NOVEL LUMINESCENT LANTHANIDE CHELATE REPORTERS, BIOSPECIFIC BINDING REACTANTS LABELLED WITH NOVEL LUMINESCENT LANTHANIDE CHELATE REPORTERS AND THEIR USE

Abstract

The present invention relates to novel luminescent lanthanide chelate reporters which are formed from two to three separate lanthanide chelate moieties covalently conjugated to each other to act as an unique labelling reactant, and which can be attached to a biospecific reactant and used in various assays.

Claims

1. A compound of formula (I) ##STR00037## or a salt thereof, wherein (i) the solid lines represent covalent bonds; (ii) the dashed line represents a covalent bond of the group -L-Z to any one of the groups Che.sub.1, A.sub.1, and Che.sub.2; and wherein L is in each case independently absent or selected from linker groups comprising from 1 to 10 moieties selected from —(CH.sub.2).sub.1-8 —, —CH═CH—, —C≡C—, —O—, —S—, —S—S—, —C(═O)—, —C(═O)NH—, —NHC(═O)—, —C(═O)N(C.sub.1-C.sub.8-alkyl)-, —N(C.sub.1-C.sub.6-alkyl)C(═O)—, —NHC(═S)NH—, —CH[(CH.sub.2).sub.0-6C(═O)O.sup.−]—, —CH[(CH.sub.2).sub.0-6C(═O)OH]—, and biradicals of 5- to 10-membered aromatic or heteroaromatic monocyclic or bicyclic rings, wherein the heteroaromatic ring contains one or more, same or different heteroatoms N, O, or S; Z is in each case independently selected from reactive groups selected from —N.sub.3, —C≡CH, —CH═CH.sub.2, —NH.sub.2, —O—NH.sub.2, —C(═O)OH, —CH(═O), —SH, —OH, maleimido and activated derivatives thereof including —NCO, —NCS, —N.sup.+≡N, bromoacetamido, iodoacetamido, reactive esters, pyridyl-2-dithio, and 6-substituted 4-chloro-1,3,5-triazin-2-ylamino and 4-chloro-1,3,5-triazin-2-yloxy; wherein the substituent in the 6-position of the 4-chloro-1,3,5-triazin-2-ylamino or 4-chloro-1,3,5-triazin-2-yloxy is selected from —H, -halogen, —SH, —NH.sub.2, —C.sub.1-C.sub.6-alkyl, —O(C.sub.1-C.sub.6-alkyl), —OAryl, —S(C.sub.1-C.sub.6-alkyl), —SAryl, —N(C.sub.1-C.sub.6-alkyl).sub.2, and N(Aryl).sub.2; wherein the carbon atoms of the aforementioned groups are unsubstituted or substituted by one or more substituents selected from —CN, -halogen, —SH, —C(═O)H, —C(═O)OH, C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-haloalkyl, —O(C.sub.1-C.sub.6-alkyl), —C(═O)(C.sub.1-C.sub.6-alkyl), —C(═O)O(C.sub.1-C.sub.6-alkyl), and phenyl; A.sub.1 is a bridging group comprising from one to three separate straight or branched, saturated or unsaturated carbon-based chains including from 1 to 12 carbon atoms, wherein the carbon-based chains are free of or comprise one to ten, same or different groups selected from —O—, —S—, —NH—, —NR.sub.1—, —C(═O)NH—, —NHC(═O)—, —C(═O)NR.sub.1—, —NR.sub.1C(═O)— and —C(═O)—, or A.sub.1 a bridging chelate moiety -Che.sub.3- of the following general formula ##STR00038## and wherein Che.sub.1 and Che.sub.2 are independently selected from the chelate moieties Che I, Che II, Che III, Che IV, Che V, Che VI, and Che VII of the following general formulae: ##STR00039## ##STR00040## and wherein R.sub.1 is in each case independently selected from C.sub.1-C.sub.6-alkyl, and from the option of representing one of the one or two groups -L-Z; R.sub.2 is in each case independently selected from —C(═O)O.sup.−, —P(═O)O.sub.2.sup.2−, P(═O)MeO.sup.−, —P(═O)PhO.sup.−, and the C.sub.1-C.sub.6-alkyl esters thereof, and from the option of representing one of the one or two groups -L-Z; R.sub.3 is in each case independently selected from the bridging groups —C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, and —P(═O)O.sub.2.sup.2−; R.sub.4 is in each case independently selected from —CH.sub.2N(CH.sub.2C(═O)O.sup.−).sub.2, —CH.sub.2N(CH.sub.2P(═O)O.sub.2.sup.2−).sub.2, —CH.sub.2N(CH.sub.2P(═O)MeO.sup.−).sub.2, —CH.sub.2N(CH.sub.2P(═O)PhO.sup.−).sub.2, and from the options defined for R.sub.2; Ln.sup.3+ is in each case independently selected from the lanthanide ions Eu.sup.3+, Tb.sup.3+, Sm.sup.3+ and Dy.sup.3+, wherein the lanthanide ion forms from seven to ten coordination bonds with the heteroatoms oxygen and nitrogen in the chelate moieties Che.sub.1, Che.sub.2, and Che.sub.3 to form from two to three separate internal chelate moieties; Ar.sub.1 is selected from the following groups ##STR00041## Ar.sub.2 is in each case independently selected from the following groups ##STR00042## and wherein G is in each case independently selected from i) a conjugating group, ii) a single bond, and iii) hydrogen; wherein each conjugating group comprises 1, 2, or 3 moieties selected from —CH═CH—, —C≡C—, —C(═O)—, and biradicals of 5 to 10-membered aromatic or heteroaromatic monocyclic or bicyclic rings, wherein the heteroaromatic ring contains one or more, same or different heteroatoms N, O, or S, and wherein the aromatic or heteroaromatic monocyclic or bicyclic rings are unsubstituted or substituted by 1 to 5 same or different substituents R.sub.5; wherein each conjugating group, if present in a terminal position, may further comprise a terminal group, which is selected from the group consisting of —H, -halogen, —CN, —CH.sub.3, and from the option of representing one of the one or two groups -L-Z; wherein R.sub.5 is independently selected from C.sub.1-C.sub.12-alkyl, —(CH.sub.2).sub.0-6—C(═O)OH, —(CH.sub.2).sub.0-6—C(═O)O.sup.−, —(CH.sub.2).sub.0-6—S(═O).sub.2OH, —(CH.sub.2).sub.0-6—S(═O).sub.2O.sup.−, —C(═O)NHR.sub.6, —C(═O)NCH.sub.3R.sub.6, —NHC(═O)NHR.sub.6, —NHC(═S)NHR.sub.6, -halogen, —OH, —SH, —OR.sub.7, —SR.sub.7, and hydrophilic groups selected from monosaccharides, disaccharides, —(CH.sub.2).sub.1-6 CH.sub.2OH, —CH(CH.sub.2OH).sub.2, —C(CH.sub.2OH).sub.3, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—H, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—C.sub.1-C.sub.4-alkyl, —O—(CH.sub.2CH.sub.2O).sub.1-6—H, and —O—(CH.sub.2CH.sub.2O).sub.1-6—C.sub.1-C.sub.4-alkyl; wherein R.sub.6 is selected from C.sub.1-C.sub.12-alkyl, —(CH.sub.2).sub.1-6C(═O)OH, —(CH.sub.2).sub.1-6C(═O)O.sup.−, —(CH.sub.2).sub.1-6 S(═O).sub.2OH, —(CH.sub.2).sub.1-6 S(═O).sub.2O.sup.− and hydrophilic groups selected from monosaccharides, disaccharides, —(CH.sub.2).sub.1-6 CH.sub.2OH, —CH(CH.sub.2OH).sub.2, —C(CH.sub.2OH).sub.3, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—H, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—C.sub.1-C.sub.4-alkyl, —O—(CH.sub.2CH.sub.2O).sub.1-6—H, and —O—(CH.sub.2CH.sub.2O).sub.1-6—C.sub.1-C.sub.4-alkyl; wherein R.sub.7 is selected from —CF.sub.3, —C.sub.1-C.sub.12-alkyl, —(CH.sub.2).sub.1-6C(═O)OH, —(CH.sub.2).sub.1-6C(═O)O.sup.−, —(CH.sub.2).sub.1-6S(═O).sub.2OH, —(CH.sub.2).sub.1-6S(═O).sub.2O.sup.−, —C(═O)NHR.sub.6, —C(═O)NCH.sub.3R.sub.6, —NHC(═O)NHR.sub.6, —NHC(═S)NH R.sub.6, (CH.sub.2).sub.1-6N(CH.sub.3).sub.2.sup.+—(CH.sub.2).sub.1-6S(═O).sub.2O.sup.−, —(CH.sub.2).sub.1-6C(═O)-(piparazin-1,4-diyl)-(CH.sub.2).sub.1-6C(═O)OH, and hydrophilic groups selected from monosaccharides, disaccharides, —(CH.sub.2).sub.1-6 CH.sub.2OH, —CH(CH.sub.2OH).sub.2, C(CH.sub.2OH).sub.3, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—H, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—C.sub.1-C.sub.4-alkyl, —O—(CH.sub.2CH.sub.2O).sub.1-6—H, and —O—(CH.sub.2CH.sub.2O).sub.1-6—C.sub.1-C.sub.4-alkyl.

