H3); 3C NMR (CDCI3, 125 MHz, 1H decoupled 500 MHz) 5 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, C'), 169.7 (1C, C = 0); MS (ES) m/z189.1 (100 %, [M + H]); HRMS (ES) m/zfound 189.1023 [M + H] C11H,30N2 requires 189.1022; C,,H,2N20 (%): calcd C 70.19, H 6.43, N 14.88; found C 70.05, H 6.49, N 15.00.

N-US)-1-(4-Bromo-phenyl)-ethyl]-2, 2, 2-trifluoro-aceta mide (27) A solution of (S)-(-)-a-methylbenzylamine (7.81 g, 39.1 mmol) in anhydrousCH2Cl2 (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 it 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 it then stir for 16 h. 1 M NaHSO3(aq) (100 ml) was introduced into the reaction mixture and the organic phase washed with H20 (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) 6 1.57 (3H, d, 3= 7.0 Hz, CH3), 5.10 (1H, q, 3= 7.0 Hz, CH), 6.57 (1H, br 5, NH), 7.20 (2H, d, 3= 8.0 Hz, H3), 7.51 (2H, d, 3= 8.0 Hz, H2); 13C NMR (CDCI3, 125 MHz, 1H decoupled 500 MHz) 6 21.2 (1C, CH3), 49.5 (1C, CII), 122.3 (1C, C1), 128.1 (2C, C3), 132.4 (2C, C2), 140.2 (1C, C4), 156.7 (1C, C = 0); 9F NMR (CDCI3, 470 MHz, 1H decoupled 500 MHz) 6 -76.2 (3F, s, CF3); MS (ES) m/z 318.1 (100 %, [M + Na]); HRMS (ES) m/z found 317.9713 [M + Na] C10H9ON79BrF323Na 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-(($)-1-(4-Cyano-phenyl)-ethyl]-2,2,2-trifluoro-acetam ide (28) NCF3

NC

A stirring solution of N-[(S)-1-(4-bromo-phenyl)-ethylj-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 HCI(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 28 as a colourless solid (2.86 g, 11.8 mmol, 60 %); RF = 0.34 (Silica, CH2CI2, 100 v); m.p. 98-99 °C; 1H NMR (CDCI3, 500 MHz) 6 1.53 (3H, d, 3= 7.5 Hz, CH3), 5.10 (1H, q, 3= 7.0 Hz, CH), 7.36 (1H, d, 3= 7.5 Hz, NH), 7.41 (2H, d, 3= 8.0 Hz, H2), 7.60 (2H, d, 3= 8.0 Hz, H3); 13C NMR (CDCI3, 125 MHz, H decoupled 500 MHz) 6 21.3 (1C, CH3), 49.9 (1C, CH), 111.7 (1C, C'), 118.7 (1C, CN), 127.1 (2C, C3), 132.9 (2C, C2), 147.1 (1C, c4), 156.9 (1C, C = 0); 9F NMR (CDCI3, 470 MHz, 1H decoupled 500 MHz) 6 -75.7 (3F, s, CF3); MS (ES-) m/z 241.2 (100 %, [M -H]); HRMS (ES) m/z found 241.0591 [M -H] C11H80N2F3 requires 241.0594; C,,H9F3N20 (%): calcd C 54.55, H 3.75, N 11.57; found C 54.48, H 3.85, N 11.65.

(S)-4-(1-Aminoethyl)benzoic acid (26) NH2.HCl Procedure A: A solution of (S)-N-ethanoyl-1-(4-cyanophenyl)ethylamine 25 (2.00 g, 10.6 mmol) in 6 M HCI(aq) (35 ml) was heated at reflux, for 72 h. The solution was allowed to cool to rt 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 (D20, 500 MHz) 6 1.52 (3H, d, 3 7.0 Hz, CH3), 4.48 (1H, q, 3= 7.0 Hz, CH), 7.37 (2H, dd, 3= 6.5; 1.5 Hz, H3), 7.86 (2H, dd, 3= 7.0; 2.0 Hz, H2); 3C NMR (D20, 125 MHz, H decoupled 500 MHz) 6 19.4 (1C, CH3), 50.8 (1C, CH), 126.9 (2C, C3), 130.6 (2C, C2), 132.3 (1C, C'), 143.1 (1C, c), 170.2 (1C, C = 0); MS (ES) m/z164.4 (100%, {M -Hf); HRMS (ES) m/zfound 164.0715 {M -Hf C9H1002N requires 164.0717.

Procedure B: An analogous procedure to that described in procedure A was followed using N.[(S)-1-(4-cyano-phenyl)-ethy-2,2,2-trifluoro-acetamide 28 (2.67 g, 11.0 mmol) in 6 M HCI(aq) (50 ml) for 72 h. The procedure yielded the hydrochloride salt of (S)-4-(1-aminoethyl)benzoic acid 26, as a colourless crystalline solid, in quantitative yield.