2. The compound of claim 1, wherein each conjugating group comprises the 1, 2 or 3 moieties in an arrangement so as to be conjugated with each other and attached to the respective pyridine in such a way that the conjugating group is conjugated with the pyridine.

3. The compound of claim 1, wherein each conjugating group comprises 1, 2, or 3 moieties selected from —CH═CH—, —C≡C—, —C(═O)—, phenylene, biphenylene, naphthylene, pyridylene, pyrazinylene, pyrimidinylene, pyridazinylene, furylene, thienylene, pyrrolylene, imidazolylene, pyrazolylene, thiazolylene, isothiazolylene, oxazolylene, isoxazolylene, fyrazanylene, 1,2,4-triazol-3,5-ylene, and oxadiazolylene, wherein the aromatic or heteroaromatic monocyclic or bicyclic rings are unsubstituted or substituted by 1 to 5 same or different substituents R.sub.5.

4. The compound of claim 1, wherein L is in each case independently absent or selected from linker groups comprising from 1 to 10 moieties selected from —(CH.sub.2).sub.1-8 —, —CH═CH—, —C≡C—, —O—, —S—, —S—S—, —C(═O)—, —C(═O)NH—, —NHC(═O)—, —C(═O)N(C.sub.1-C.sub.6-alkyl)-, —N(C.sub.1-C.sub.6-alkyl)C(═O)—, —NHC(═S)NH—, —CH[(CH.sub.2).sub.0-6C(═O)O.sup.−]—, —CH[(CH.sub.2).sub.0-6C(═O)OH]—, phenylene, pyridylene, and triazolene.

5. The compound of claim 1, wherein Z is in each case independently selected from reactive groups selected from —N.sub.3, —C≡CH, —CH═CH.sub.2, —NH.sub.2, —O—NH.sub.2, —C(═O)OH, —CH(═O), —SH, —OH, maleimido, —NCO, —NCS, —N.sup.+≡N, bromoacetamido, iodoacetamido, aromatic esters based on p-nitrophenol, pentafluorophenol, 2,4,5-trichlorophenol, N-hydroxy-5-norbornene-endo-2,3-dicarboxyimide, hydroxybenzotriazole, 1-hydroxy-7-azabenzoptriazole, sulfo-N-hydroxysuccinimide, or N-hydroxysuccinimide, esters based on phosphonium-, uronium-, or guanidinium-based coupling reagents, triazinyl or pyridinium esters, pyridyl-2-dithio, and 6-substituted 4-chloro-1,3,5-triazin-2-ylamino and 4-chloro-1,3,5-triazin-2-yloxy; wherein the substituent in the 6-position of the 4-chloro-1,3,5-triazin-2-ylamino or 4-chloro-1,3,5-triazin-2-yloxy is selected from —H, -halogen, —SH, —NH.sub.2, —C.sub.1-C.sub.6-alkyl, —O(C.sub.1-C.sub.6-alkyl), —OAryl, —S(C.sub.1-C.sub.8-alkyl), —SAryl, —N(C.sub.1-C.sub.8-alkyl).sub.2, and N(Aryl).sub.2; wherein the carbon atoms of the aforementioned groups are unsubstituted or substituted by one or more substituents selected from —CN, -halogen, —SH, —C(═O)H, —C(═O)OH, C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-haloalkyl, —O(C.sub.1-C.sub.6-alkyl), —C(═O)(C.sub.1-C.sub.6-alkyl), —C(═O)O(C.sub.1-C.sub.6-alkyl), and phenyl.

6. The compound of claim 1, wherein A.sub.1 is a bridging chelate moiety -Che.sub.3-, wherein Ar.sub.1 is the following group: ##STR00043##

7. The compound of claim 1, wherein Che.sub.1 and Che.sub.2 are independently selected from the chelate moieties Che I and Che IV, wherein Ar.sub.2 is the following group: ##STR00044##

8. The compound of claim 1, which is any one of the compounds 6, 7, 26, 27, 28, 29, 35, 36, 45, 46, 47, 48, 56, 57, 58, 59, 65, and 66.

9. A compound of formula (II) ##STR00045## or a salt thereof, wherein L is in each case independently absent or selected from linker groups comprising from 1 to 10 moieties selected from —(CH.sub.2).sub.1-8—, —CH═CH—, —C≡C—, —O—, —S—, —S—S—, —C(═O)—, —C(═O)NH—, —NHC(═O)—, —C(═O)N(C.sub.1-C.sub.6-alkyl)-, —N(C.sub.1-C.sub.6-alkyl)C(═O)—, —NHC(═S)NH—, —CH[(CH.sub.2).sub.0-6C(═O)O.sup.−]—, —CH[(CH.sub.2).sub.0-6C(═O)OH]—, and biradicals of 5- to 10-membered aromatic or heteroaromatic monocyclic or bicyclic rings, wherein the heteroaromatic ring contains one or more, same or different heteroatoms N, O, or S; Z is in each case independently selected from reactive groups selected from —N.sub.3, —C≡CH, —CH═CH.sub.2, —NH.sub.2, —O—NH.sub.2, —C(═O)OH, —CH(═O), —SH, —OH, maleimido and activated derivatives thereof including —NCO, —NCS, —N.sup.+≡N, bromoacetamido, iodoacetamido, reactive esters, pyridyl-2-dithio, and 6-substituted 4-chloro-1,3,5-triazin-2-ylamino and 4-chloro-1,3,5-triazin-2-yloxy; wherein the substituent in the 6-position of the 4-chloro-1,3,5-triazin-2-ylamino or 4-chloro-1,3,5-triazin-2-yloxy is selected from —H, -halogen, —SH, —NH.sub.2, —C.sub.1-C.sub.6-alkyl, —O(C.sub.1-C.sub.6-alkyl), —OAryl, —S(C.sub.1-C.sub.6-alkyl), —SAryl, —N(C.sub.1-C.sub.6-alkyl).sub.2, and N(Aryl).sub.2; wherein the carbon atoms of the aforementioned groups are unsubstituted or substituted by one or more substituents selected from —CN, -halogen, —SH, —C(═O)H, —C(═O)OH, C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-haloalkyl, —O(C.sub.1-C.sub.6-alkyl), —C(═O)(C.sub.1-C.sub.6-alkyl), —C(═O)O(C.sub.1-C.sub.6-alkyl), and phenyl; and wherein A.sub.1 is a bridging group comprising from one to three separate straight or branched, saturated or unsaturated carbon-based chains including from 1 to 12 carbon atoms, wherein the carbon-based chains are free of or comprise one to ten, same or different groups selected from —O—, —S—, —NH—, —NR.sub.1—, —C(═O)NH—, —NHC(═O)—, —C(═O)NR.sub.1—, —NR.sub.1C(═O)— and —C(═O)—, or A.sub.1 a bridging chelate moiety -Che*.sub.3- of the following general formula ##STR00046## and wherein Che*.sub.1 and Che*.sub.2 are independently selected from the chelate moieties Che* I, Che* II, Che* III, Che* IV, Che* V, Che* VI, and Che* VII of the following general formulae: ##STR00047## ##STR00048## and wherein R.sub.1 is in each case independently selected from C.sub.1-C.sub.6-alkyl, and from the option of representing one of the one or two groups -L-Z; R.sub.2 is in each case independently selected from —C(═O)O.sup.−, —P(═O)O.sub.2.sup.2−, P(═O)MeO.sup.−, —P(═O)PhO.sup.−, and the C.sub.1-C.sub.6-alkyl esters thereof, and from the option of representing one of the one or two groups -L-Z; R.sub.3 is in each case independently selected from the bridging groups —C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, and —P(═O)O.sub.2.sup.2−; R.sub.4 is in each case independently selected from —CH.sub.2N(CH.sub.2C(═O)O.sup.−).sub.2, —CH.sub.2N(CH.sub.2P(═O)O.sub.2.sup.2−).sub.2, —CH.sub.2N(CH.sub.2P(═O)MeO.sup.−).sub.2, —CH.sub.2N(CH.sub.2P(═O)PhO.sup.−).sub.2, and from the options defined for R.sub.2; Ar.sub.1 is selected from the following groups ##STR00049## Ar.sub.2 is in each case independently selected from the following groups ##STR00050## and wherein G is in each case independently selected from i) a conjugating group, ii) a single bond, and iii) hydrogen; wherein each conjugating group comprises 1, 2, or 3 moieties selected from —CH═CH—, —C≡C—, —C(═O)—, and biradicals of 5 to 10-membered aromatic or heteroaromatic monocyclic or bicyclic rings, wherein the heteroaromatic ring contains one or more, same or different heteroatoms N, O, or S, and wherein the aromatic or heteroaromatic monocyclic or bicyclic rings are unsubstituted or substituted by 1 to 5 same or different substituents R.sub.5; wherein each conjugating group, if present in a terminal position, may further comprise a terminal group, which is selected from the group consisting of —H, -halogen, —CN, —CH.sub.3, and from the option of representing one of the one or two groups -L-Z; wherein R.sub.5 is independently selected from C.sub.1-C.sub.12-alkyl, —(CH.sub.2).sub.0-6—C(═O)OH, —(CH.sub.2).sub.0-6—C(═O)O.sup.−, —(CH.sub.2).sub.0-6—S(═O).sub.2OH, —(CH.sub.2).sub.0-6—S(═O).sub.2O.sup.−, —C(═O)NHR.sub.6, —C(═O)NCH.sub.3R.sub.6, —NHC(═O)NHR.sub.6, —NHC(═S)NHR.sub.6, -halogen, —OH, —SH, —OR.sub.7, —SR.sub.7, and hydrophilic groups selected from monosaccharides, disaccharides, —(CH.sub.2).sub.1-6CH.sub.2OH, —CH(CH.sub.2OH).sub.2, —C(CH.sub.2OH).sub.3, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—H, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—C.sub.1-C.sub.4-alkyl, —O—(CH.sub.2CH.sub.2O).sub.1-6—H, and —O—(CH.sub.2CH.sub.2O).sub.1-6—C.sub.1-C.sub.4-alkyl; wherein R.sub.6 is selected from C.sub.1-C.sub.12-alkyl, —(CH.sub.2).sub.1-6C(═O)OH, —(CH.sub.2).sub.1-6C(═O)O.sup.−, —(CH.sub.2).sub.1-6S(═O).sub.2OH, —(CH.sub.2).sub.1-6S(═O).sub.2O.sup.− and hydrophilic groups selected from monosaccharides, disaccharides, —(CH.sub.2).sub.1-6CH.sub.2OH, —CH(CH.sub.2OH).sub.2, —C(CH.sub.2OH).sub.3, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—H, —(CH.sub.2).sub.1-3—O—(CH.sub.2 CH.sub.2O).sub.0-5—C.sub.1-C.sub.4-alkyl, —O—(CH.sub.2CH.sub.2O).sub.1-6—H, and —O—(CH.sub.2CH.sub.2O).sub.1-6—C.sub.1-C.sub.4-alkyl; wherein R.sub.7 is selected from —CF.sub.3, —C.sub.1-C.sub.12-alkyl, —(CH.sub.2).sub.1-6C(═O)OH, —(CH.sub.2).sub.1-6C(═O)O.sup.−, (CH.sub.2).sub.1-6S(═O).sub.2OH, —(CH.sub.2).sub.1-6S(═O).sub.2O.sup.−, —C(═O)NHR.sub.6, —C(═O)NCH.sub.3R.sub.6, —NHC(═O)NHR.sub.6, —NHC(═S)NH R.sub.6, —(CH.sub.2).sub.1-6N(CH.sub.3).sub.2.sup.+—(CH.sub.2).sub.1-6S(═O).sub.2O.sup.−—, —(CH.sub.2).sub.1-6C(═O)-(piparazin-1,4-diyl)-(CH.sub.2).sub.1-6C(═O)OH, and hydrophilic groups selected from monosaccharides, disaccharides, —(CH.sub.2).sub.1-6CH.sub.2OH, —CH(CH.sub.2OH).sub.2, C(CH.sub.2OH).sub.3, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—H, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—C.sub.1-C.sub.4-alkyl, —O—(CH.sub.2CH.sub.2O).sub.1-6—H, and —O—(CH.sub.2CH.sub.2O).sub.1-6—C.sub.1-C.sub.4-alkyl.