Character/sat/on was Identical to that reported in procedure A. ($)-Methyl-4-( 1-aminoethyl)benzoate (29) 2fNH Concentrated HCI(aq) (12 M, 2.00 ml) was added to a stirring solution of (S)-4-(1-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. H NMR (CH3OD, 500 MHz) 5 1.66 (3H, d, 3 = 7.0 Hz, CH3), 3.93 (3H, s, CO2CH3), 4.58 (1H, q, 3= 7.0 Hz, CH), 7.61 (2H, d, 3= 8.5 Hz, H3), 8.10 (2H, d, J = 8.5 Hz, H2); 3C NMR (CH3OD, 125 MHz, H decoupled 500 MHz) 5 19.5 (1C, CH3), 50.8 (1C, CH), 51.7 (1C, CO2CH3), 126.9 (2C, C3), 130.2 (2C, C2), 133.0 (1C, C'), 143.5 (1C, C4), 166.7 (1C, C = 0); MS (ES) m/z 180.0 (100 %, [M + H]); HRMS (ES) m/zfound 180.1019 [M + H]C,oH,4NO2 requires 180.1019.

4-[(S)-1-(2-Chloro-acetylami no)-ethyl]-benzoic acid methyl ester (30) 2N)cl 0 Chloroacetyl chloride (0.540 g, 0.380 ml, 4.78 mmol) was added dropwise to a stirring solution of (S)-methyl-4-(1-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 H20 (150 ml) and then 0.1 M HCI (aq) (150 ml). The organic phase was separated, dried over K2C03, filtered and the filtrate concentrated under reduced pressure to afford a crude solid. Recrystallisation from warm diethyl ether yielded the title compound3o as colourless solid (0.704 g, 2.76 mmol, 75 %); H NMR(CDCI3, 500 MHz) 5 1.55 (3H, d, 3= 7.5 Hz, CH3), 3.92 (3H, s, CO2CH3), 4.08 (2H, dd, 3= 15; 6.5 Hz, CH2), 5.18 (1H, q, 3= 7.0 Hz, CH), 6.85 (lI-f, d, 3= 6.0 Hz, Nt-f), L39 (21-f, d, 3= 8.5 Hz, H3), 8.03 (2H, d, 3 = 8.0 Hz, H2); 13C NMR (CDCI3, 125 MHz, H decoupled 500 MHz) 5 22.0 (1C, CH3), 42.8 (1C, CH2), 49.3 (1C, CH), 52.4 (1C, CO2CH3), 1263 (2C, C3), 129.7 (1C, C4), 130.4 (2C, C2), 147.7 (1C, C'), 165.4 (1C, C(0)CH2), 167.0 (1C, C(0)CH3); MS (ES) m/z256.0 (100 %, [M + H]); HRMS (ES) m/zfound 256.0735 [M + H] C,2H,503N,35Cl, requires 256.0735.

1,4,7,1O-Tetraaza-cyclododecane-1,7-dicarboxylic acid dibenzyl ester (31) This cyclen derivative was prepared according to the procedure described by Z. Kovacs et al., J. Chein. Soc., Che,n. 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 H20 -1,4-dioxane (50: 20 v/v1 70 ml) and the pH adjusted to pH 2.5 by the addition of conc. HCI(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 H20 (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) 6 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, 5, Cbz CH2), 7.33-7.40 (1OH, m, Ph); 3C NMR (CDCI3, 125 MHz, H decoupled 500 MHz) 6 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 CU2), 50.9 (1C, cyclen CH2), 68.1 (2C, Cbz CH2), 68.2 (1C, cyclen CU2), 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 = 0), 156.5 (1C, C = 0); MS (ES) m/z441.4 (100 %, {M + H]).

4,1O-bis-(((S)-1-Phenyl-ethylcarba moyl)-methyl] -1,4,7, 1O-tetraaza-cyclododecane-1,7-dicarboxylic acid dibenzyl ester (32) HN0 (OBn BnOI

NH

A stirred mixture of 1,4,7, 10-tetraaza-cyclododecane-1, 7-dica rboxylic acid dibenzyl ester 31 (1.34 g, 3.05 mmol), (5)-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 H20 (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; H NMR (CDCI3, 500 MHz) 6 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 (20H, m, Ph), 7.59 (2H, br s, NH); 3C NMR (CDCI3, 125 MHz, H decoupled 500 MHz) 6 22.0 (2C, CH3), 48.1 (1C, CH), 48.7 (1C, CH), 55.0 -56.4 (4C, br 5, 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/z763.0 (100 %, [M + HJ); HRMS (ES) m/zfound 763.4187 [M + HJ GH55N6O6 requires 763.4178.