10. A detection agent comprising a biospecific binding reactant conjugated to a compound of claim 1.

11. The detection agent of claim 10, wherein the biospecific binding reactant is selected from i) an antibody, an antigen, a receptor ligand, a specific binding protein, a DNA probe, a RNA probe, a hapten, a drug, and lectin; or ii) an oligopeptide, an oligonucleotide, a modified oligonucleotide, a modified polynucleotide, a protein, an oligosaccaride, a polysaccharide, a phospholipid, a PNA and a steroid.

12. A method of detecting an analyte in a biospecific binding assay, said method comprising: a) forming a complex between the analyte and a compound of claim 1; b) exciting said complex with a radiation having an excitation wavelength of the compound, thereby forming an excited complex; and c) detecting emission radiation emitted from said excited complex.

13. A method of labelling a biospecific binding reactant with a compound of claim 1, comprising: a) providing a biospecific binding reactant; and b) conjugating the biospecific binding reactant with the compound.

14-16. (canceled)

17. A solid support material conjugated with a compound of claim 1.

18. The compound of claim 3, wherein each conjugating group is independently selected from phenylene-C≡C—, phenylene, thienylene, and furylene.

19. The compound of claim 4, wherein: L is in each case independently absent or selected from linker groups comprising from 1 to 10 moieties selected from —(CH.sub.2).sub.1-8 —, —C≡C—, —O—, —C(═O)—, —C(═O)NH—, —NHC(═O)—, phenylene, pyridylene, and triazolene.

20. The compound of claim 5, wherein: Z is in each case independently —NCS or —NH.sub.2.

21. The compound of claim 6, wherein: R.sub.2 is in each case —C(═O)O.sup.−; and R.sub.3 is in each case selected from the bridging groups —C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-S—.

22. The compound of claim 7, wherein: R.sub.2 is in each case —C(═O)O.sup.−; R.sub.3 is in each case selected from the bridging groups —C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-S—; and R.sub.4 is in each case —C(═O)O.sup.−.

23. The detection agent of claim 11, wherein the biospecific binding reactant is an antibody.

Description

FIGURES

[0091] FIG. 1 shows ligand esters 24 and 25, which are preferred compounds of formula (II) as described herein.

[0092] FIG. 2 shows Eu chelates 26, 27, 28, and 29, which are preferred compounds of formula (I) as described herein.

[0093] FIG. 3 shows ligand ester 34, which is a preferred compound of formula (II) as described herein.

[0094] FIG. 4 shows Eu chelates 35 and 36, which are preferred compounds of formula (I) as described herein.

[0095] FIG. 5 shows ligand esters 43 and 44, which are preferred compounds of formula (II) as described herein.

[0096] FIG. 6 shows Eu chelates 45, 46, 47, and 48, which are preferred compounds of formula (I) as described herein.

[0097] FIG. 7 shows ligand ester 55, which is a preferred compound of formula (II) as described herein.

[0098] FIG. 8 shows Eu chelates 56 and 57, which are preferred compounds of formula (I) as described herein.

[0099] FIG. 9 shows Eu chelates 58 and 59, which are preferred compounds of formula (I) as described herein.

[0100] FIG. 10 shows reference Eu chelates Ref 1, Ref 2 and Ref 3, which comprise only one chelate moiety.

DETAILED DESCRIPTION

[0101] As indicated above, the present invention relates to a compound of formula (I)

##STR00014##

or a salt thereof, wherein [0102] (i) the solid lines represent covalent bonds; [0103] (ii) the dashed line represents a covalent bond of the group -L-Z to any one of the groups Che.sub.1, A.sub.1, and Che.sub.2;
and wherein [0104] L is in each case independently absent or selected from linker groups comprising from 1 to 10 moieties selected from —(CH.sub.2).sub.1-8 —, —CH═CH—, —C≡C—, —O—, —S—, —S—S—, —C(═O)—, —C(═O)NH—, —NHC(═O)—, —C(═O)N(C.sub.1-C.sub.6-alkyl)-, —N(C.sub.1-C.sub.6-alkyl)C(═O)—, —NHC(═S)NH—, —CH[(CH.sub.2).sub.0-6C(═O)O.sup.−]—, —CH[(CH.sub.2).sub.0-6C(═O)OH]—, and biradicals of 5- to 10-membered aromatic or heteroaromatic monocyclic or bicyclic rings, wherein the heteroaromatic ring contains one or more, same or different heteroatoms N, O, or S; [0105] Z is in each case independently selected from reactive groups selected from —N.sub.3, —C≡CH, —CH═CH.sub.2, —NH.sub.2, —C(═O)OH, —CH(═O), —SH, —OH, maleimido and activated derivatives thereof including —NCO, —NCS, —N.sup.+≡N, bromoacetamido, iodoacetamido, reactive esters, pyridyl-2-dithio, and 6-substituted 4-chloro-1,3,5-triazin-2-ylamino and 4-chloro-1,3,5-triazin-2-yloxy; wherein the substituent in the 6-position of the 4-chloro-1,3,5-triazin-2-ylamino or 4-chloro-1,3,5-triazin-2-yloxy is selected from —H, -halogen, —SH, —NH.sub.2, —C.sub.1-C.sub.6-alkyl, —O(C.sub.1-C.sub.6-alkyl), —OAryl, —S(C.sub.1-C.sub.6-alkyl), —SAryl, —N(C.sub.1-C.sub.6-alkyl).sub.2, and N(Aryl).sub.2; [0106] wherein the carbon atoms of the aforementioned groups are unsubstituted or substituted by one or more substituents selected from —CN, -halogen, —SH, —C(═O)H, —C(═O)OH, C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-haloalkyl, —O(C.sub.1-C.sub.6-alkyl), —C(═O)(C.sub.1-C.sub.6-alkyl), —C(═O)O(C.sub.1-C.sub.6-alkyl), and phenyl; [0107] A.sub.1 is a bridging group comprising from one to three separate straight or branched, saturated or unsaturated carbon-based chains including from 1 to 12 carbon atoms, wherein the carbon-based chains are free of or comprise one to ten, same or different groups selected from —O—, —S—, —NH—, —NR.sub.1—, —C(═O)NH—, —NHC(═O)—, —C(═O)NR.sub.1—, —NR.sub.1C(═O)— and —C(═O)—,
or [0108] A.sub.1 a bridging chelate moiety -Che.sub.3- of the following general formula

##STR00015##

and wherein [0109] Che.sub.1 and Che.sub.2 are independently selected from the chelate moieties Che I, Che II, Che III, Che IV, Che V, Che VI, and Che VII of the following general formulae:

##STR00016## ##STR00017##

and wherein [0110] R.sub.1 is in each case independently selected from C.sub.1-C.sub.6-alkyl, and from the option of representing one of the one or two groups -L-Z; [0111] R.sub.2 is in each case independently selected from —C(═O)O.sup.−, —P(═O)O.sub.2.sup.2−, P(═O)MeO.sup.−, —P(═O)PhO.sup.−, and the C.sub.1-C.sub.6-alkyl esters thereof, and from the option of representing one of the one or two groups -L-Z; [0112] R.sub.3 is in each case independently selected from the bridging groups —C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, and —P(═O)O.sub.2.sup.2-; [0113] R.sub.4 is in each case independently selected from —CH.sub.2N(CH.sub.2C(═O)O.sup.−).sub.2, —CH.sub.2N(CH.sub.2P(═O)O.sub.2.sup.2−).sub.2, —CH.sub.2N(CH.sub.2P(═O)MeO).sub.2, —CH.sub.2N(CH.sub.2P(═O)PhO.sup.−).sub.2, and from the options defined for R.sub.2; [0114] Ln.sup.3+ is in each case independently selected from the lanthanide ions Eu.sup.3+, Tb.sup.3+, Sm.sup.3+ and Dy.sup.3+, wherein the lanthanide ion forms from seven to ten coordination bonds with the heteroatoms oxygen and nitrogen in the chelate moieties Che.sub.1, Che.sub.2, and Che.sub.3 to form from two to three separate internal chelate moieties; [0115] Ar.sub.1 is selected from the following groups

##STR00018## [0116] Ar.sub.2 is in each case independently selected from the following groups

##STR00019##

and wherein [0117] G is in each case independently selected from i) a conjugating group, ii) a single bond, and iii) hydrogen; [0118] wherein each conjugating group comprises 1, 2, or 3 moieties selected from —CH═CH—, —C≡C—, —C(═O)—, and biradicals of 5 to 10-membered aromatic or heteroaromatic monocyclic or bicyclic rings, wherein the heteroaromatic ring contains one or more, same or different heteroatoms N, O, or S, and wherein the aromatic or heteroaromatic monocyclic or bicyclic rings are unsubstituted or substituted by 1 to 5 same or different substituents R.sub.5; [0119] wherein each conjugating group, if present in a terminal position, may further comprise a terminal group, which is selected from the group consisting of —H, -halogen, —CN, —CH.sub.3, and from the option of representing one of the one or two groups -L-Z. [0120] wherein R.sub.5 is independently selected from C.sub.1-C.sub.12-alkyl, —(CH.sub.2).sub.0-6—C(═O)OH, —(CH.sub.2).sub.0-6—C(═O)O.sup.−, —(CH.sub.2).sub.0-6—S(═O).sub.2OH, —(CH.sub.2).sub.0-6—S(═O).sub.2O.sup.−, —C(═O)NHR.sub.6, —C(═O)NCH.sub.3R.sub.6, —NHC(═O)NHR.sub.6, —NHC(═S)NHR.sub.6, -halogen, —OH, —SH, —OR.sub.7, —SR.sub.7, and hydrophilic groups selected from monosaccharides, disaccharides, —(CH.sub.2).sub.1-6 CH.sub.2OH, —CH(CH.sub.2OH).sub.2, —C(CH.sub.2OH).sub.3, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—H, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—C.sub.1-C.sub.4-alkyl, —O—(CH.sub.2CH.sub.2O).sub.1-6—H, and —O—(CH.sub.2CH.sub.2O).sub.1-6—C.sub.1-C.sub.4-alkyl; [0121] wherein R.sub.6 is selected from C.sub.1-C.sub.12-alkyl, —(CH.sub.2).sub.1-6C(═O)OH, —(CH.sub.2).sub.1-6C(═O)O.sup.−, —(CH.sub.2).sub.1-6 S(═O).sub.2OH, —(CH.sub.2).sub.1-6 S(═O).sub.2O.sup.− and hydrophilic groups selected from monosaccharides, disaccharides, —(CH.sub.2).sub.1-6CH.sub.2OH, —CH(CH.sub.2OH).sub.2, —C(CH.sub.2OH).sub.3, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—H, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—C.sub.1-C.sub.4-alkyl, —O—(CH.sub.2CH.sub.2O).sub.1-6—H, and —O—(CH.sub.2CH.sub.2O).sub.1-6—C.sub.1-C.sub.4-alkyl; [0122] wherein R.sub.7 is selected from —CF.sub.3, —C.sub.1-C.sub.12-alkyl, —(CH.sub.2).sub.1-6C(═O)OH, —(CH.sub.2).sub.1-6C(═O)O.sup.−, —(CH.sub.2).sub.1-6 S(═O).sub.2OH, —(CH.sub.2).sub.1-6 S(═O).sub.2O.sup.−, —C(═O)NHR.sub.6, —C(═O)NCH.sub.3R.sub.6, —NHC(═O)NHR.sub.6, —NHC(—S)NHR.sub.6, —(CH.sub.2).sub.1-6 N(CH.sub.3).sub.2.sup.+—(CH.sub.2).sub.1-6 S(═O).sub.2O.sup.−, —(CH.sub.2).sub.1-6C(═O)-(piparazin-1,4-diyl)-(CH.sub.2).sub.1-6C(═O)OH, and hydrophilic groups selected from monosaccharides, disaccharides, —(CH.sub.2).sub.1-6CH.sub.2OH, —CH(CH.sub.2OH).sub.2, —C(CH.sub.2OH).sub.3, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—H, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—C.sub.1-C.sub.4-alkyl, —O—(CH.sub.2CH.sub.2O).sub.1-6—H, and —O—(CH.sub.2CH.sub.2O).sub.1-6—C.sub.1-C.sub.4-alkyl.

[0123] Preferred embodiments of the compounds of formula (I) are defined hereinafter. It is to be understood that the preferred embodiments regarding the groups and substituents of the compounds of formula (I) are also preferred in connection with the compounds of formula (II), as well as in connection with the detection agent of the invention, the methods and uses of the invention, and the solid support material of the invention.

[0124] The compounds of formula (I) comprise from two to three separate lanthanide chelate moieties, which are covalently tethered to each other. In particular, Che.sub.1 and Che.sub.2 are independently selected from the chelate moieties Che I, Che II, Che III, Che IV, Che V, Che VI, and Che VII as defined above. In one embodiment of the invention, A.sub.1 represents a bridging group comprising from one to three separate straight or branched, saturated or unsaturated carbon-based chains including from 1 to 12 carbon atoms, wherein the carbon-based chains are free of or comprise one to ten, same or different groups selected from —O—, —S—, —NH—, —NR.sub.1—, —C(═O)NH—, —NHC(═O)—, —C(═O)NR.sub.1—, —NR.sub.1C(═O)— and —C(═O)—. This results in compounds of formula (I) comprising only the two lanthanide chelate moieties Che.sub.1 and Che.sub.2. In another embodiment of the invention, A.sub.1 represents a bridging chelate moiety -Che.sub.3- as defined above. This results in compounds of formula (I) comprising three lanthanide chelate moieties, i.e. Che.sub.1, Che.sub.2, and Che.sub.3.

[0125] The lanthanide chelate moieties preferably comprise from one to three individual chromophore moieties around an emitting lanthanide ion. Preferably, the lanthanide ion in each case forms from seven to ten coordination bonds with the heteroatoms oxygen and nitrogen in the chelate moieties Che.sub.1, Che.sub.2, and Che.sub.3.