N-((S)-1-Phenyl-ethyl)-2-(7-(((5)-1-phenyl-ethylcarbamoyl)-methyl]- 1,4,7,1O-tetraaza-cyclododec-1-yI)-acetam ide (33) 1-NN

N H'

ONH

4, 10-bis-[((S)-1-Phenyl-ethylcarbamoyl)-methyljJ-1,4,7, 10-tetraaza-cyclododecane-1,7-dicarboxylic acid dibenzyl ester 32 (3.14 g, 4.12 mmol) in CH3OH -H20 (40: 20 v/v, ml) was shaken in a Parr hydrogenation flask at 40 psi H2 over Pd(OH)ilC (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; H NMR (CD3OD, 500 MHz) 6 1.46 (6H, d, 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); 3C NMR (CDCI3, 125 MHz, 1H decoupled 500 MHz) 6 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(olm)), 130.5 (2C, Ph(p)), 145.8 (2C, Ph(q)), 172.7 (2C, C = 0); MS (ES) m/z495.3 (100 %, [M + H]); HRMS (ES) m/zfound 495.3447 {M + H] C28H43N602 requires 495.3442.

4,1O-bis-[((S)-1-Phenyl-ethylcarbamoyl)-methyl] -1,4,7, 1O-tetraaza-cyclododecane-1-carboxylic acid tet-butyI ester (37)

N N

This cyclen derivative was prepared following the procedure described by L-O. Pälsson et al., Dalton Trans., 2007, 5726.

A solution of di-tertbutyl 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)- 1-phenyl-ethyl)-2-(7-[(($)-1-phenyl-ethylcarbamoyl)-methylj-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 %); RF = 0.46 (Alumina, CH2CI2 -CH3OH, 49:1 v/v); m.p. 98-101 °C; H NMR (CDCI3, 500 MHz) 6 1.39 (9H, s, tBoc 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) 6 22.0 (2C, CFI3), 28.8 (3C, tBoc CH3), 48.7 (2C, CH), 49.0-53.8 (8C, br m, cyclen CH2), 59.6 (2C, CH2CO), 80.3 (1C, CtBU), 126.8 (4C, Ph(o/m)), 127.5 (4C, Ph(o/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] C33H5104N6 requires 595.3966.

4, lO-bis-[((S)-1-Phenyl-ethylcarbamoyl)-methyl]-1,4,7, 1O-tetraaza-cyclododecane-1,7-dicarboxylic acid di-te*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.

ExamDle 4: Synthesis of EuL8 (see scheme 7) 7-(6-(6-tert-Butyl-1O-oxo-1OH-9-oxa-1-aza-anthracen-2-yI)-pyridin- 2ylmethyl] -4,1O-bis-(((S-1-phenyI-ethylcarbamOyI)-methyI]-1,4,7,1O-tetraaza-cyclo dodecane-1-carboxylic acid te,t-butyl ester (39) HN0 NN N<

ND

A stirring mixture of 4, 10-bis-[((S)-1-phenyl-ethylcarbamoyl)-methyl]-1,4,7, 10- tetraaza-cyclododecane-1-carboxylic acid tert-butyl ester 33 (0.325 g, 0.547 mmol), 2- (6-bromomethyl-pyridin-2-yl)-6-teiDbutyl-9-oxa-1-aza-anthracen-10-one 9 (0.254 g, 0.601 mmol) and K2C03 (0.113 g, 0.821 mmol) in an/iydrous 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 39 as an orange crystalline solid (0.379 g, 0.404 mmol, 74 %); RF = 0.74 (Alumina, CH2CI2 -CH3OH, 49 1 v/v); m.p. 87-89 °C; 1H NMR (CDCI3, 500 MHz) ô 1.38 (18H, br s, tBu CH3; tBoc CH3), 1.46 (611, d, I = 4.0 Hz, CH3), 2.61 (8H, br 5, cyclen CH2), 2.82 (4H, br s, cyclen CH2), 3.07 (4H, br s, cyclen CH2), 3.33 (2H, br s, CH2CO), 3.40 (211, br s, CH2CO), 3.61 (2H, br 5, CH2 pyridine), 5.12 (2H, br S. CH), 7.19 (1H, d, 3-8.0 Hz, H5'), 7.26 (1OH, br s, Ph), 7.46 (1H, br s, NH), 7.58 (1H, d, J= 8.5 Hz, H'°), 7.64 (1H, t, J= 8.0 Hz, H4'), 7.74 (1H, br s, NH), 7.82 (1H, dd, 3= 9.0; 2.5 Hz, H9), 8.29 (1H, d, J= 2.5 Hz, H7), 8.41 (1H, d, 3 = 8.0 Hz, H3), 8.46 (1H, d, 3 = 8.0 Hz, H2), 8.78 (1H, d, 3 = 8.0 Hz, H3); 13C NMR (CDCI3, 125 MHz, H decoupled 500 MHz) ó 21.3 (1C, CH3), 22.0 (1C, CH3), 28.7 (3C, tBOC 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, tBOC(q)), 116.6 (1C, C4), 118.3 (1C, C10), 121.1 (1C, C3'), 121.3 (1C, C6), 122.7 (1C, C7), 124.8 (1C, C5'), 126.5 (1C, P(cj/m)), 126.8 (1C, P(o/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, C4'), 138.5 (1C, C3), 143.0 (1C, Ph(q)), 143.8 (1C, Ph(q)), 148.2 (1C, C8), 153.8 (1C, C"), 154.2 (1C, C'1), 156.1 (1C, BOC C = 0), 158.0 (1C, C6'), 160.1 (1C, C1), 160.3 (1C, C'2), 170.1 (2C, amide C = 0), 177.8 (1C, C5); MS (ES) m/z937.5 (100 %, [M + t-1]); HRMS (ES) m/zfound 937.5331 [M + HJ C55H6905N8 requires 937.5334.

2-{4-(6-(6-tert'Butyl-10-oxo-1OH-9-oxa-1-aza-anthracen-2-yl)-pyridin- 2ymethyl]-7-(((S)-1-phenyl-ethylcarbamoy)-niethyl]-1,4,7, 10-tetraaza-cyclododec-1-yI}-N-((S)-1-phenyI-ethy)-acetamide (40) (also named L8)

HN

IN N

N N

H'\_J), N" \

ONH

A solution of 7-[6-(6-tertbutyl-lO-oxo-10H9-oxa-1-aza-anthracen-2-yl)-pyridin- 2ylmethyl]-4, 10-bis-[((S)-1-phenyl-ethylcarbamoyl)-methyl] 1,4,7, 10-tetraaza-cyclo dodecane-1-carboxylic acid terbutyl ester 39 (0.320 g, 0.341 mmcl) 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 te,Dbutyl 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 K2C03, filtered and the filtrate concentrated under reduced pressure to yield the title compound 40 as an orange coloured crystalline solid (0.254 g, 0.304 mmol, 89 %); m.p. °C; H NMR (CDCI3, 500 MHz) 6 1.38 (9H, s, tBu CH3), 1.47 (6H, d, 3= 7.0 Hz, CH3), 2.58 (8H, br s, cyclen CH2), 2.64 (8H, br s, cyclen CH2), 3.09 (4H, q, 3= 17.0 Hz, CH2CO), 3.60 (1H, d, 3 = 15.0 Hz, CH2 pyridine), 3.68 (1H, d, 3 = 15.0 Hz, CH2 pyridine), 5.14 (2H, q, 3= 7.5 Hz, CH), 7.16 (3H, q, 3= 8.0 Hz, H5': Ph(p)), 7.24 (4H, t, 3 = 7.5 Hz, Ph(m)), 7.30 (4H, d, 3 = 7.5 HZ, Ph(O)), 7.56 (1H, d, 3 9.0 Hz, H'°), 7.62 (1H, t, 3= 7.5 Hz, H4'), 7.82 (1H, dd, 3= 8.5; 2.0 Hz, H9), 8.06 (2H, d, 3= 8.5 Hz, NH), 8.28 (1H, d, 3= 2.5 Hz, H7), 8.37 (1H, d, 3= 7.5 Hz, H3'), 8.44 (1H, d, J= 8.5 Hz, H2), 8.77 (1H, d, 3 = 8.5 Hz, H3); 3C (CDCI3, 125 MHz, H decoupled 500 MHz) 6 21.7 (2C, CH3), 31.5 (3C, C'4), 35.0 (1C, C'3), 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; C'°), 121.0 (1C, C3'). 121.3 (1C, c6), 122.7 (1C, C7), 125.0 (1C, C5'), 126.7 (4C, Ph(O)), 127.5 (2C, Ph(p)), 128.8 (4C, Ph(m)), 133.8 (1C, C9), 137.6 (1C, C4'), 138.5 (1C, C3), 143.5 (2C, Ph(q)), 148.2 (1C, C8), 153.6 (1C, C"), 154.2 (1C, C"), 158.0 (1C, C6'), 160.2 (1C, C'), 160.3 (1C, C12), 170.8 (2C, amide C=O), 177.8 (1C, C5); MS (ES) m/z837.5 (100 %, [M + H]); HRMS (ES) m/zfound 837.4817 [M + H] C50H6104N8 requires 837.4810.