[0126] In one embodiment, the lanthanide ion Ln.sup.3+ is in each case independently selected from the lanthanide ions Eu.sup.3+, Tb.sup.3+, Sm.sup.3+ and Dy.sup.3+, wherein the lanthanide ion forms from seven to ten coordination bonds with the heteroatoms oxygen and nitrogen in the chelate moieties Che.sub.1, Che.sub.2, and Che.sub.3. In a preferred embodiment, the lanthanide ion Ln.sup.3+ is in each case Eu.sup.3+, which forms from seven to ten coordination bonds with the heteroatoms oxygen and nitrogen in the chelate moieties Che.sub.1, Che.sub.2, and Che.sub.3.

[0127] The compounds of formula (I) further preferably comprise one or more conjugating groups as substituents G, wherein each conjugating group comprises 1, 2, or 3 moieties selected from —CH═CH—, —C≡C—, —C(═O)—, and biradicals of 5 to 10-membered aromatic or heteroaromatic monocyclic or bicyclic rings, wherein the heteroaromatic ring contains one or more, same or different heteroatoms N, O, or S, and wherein the aromatic or heteroaromatic monocyclic or bicyclic rings are unsubstituted or substituted by 1 to 5 same or different substituents R.sub.5, wherein R.sub.5 is defined as above; and wherein each conjugating group, if present in a terminal position, may further comprise a terminal group, which is selected from the group consisting of —H, -halogen, —CN, —CH.sub.3, and from the option of representing one of the one or two groups -L-Z. In a preferred embodiment, each conjugating group comprises 1, 2, or 3 moieties selected from —CH═CH—, —C≡C—, —C(═O)—, phenylene, biphenylene, naphthylene, pyridylene, pyrazinylene, pyrimidinylene, pyridazinylene, furylene, thienylene, pyrrolylene, imidazolylene, pyrazolylene, thiazolylene, isothiazolylene, oxazolylene, isoxazolylene, fyrazanylene, 1,2,4-triazol-3,5-ylene, and oxadiazolylene, wherein the aromatic or heteroaromatic monocyclic or bicyclic rings are unsubstituted or substituted by 1 to 5 same or different substituents R.sub.5, and more preferably each conjugating group is independently selected from phenylene-C≡C—, phenylene, thienylene, and furylene.

[0128] Typically, the conjugating groups are attached to the pyridine groups of the chelate moieties. This may enhance the luminescence intensity by increasing the chromophore's molar absorptivity due to an increased π-electron conjugation of the aromatic chromophore. Therefore, in a preferred embodiment, each conjugating group comprises the 1, 2 or 3 moieties in an arrangement so as to be conjugated with each other and attached to the respective pyridine in such a way that the conjugating group is conjugated with the pyridine.

[0129] The compounds of formula (I) further comprise one or two reactive groups Z in order to allow a covalent attachment to a biospecific binding reactant. The reactive group Z is connected to any one of the groups Che.sub.1, A.sub.1, and Che.sub.2 of the compounds of formula (I) via a group L, which is a linker group, also referred to as spacer, i.e. a distance-making biradical, so as—if necessary or desirable—to position the reactive group Z in a position accessible for reaction with the biospecific binding reactant.

[0130] In one embodiment of the invention, [0131] L is in each case independently absent or selected from linker groups comprising from 1 to 10 moieties selected from —(CH.sub.2).sub.1-8 —, —CH═CH—, —C≡C—, —O—, —S—, —S—S—, —C(═O)—, —C(═O)NH—, —NHC(═O)—, —C(═O)N(C.sub.1-C.sub.6-alkyl)-, —N(C.sub.1-C.sub.6-alkyl)C(═O)—, —NHC(═S)NH—, —CH[(CH.sub.2).sub.0-6C(═O)O.sup.−]—, —CH[(CH.sub.2).sub.0-6C(═O)OH]—, and biradicals of 5- to 10-membered aromatic or heteroaromatic monocyclic or bicyclic rings, wherein the heteroaromatic ring contains one or more, same or different heteroatoms N, O, or S.

[0132] In a preferred embodiment of the invention, [0133] L is in each case independently absent or selected from linker groups comprising from 1 to 10 moieties selected from —(CH.sub.2).sub.1-8 —, —CH═CH—, —C≡C—, —O—, —S—, —S—S—, —C(═O)—, —C(═O)NH—, —NHC(═O)—, —C(═O)N(C.sub.1-C.sub.6-alkyl)-, —N(C.sub.1-C.sub.6-alkyl)C(═O)—, —NHC(═S)NH—, —CH[(CH.sub.2).sub.0-6C(═O)O.sup.−]—, —CH[(CH.sub.2).sub.0-6C(═O)OH]—, phenylene, pyridylene, and triazolene.

[0134] In a more preferred embodiment of the invention, [0135] L is in each case independently absent or selected from linker groups comprising from 1 to 10 moieties selected from —(CH.sub.2).sub.1-8 —, —C≡C—, —O—, —C(═O)—, —C(═O)NH—, —NHC(═O)—, phenylene, pyridylene, and triazolene.

[0136] In an even more preferred embodiment of the invention, [0137] L is in each case independently absent or selected from linker groups comprising from 1 to 10 moieties selected from —(CH.sub.2).sub.1-8 —, —O—, —C(═O)—, —NHC(═O)—, —C(═O)NH— and phenylene.

[0138] In one embodiment of the invention, [0139] Z is in each case independently selected from reactive groups selected from —N.sub.3, —C≡CH, —CH═CH.sub.2, —NH.sub.2, —O—NH.sub.2, —C(═O)OH, —CH(═O), —SH, —OH, maleimido and activated derivatives thereof including —NCO, —NCS, —N.sup.+≡N, bromoacetamido, iodoacetamido, reactive esters, pyridyl-2-dithio, and 6-substituted 4-chloro-1,3,5-triazin-2-ylamino and 4-chloro-1,3,5-triazin-2-yloxy; wherein the substituent in the 6-position of the 4-chloro-1,3,5-triazin-2-ylamino or 4-chloro-1,3,5-triazin-2-yloxy is selected from —H, -halogen, —SH, —NH.sub.2, —C.sub.1-C.sub.6-alkyl, —O(C.sub.1-C.sub.6-alkyl), —OAryl, —S(C.sub.1-C.sub.6-alkyl), —SAryl, —N(C.sub.1-C.sub.6-alkyl).sub.2, and N(Aryl).sub.2; [0140] wherein the carbon atoms of the aforementioned groups are unsubstituted or substituted by one or more substituents selected from —CN, -halogen, —SH, —C(═O)H, —C(═O)OH, C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-haloalkyl, —O(C.sub.1-C.sub.6-alkyl), —C(═O)(C.sub.1-C.sub.6-alkyl), —C(═O)O(C.sub.1-C.sub.6-alkyl), and phenyl;

[0141] Suitable reactive esters are described in the article by Christian A. G. N. Montalbetti and V. Falque in Tetrahedron 61 (2005) 10827 and preferably include aromatic esters based on p-nitrophenol, pentafluorophenol, 2,4,5-trichlorophenol, N-hydroxy-5-norbornene-endo-2,3-dicarboxyimide, hydroxybenzotriazole, 1-hydroxy-7-azabenzoptriazole, sulfo-N-hydroxysuccinimide, or N-hydroxysuccinimide, esters based on phosphonium-, uronium-, or guanidinium-based coupling reagents, and triazinyl or pyridinium esters. Preferred reactive esters are selected from the following reactive esters:

##STR00020##

[0142] In a preferred embodiment of the invention, [0143] Z is in each case independently selected from reactive groups selected from —N.sub.3, —C≡CH, —CH═CH.sub.2, —NH.sub.2, —O—NH.sub.2, —C(═O)OH, —CH(═O), —SH, —OH, maleimido, —NCO, —NCS, —N.sup.+≡N, bromoacetamido, iodoacetamido, aromatic esters based on p-nitrophenol, pentafluorophenol, 2,4,5-trichlorophenol, N-hydroxy-5-norbornene-endo-2,3-dicarboxyimide, hydroxybenzotriazole, 1-hydroxy-7-azabenzoptriazole, sulfo-N-hydroxysuccinimide, or N-hydroxysuccinimide, esters based on phosphonium-, uronium-, or guanidinium-based coupling reagents, triazinyl or pyridinium esters, pyridyl-2-dithio, and 6-substituted 4-chloro-1,3,5-triazin-2-ylamino and 4-chloro-1,3,5-triazin-2-yloxy; wherein the substituent in the 6-position of the 4-chloro-1,3,5-triazin-2-ylamino or 4-chloro-1,3,5-triazin-2-yloxy is selected from —H, -halogen, —SH, —NH.sub.2, —C.sub.1-C.sub.6-alkyl, —O(C.sub.1-C.sub.6-alkyl), —OAryl, —S(C.sub.1-C.sub.6-alkyl), —SAryl, —N(C.sub.1-C.sub.6-alkyl).sub.2, and N(Aryl).sub.2; [0144] wherein the carbon atoms of the aforementioned groups are unsubstituted or substituted by one or more substituents selected from —CN, -halogen, —SH, —C(═O)H, —C(═O)OH, C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-haloalkyl, —O(C.sub.1-C.sub.6-alkyl), —C(═O)(C.sub.1-C.sub.6-alkyl), —C(═O)O(C.sub.1-C.sub.6-alkyl), and phenyl;

[0145] In a more preferred embodiment, [0146] Z is in each case independently —NCS or —NH.sub.2.

[0147] As indicated above, A.sub.1 may in one embodiment be a bridging group comprising from one to three separate straight or branched, saturated or unsaturated carbon-based chains including from 1 to 12 carbon atoms, wherein the carbon-based chains are free of or comprise one to ten, same or different groups selected from —O—, —S—, —NH—, —NR.sub.1—, —C(═O)NH—, —NHC(═O)—, —C(═O)NR.sub.1—, —NR.sub.1C(═O)— and —C(═O)—.

[0148] In a preferred embodiment, [0149] A.sub.1 is a bridging group comprising from one to three separate straight or branched, saturated or unsaturated carbon-based chains including from 1 to 12 carbon atoms, wherein the carbon-based chains are free of or comprise one to ten, same or different groups selected from —NR.sub.1—, —C(═O)NH—, —NHC(═O)—, —C(═O)NR.sub.1—, —NR.sub.1C(═O)— and —C(═O)—.

[0150] In connection with the above embodiments regarding A.sub.1, it is preferred that [0151] R.sub.1 is in each case independently selected from C.sub.1-C.sub.6-alkyl, and from the option of representing one of the one or two groups -L-Z.

[0152] Preferably, R.sub.1 in each case represents one of the one or two groups -L-Z.

[0153] As indicated above, A.sub.1 may in another embodiment be a bridging chelate moiety -Che.sub.3- as defined above. In this connection, it is preferred that Ar.sub.1 is any one of the above defined groups. Particularly preferably, Ar.sub.1 is the following group:

##STR00021##

[0154] Furthermore, it is preferred in connection with -Che.sub.3- and the preferred option regarding Ar.sub.1 as defined above that [0155] R.sub.2 is in each case independently selected from —C(═O)O.sup.−, —P(═O)O.sub.2.sup.2−, P(═O)MeO.sup.−, —P(═O)PhO.sup.−, and the C.sub.1-C.sub.6-alkyl esters thereof, and from the option of representing one of the one or two groups -L-Z; [0156] R.sub.3 is in each case independently selected from the bridging groups —C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, and —P(═O)O.sub.2.sup.2-.

[0157] More preferably, [0158] R.sub.2 is in each case —C(═O)O.sup.−; [0159] R.sub.3 is in each case selected from the bridging groups —C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-S—.

[0160] Furthermore, it is preferred in connection with -Che.sub.3- and the preferred option regarding Ar.sub.1 as defined above that [0161] G is in each case independently selected from i) a conjugating group, ii) a single bond, and iii) hydrogen; [0162] wherein each conjugating group comprises 1, 2, or 3 moieties selected from —CH═CH—, —C≡C—, —C(═O)—, and biradicals of 5 to 10-membered aromatic or heteroaromatic monocyclic or bicyclic rings, wherein the heteroaromatic ring contains one or more, same or different heteroatoms N, O, or S, and wherein the aromatic or heteroaromatic monocyclic or bicyclic rings are unsubstituted or substituted by 1 to 5 same or different substituents R.sub.5, wherein R.sub.5 is defined as above; [0163] wherein each conjugating group, if present in a terminal position, may further comprise a terminal group, which is selected from the group consisting of —H, -halogen, —CN, —CH.sub.3, and from the option of representing one of the one or two groups -L-Z.

[0164] Preferably, [0165] G is in each case independently selected from i) a conjugating group, ii) a single bond, and iii) hydrogen; [0166] wherein each conjugating group comprises 1, 2, or 3 moieties selected from —CH═CH—, —C(═O)—, phenylene, biphenylene, naphthylene, pyridylene, pyrazinylene, pyrimidinylene, pyridazinylene, furylene, thienylene, pyrrolylene, imidazolylene, pyrazolylene, thiazolylene, isothiazolylene, oxazolylene, isoxazolylene, fyrazanylene, 1,2,4-triazol-3,5-ylene, and oxadiazolylene, wherein the aromatic or heteroaromatic monocyclic or bicyclic rings are unsubstituted or substituted by 1 to 5 same or different substituents R.sub.5.

[0167] More preferably, [0168] G is in each case independently selected from i) a conjugating group, ii) a single bond, and iii) hydrogen; [0169] wherein each conjugating group is independently selected from phenylene-C≡C—, phenylene, thienylene, and furylene.

[0170] As indicated above, Che.sub.1 and Che.sub.2 are independently selected from the chelate moieties Che I, Che II, Che III, Che IV, Che V, Che VI, and Che VII as defined above. In a preferred embodiment, Che.sub.1 and Che.sub.2 are independently selected from the chelate moieties Che I and Che IV. In this connection with the aforementioned chelate moieties, in particular in connection with the chelate moieties Che I and Che IV, it is preferred that Ar.sub.2 is any one of the above defined groups. Particularly preferably, Ar.sub.2 is the following group:

##STR00022##

[0171] It is to be understood that -Che.sub.1- and Che.sub.2- may be identical or different from each other. Preferably, -Che.sub.1- and Che.sub.2- are identical.