[EuL8] 3+ H20 ___L-N-' 3 C1 H' O\NH)K' \/ A stirring solution of 2-{4-{6-(6-tert-butyl-10-oxo-10H9-oxa-1-aza-anthracen-2-yl)- pyridin-2ylmethyl] -7-[((S)-1-phenyl-ethylca rbamoyl)-methyl]-1,4,7, 10-tetraaza-cyclododec-1-yl}-Ab((S)-1-phenyl-ethyl)-acetamide 40 (0.020 g, 0.024 mmol) and Eu(OTf)3.6H20 (0.017 g, 0.024 mmol) in an/iydrousCH3CN (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 mm. 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 DOWEX 1 x 8 200-400 mesh Cl' resin. The solid material was dissolved in a mixture of 1-120 -CFI3OH (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 %); Amax (F120) 355 nm; T (1120) = 0.36 ms; HPLC (Method A) = 11.0 mm.

Example 5: Synthesis of L! and EuL (see scheme 7) 4-(($)-1-(2-{7-(6-(6-tert-Butyl-1O-oxo-1OH-9-oxa-1-aza-anthracen-2-yl)- pyridin-2-ylmethyl]-4, 1O-bis-(((S)-1-phenyl-ethylcarbamoyl)-methyl]- 1,4,7, 1O-tetraaza-cyclododec-1-yl}-acetylamino)-ethyl]-benzoic acid methyl ester ((L9]) 0P HN0 r_N N-\\ / NIL'I)\ N \

HO

A stirring mixture of 2-{4-[6-(6-tetbutyl-10-oxo-1OH-9-oxa-1-aza-anthracen-2-yl)- pyridin-2ylmethyl]-7-{((S)-1-phenyl-ethylcarbamoyl)-methyl]-1,4,7, 10-tetraaza- cyclododec-1-yl}-/V-(($)-1-phenyl-ethyl)-acetamide 40 (0.131 g, 0.157 mmcl), 4-{(S)- 1-(2-chloro-acetylamino)-ethyl]-benzoic acid methyl ester 30 (0.050 g, 0.196 mmol) and K2C03 (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 %); RF = 0.27 (Alumina, CH2CI2 -CH3OH, 49: 1 v/v); m.p. 87-89 °C; H NMR (CDCI3, 500 MHz) ó L40 (9H, 5, tBU CF-I3), 1.44 (9H, d, 3= 7.0 Hz, Cl-i3), 2.71 (16 H, br s, cyclen CH2), 3.25 (6H, br s, CH2CO), 375 (2H, 5, CH pyridine), 3.84 (3H, s, C(O)OCH3), 5.05 (3H, q, CH), 7.16 (2H, d, 3= 8.0 Hz, H5'; Ph(p)), 7.28 (10Ff, m, Ph), 7.33 (2H, d, J 8.0 Hz, Ph(o,'m)), 7.60 (1H, d, 3= 83 Hz, H'°), 7.73 (1H, t, 3= 8.0 Hz, H4'), 7.85 (1H, dd, 3= 8.5; 2.5 Hz, H9), 7.89 (2H, d, 3 8.0 Hz, Ph(o/m)), 8.30 (1H, d, 3 = 2.5 Hz, H7), 8.43 (2H, d, 3 = 8.0 Hz, H2; H3'), 8.77 (1H, d, 3 = 8.0 Hz, H3); 3C (CDCI3, 125 MHz, 1H decoupled 500 MHz) O 21.9 (3C, CH3), 31.5 (3C, C'4), 35.0 (1C, C'3), 48.9 (3C, CM), 51.5 (2C, cyclen CM2), 52.3 (1C, C(O)OCH3), 52.5 (2C, cyclen Cl-I2), 53.0 (2C, cyclen CM2), 53.7 (2C, cyden CM2), 58.5 (3C, CH2CO), 60.2 (1C, CH2 pyridine), 116.6 (1C, C4), 118.3 (2C, C2; C'°), 121.3 (1C, C3'). 121.4 (1C, C6), 122.7 (1C, C7), 125.4 (2C, Ph), 126.2 (2C, Ph), 126.5 (4C, Ph), 127.5 (1C, C5'), 128.8 (4C, Ph), 129.2 (1C, Ph), 130.1 (2C, Ph), 133.8 (1C, C9), 137.8 (1C, C4'), 138.6 (1C, C3), 143.5 (2C, Ph(q)), 148.2 (1C, C8), 153.6 (1C, C"), 154.2 (1C, C11), 159.9 (1C, C6'), 160.2 (1C, C'), 160.3 (1C, C'2), 166.9 (1C, C(O)OCH3), 170.8 (3C, amide C = 0), 177.8 (1C, C5); MS (ES) m/z1056.6 (100 %, {M + HJ); HRMS (ES) m/zfound 1056.5733 [M + HJC62H74O7N9 requires 1056.5706.

[EuL9] 3+ /N 3CI A stirred solution of 4-[(S)-1-(2-{7-[6-(6-tertbutyl-10-oxo-10H-9-oxa-1-aza-anthracen- 2-yl)-pyridin-2-ylmethy?]-4, 10-bis-[((S)-1-phenyl-ethylcarbamoyl)-methylj-1,4,7, 10-tetraaza-cyclododec-1-yl}-acetylamino)-ethylj-benzoic acid methyl ester [La] (0.061 g, 0.057 mmol) and Eu(OTf)3.6H20 (0.045 g, 0.063 mmol) in anhiydrousCH3CN (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 mm. 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 DOWEX 1 x 8 200-400 mesh Cl' resin. The solid material was dissolved in a mixture of H20 -CH3OH (1:1 v/v1 20 ml) and 0.8 g of prepared resin added to the solution, which was stirred at it, 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 mmcl, 64 %); Amax (H20) = 348 nm; T (H20) = 1.02 ms; T (D20) = 1.34 ms; I-PLC (Method A) 4. = 10.9 mm.

ExamDle 6: Activation and conjugation of EuL2 with BG-NH2 (EuL9_CO2H] 3+

HN 3CV NHO/

A solution of (EuL9] (1.5 tmol) in CH3OH -0.02 M KOH(aq) (1:1 v/v1 2 ml) was stirred at it. The progress of hydrolysis was monitored by reverse phase HPLC using a Chromolith performance RP18e 100 x 4.6 mm column. (Method � tR (ester) = 6.40 mins, tR (acid) = 6.24 mins).

The reaction ran to completion in approximately 2 h and was neutralised using a dilute HCl() solution. The solvent was removed under reduced pressure and the crude residue used directly in the next step without further purification.

(EuL9_CONHBG] H2N 3+ HN&/ 1_N/NTh / / NHo To a stirred solution of [EuL9_CO2H] in anhydrous DMF (2 ml) was added incremential equivalents of TSTU and DIPEA (50 pi of a 100 mM solution in DMF in each case, 5 pmol). 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 tmol) in anhydrous DMF (400 p1). The mixture was stirred at it and the progress of the reaction was monitored by reverse phase HPLC using a Chromolith performance RP18e 100 x 4.6mm column. (MethodD;tR(NHS)=6.28mins, tR(BG)-5.97mins).

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; tR (BG) = 6.67 mins,L The product was obtained as a colourless solid (320 nmol, 34 %) Exam�le 7: Synthesis of L and EuL (see schema 8) (S)-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 mmcl) in H20 (50 ml) was added dropwise over 30 mm to a stirred solution of (S)-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 H2SO4(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 %); RF = 0.25 (Silica, Hexane -EtOAc 17: 3 v/v); H NMR (CDCI3, 700 MHz) 6 2.30 (1H, m, CH2CHBr), 2.42 (1H, m, CH2CHBr), 2.60 (2H, m, CH2CH,CHBr), 4.41 (1H, dd, J= 9.0; 6.0 Hz, CH), 5.14 (2H, 5, CH2Ph), 7.33-7.39 (5H, m, Ph); 13C NMR (CDCI3, 176 MHz, 1H decoupled 700 MHz) 6 29.7 (1C, CH2CH2CHBr), 31.6 (1C, CH2CHBr), 44.2 (1C, CH), 66.9 (1C, CH2Ph), 128.4 (1C, Ph(o/m)), 128.5 (1C, Ph(o/m)), 128.6 (1C, Ph(o/m)), 128.7 (1C, Ph(olm)), 128.8 (1C, Ph(p)), 135.8 (1C, Ph(q)), 172.1 (1C, Cbz C = 0), 173.4 (1C, Acid C = 0); MS (ES) m/z323.2 (100 %, [M + H]).

(S)-2-Bromo-pentanedioic acid 5-benzyl ester 1-tert-butyl ester COLtOk A solution of (S)-2-bromo-pentanedioic acid 5-benzyl ester 44 (2.00 g, 6.85 mmol) in tertbutyl acetate (25 ml) and HCIO4 in H20 (70 %, 0.34 mmcl) was stirred, at it, for 16 h. H20 (35 ml) was added to the reaction mixture, and the organic phase separated. The organic phase was washed with H20 (25 ml), followed by 5 % Na2CO3 (aq) (25 ml). The solvent was removed under reduced pressure to yield the title compound45 as a yellow coloured oil (2.15 g, 6.03 mmcl, 88 %); 1H NMR (CDCI3, 700 MHz) 6 1.46 (9H, 5, tBoc CH3), 2.25 (1H, m, CH2CHBr), 2.35 (1H, m, CH2CHBr), 2.55 (2H, m, CH21CH2,CHBr), 4.23 (1H, q, J= 5.5; 2.0 Hz, CH), 5.12 (2H, s, CH2Ph), 7.31- 7.36 (5H, m, Ph); 3C NMR (COd3, 176 MHz, H decoupled 700 MHz) 6 27.9 (3C, tBOC CH3), 29.9 (1C, CH2CH), 31.8 (1C, CH2CH2), 46.9 (1C, CH), 66.7 (1C, CH,Ph), 82.8 (1C, tBOC(q)), 128.4 (1C, Ph(o/m)), 128.5 (1C, Ph(o/m), 128.6 (1C, Ph(o/m)), 128.7 (1C, Ph(o/m)), 128.8 (1C, Ph(p)), 136.0 (1C, Ph(q)), 168.5 (1C, tBoc C = 0), 172.2 (1C, Cbz C = 0); MS (ES) m/z379.0 (100 %, [M + Na]).

4, 10-bis-tert'Butoxycarbonylmethyl-1,4,7, 1O-tetraaza-cycloclodecane-1,7-dicarboxytic 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-1,7-dica rboxylic acid dibenzyl ester 31 (2.65 g, 6.02 mmol), tert-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 46 as a yellow coloured oil (2.46 g, 3.68 mmol, 61 %); RF = 0.53 (Silica, CH2CI2 -CH3OH, 39: 1 v/v); 1H NMR (CDCI3, 700 MHz) 5 1.46 (18H, s, tBoc CH3), 2.86 (8H, br s, cyclen CH2), 3.31 (4H, br s, CH2CO), 3.42 (8H, br 5, cyclen CH2), 5.12 (4H, s, CH2Ph), 7.25-7.33 (1OH, br s, Ph); 3C NMR (CDCI3, 176 MHz, 1H decoupled 700 MHz) 5 28.0 (6C, tBOC CH3), 46.7 (2C, cyclen CH2), 46.8 (2C, cyclen CH2), 54.2 (2C, cyden CH2), 54.5 (2C, cyclen CH2), 55.9 (2C, CH2CO), 66.8 (2C, CH2Ph), 80.7 (2C, tBOC(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 = 0), 170.4 (2C, tBoc C 0), MS (ES) m/z 669.4 (100 %, [M + HJ); HRMS (ES) m/z found 669.3860 [M + H] C35H5303N4 requires 669.3858.

(7-tertButoxycarbonylmethyl-1,4,7, 1O-tetraaza-cyclododec-1-yI)-acetic acid teit-butyl ester (47)

N ND \I H

4, 10-Bis-te,Dbutoxycarbonylmethyl-1,4,7, 10-tetraaza-cydododecane-1,7-dicarboxylic acid dibenzyl ester (2.46 g, 3.68 mmol) in CH3OH -H20 (3:1 v/v, 20 ml) was shaken in a Parr hydrogenation flask at 40 psi H2 over Pd(OH)2/C (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 %); H NMR (CDCI3, 500 MHz) 5 1.46 (18H, s, tBOC CH3), 2.65 (8H, br s, cyclen CH2), 2.84 (8H, br s, cyclen CH2), 3.33 (4H, 5, CH2CO); 3C (CDCI3, 125 MHz, H decoupled 500 MHz) 5 27.3 (6C, tBoc CH3), 51.8-53.4 (8C, cyclen CH2), 82.7 (2C, tBOC(q)), 171.9 (2C, C = 0); MS (ES) m/z401.4 (100 %, [M + HJ); HRMS (ES) m/zfound 401.3121 [M + H] C20H4104N4 requires 401.3122.

2-(4, lO-bis-te,t-Butoxycarbonylmethyl-1,4,7,1O-tetraaza-cyclododec-1-yl) -pentanedioic acid 5-benzyl ester 1-te*butyl ester (49) O\Q

D oo d

A stirred mixture of (7-tert-butoxycarbonylmethyl-1,4,7, 10-tetraaza-cyclododec-1-yl)-acetic acid tert-butyl ester 47 (0.369 g, 0.925 mmol), (S)-2-bromo-pentanedioic acid 5-benzyl ester 1-te,butyl ester (0.325 g, 0.923 mrnol) 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 conipound49 as a yellow coloured solid (0.373 g, 0.552 mmol, 60 %); RF = 0.28 (Alumina, CH2CI2 -CH3OH, 49: 1 v/v); H NMR (CDCI3, 700 MHz) 6 1.34 (27H, s, tBoc CH3), 1.80 (2H, m, arm CH2), 2.41 (4H, m, cyclen CH2; arm Cl-I2), 230 (6H, m, cyclen Cl-I2), 3.05 (8H, m, cyclen Cl-I2), 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); 3C NMR (CDCI3, 176 MHz, H decoupled 700 MHz) 6 25.0 (1C, CH2), 28.3 (9C, tBOC 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, tBoc(q)), 82.2 (1C, tBOC(q)), 128.4 (2C, Ph(o/m)), 128.5 (1C, Ph(p)), 128.7(2C, Ph(o/m)), 136.0 (1C, Ph(q)), 170.4 (2C, Boc C = 0), 171.4 (1C, tBOC C = 0), 172.9 (1C, Cbz C = 0); MS (ES) m/z 677.4 (100 %, [M + H]); HRMS (ES) m/z found 677.4484 [M + H] C36H6108N4 requires 677.4484.

2-{4,1O-bis-teit-Butoxycarbonylmethyl-7-[6-(6-teii'butyl-1O-oxo-1OH-9- oxa-1-aza-anthracen-2-yl)-pyridin-2-ylmethyl]-1,4,7, 1O-tetraaza-cyclododec-1-yI}-pentanedioic acid 5-benzyl ester 1-tet-butyI ester ((L13]) A stirred mixture of 2-(6-bromomethyl-pyridin-2-yl)-6-tertbutyl-9-oxa-1-aza- anthracen-lO-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-tertbutyl ester 49 (0.195 g, 0.289 mmol) and K2C03 (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 °h CH3OH increments) to yield the title compound (112] as a yellow oil (0.188 g, 0.116 mmol, 40 %); RF = 0.37 (Alumina, CH2CI2 -CH3OH, 19: 1 v/v); H NMR (CDCI3, 700 MHz) ö 1.34 (27H, s, tBoc CH3), 1.38 (9H, s tBu 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; H5'), 7.54 (1H, d, J 8.5 Hz, H'°), 7.72-7.78 (2H, br s, H4'; H9), 8.24 (1H, d, i = 3.0 Hz, H7), 8.32 (1H, d, J = 8.0 Hz, H3'), 8.40 (1H, br s, cyclen NH), 8.48 (1H, d, J = 7.0 Hz, H2), 8.75 (1H, d, J 8.0 Hz, H3); 3C NMR (CDCI3, 176 MHz, H decoupled 700 MHz) 6 25.0 (1C, CH2), 28.3 (9C, tBoc CH3), 30.9 (1C, CH2), 31.5 (3C, C'4), 34.9 (1C, C'3), 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, tBoc(g)), 82.2 (1C, tBOC(q)), 116.4 (1C, C"), 118.3 (1C, C10), 118.5 (1C, C2), 119.7 (1C, C5'), 121.3 (1C, C6), 122.7 (1C, C7), 123.8 (1C, C2'), 128.4 (2C, Ph(olm)), 128.5 (1C, Ph(p)), 128.7(2C, Ph(olm)), 133.6 (1C, C9), 136.1 (1C, Ph(q)), 137.9 (1C, C4'), 138.4 (1C, C3), 148.1 (1C, C8), 153.6 (1C, C"), 154.2 (1C, C'1), 158.6 (1C, C6'), 160.2 (1C, C'), 160.6 (1C, C'2), 170.4 (2C, tBoc C = 0), 171.4 (1C, tBoc C = 0), 172.9 (1C, Cbz C = 0), 177.8 (1C, C5); MS (ES) m/z 1019.6 (100 %, [M + HJ); HRMS (ES) in/z found 1019.5866 [M + H] C58H790,0N6 requires 1019.5852; HPLC (Method A) 4 = 12.0 mm.

(EuL'3] O\o (rh NN \\ A stirred mixture of 2-{4, lO-bis-tert-butoxycarbonylmethyl-7-[6-(6-tetbutyI-lO-oxo- 10H9-oxa-1-aza-anthracen-2-yl)-pyridin-2-ylmethyl]-1,4,7, 10-tetraaza-cyclododec-1-yl}-pentanedioic acid 5-benzyl ester 1-teiDbutyl 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 111 NMR to ensure complete deprotection, with the material used immediately for complexation.

HPLC (Method A) tj,. = 10.83 mm.

The deprotected ligand was dissolved in CH3OH -H20 (2: 2 v/v, 4 ml) and Eu(OAc)3.6H20 (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 HCI (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 %); Amax (H20) = 355 nm.

ExamDle A: AbsorDtion spectra The absorption spectrum of the complex EuL1 prepared in Example 1 was recorded in water at 295 K. For purpose of comparison, the absorption spectrum for the complex TbL1 having the following formula was also recorded.

[TbL1] 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 Amax was observed to a wavelength 350 nm, while a gradual decrease in absorption was observed above 350 nm.

The pyridyl-azaxanthone complex EuL' is more suitable than the pyrazoyl-azaxanthone complex ThL' 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.

Examnie B: Emission sDectra Emission from complex [EuL'] (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 (c1m 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.

ExamDle 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 3to9.