[0172] Furthermore, it is preferred in connection with -Che.sub.1- and -Che.sub.2- as well as the above mentioned preferred embodiments in this connection that [0173] R.sub.2 is in each case independently selected from —C(═O)O.sup.−, —P(═O)O.sub.2.sup.2−, P(═O)MeO.sup.−, —P(═O)PhO.sup.−, and the C.sub.1-C.sub.6-alkyl esters thereof, and from the option of representing one of the one or two groups -L-Z; [0174] R.sub.3 is in each case independently selected from the bridging groups —C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-S—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, and —P(═O)O.sub.2.sup.2-; [0175] R.sub.4 is in each case independently selected from —CH.sub.2N(CH.sub.2C(═O)O.sup.−).sub.2, —CH.sub.2N(CH.sub.2P(═O)O.sub.2.sup.2−).sub.2, —CH.sub.2N(CH.sub.2P(═O)MeO.sup.−).sub.2, —CH.sub.2N(CH.sub.2P(═O)PhO.sup.−).sub.2, and from the options defined for R.sub.2.

[0176] More preferably, [0177] R.sub.2 is in each case —C(═O)O.sup.−; [0178] R.sub.3 is in each case selected from the bridging groups —C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-NHC(═O)—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-O—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-C(═O)NH—, —C(═O)NH—(C.sub.1-C.sub.6-alkylene)-S—; [0179] R.sub.4 is in each case —C(═O)O.sup.−.

[0180] Furthermore, it is preferred in connection with -Che.sub.1- and -Che.sub.2- as well as the above mentioned preferred embodiments in this connection that [0181] G is in each case independently selected from i) a conjugating group, ii) a single bond, and iii) hydrogen; [0182] wherein each conjugating group comprises 1, 2, or 3 moieties selected from —CH═CH—, —C≡C—, —C(═O)—, and biradicals of 5 to 10-membered aromatic or heteroaromatic monocyclic or bicyclic rings, wherein the heteroaromatic ring contains one or more, same or different heteroatoms N, O, or S, and wherein the aromatic or heteroaromatic monocyclic or bicyclic rings are unsubstituted or substituted by 1 to 5 same or different substituents R.sub.5, wherein R.sub.5 is defined as above; [0183] wherein each conjugating group, if present in a terminal position, may further comprise a terminal group, which is selected from the group consisting of —H, -halogen, —CN, —CH.sub.3, and from the option of representing one of the one or two groups -L-Z.

[0184] Preferably, [0185] G is in each case independently selected from i) a conjugating group, ii) a single bond, and iii) hydrogen; [0186] wherein each conjugating group comprises 1, 2, or 3 moieties selected from —CH═CH—, —C≡C—, —C(═O)—, phenylene, biphenylene, naphthylene, pyridylene, pyrazinylene, pyrimidinylene, pyridazinylene, furylene, thienylene, pyrrolylene, imidazolylene, pyrazolylene, thiazolylene, isothiazolylene, oxazolylene, isoxazolylene, fyrazanylene, 1,2,4-triazol-3,5-ylene, and oxadiazolylene, wherein the aromatic or heteroaromatic monocyclic or bicyclic rings are unsubstituted or substituted by 1 to 5 same or different substituents R.sub.5.

[0187] More preferably, [0188] G is in each case independently selected from i) a conjugating group, ii) a single bond, and iii) hydrogen; [0189] wherein each conjugating group is independently selected from phenylene-C≡C—, phenylene, thienylene, and furylene.

[0190] The conjugating groups in the compounds of the present invention may be modified by a hydrophilic group as defined in connection with R.sub.5, R.sub.6, and R.sub.7. Examples of hydrophilic groups are mono- and oligosaccharides, such as monosaccharides and disaccharides, oligoalkylene glycols (e.g. those having 1-20 repeating units) such as oligoethylene glycol and oligopropylene glycol, etc. In a preferred embodiment, the hydrophilic group is selected from monosaccharides, disaccharides, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—H, —(CH.sub.2).sub.1-3—O—(CH.sub.2CH.sub.2O).sub.0-5—C.sub.1-4-alkyl, —O—(CH.sub.2CH.sub.2O).sub.1-6—H, and —O—(CH.sub.2CH.sub.2O).sub.1-6—C.sub.1-4-alkyl, in particular monosaccharides.

[0191] In the present context, the term “monosaccharide” is intended to mean C.sub.5-C.sub.7 carbohydrates being either in the acyclic or in cyclic form. Examples of monosaccharides are C.sub.6 carbohydrates, e.g. those selected from

##STR00023##

[0192] In the present context, the term “disaccharide” is intended to mean two monosaccharides (cf. above) linked together, preferably via glycosidic bonds.

[0193] It is to be understood that the hydrophilic group may also comprise a spacer, i.e. a distance-making biradical, such as the ones defined in connection with the group L.

[0194] In a particularly preferred embodiment, the compound of formula (I) is any one of the compounds 6, 7, 26, 27, 28, 29, 35, 36, 45, 46, 47, 48, 56, 57, 58, 59, 65, and 66 as defined herein (see FIGS. 2, 4, 6, 8, 9, and Schemes 2 and 13).

[0195] As indicated above, the present invention also relates to compounds of formula (II), also referred to as ligands and ligand esters, which may be used as precursors for the compounds of formula (I), i.e. the (lanthanide) chelates. The above defined preferred embodiments regarding groups and substituents of the compounds of formula (I) are also preferred for the compounds of formula (II). However, the chelate moieties do not contain a lanthanide ion.

[0196] In yet another aspect, the present invention relates to a detection agent comprising a biospecific binding reactant conjugated to a compound of formula (I) or a salt thereof or a compound of formula (II) or a salt thereof. The detection agent is a detectable molecule comprising a biospecific binding reactant conjugated to a luminescent lanthanide chelate of formula (I) or a precursor of formula (II) of the present invention. Conjugation, i.e. the formation of a covalent bond, is typically achieved by means of the reactive group Z of said chelate. The biospecific binding reactant should be capable of specifically binding an analyte of interest for the purpose of quantitative or qualitative analysis of said analyte in a sample.

[0197] Examples of biospecific binding reactants are those selected from an antibody, an antigen, a receptor ligand, a specific binding protein, a DNA probe, a RNA probe, an oligopeptide, an oligonucleotide, a modified oligonucleotide (e.g. a locked nucleic acid (LNA) modified oligonucleotide), a modified polynucleotide (e.g. an LNA modified polynucleotide), a protein, an oligosaccaride, a polysaccharide, a phospholipid, a PNA, a steroid, a hapten, a drug, a receptor binding ligand, and lectin. In a preferred embodiment, the biospecific binding reactant is selected from antibodies, e.g. Troponin I antibodies (anti-TnI).

[0198] In another aspect, the present invention relates to a method of detecting an analyte in a biospecific binding assay, said method comprising the steps of:

a) forming a complex between the analyte and the compound of formula (I) or formula (II) or the detection agent of the invention;
b) exciting said complex with a radiation having an excitation wavelength of the compound of formula (I) or formula (II) or the detection agent of the invention, thereby forming an excited complex; and
c) detecting emission radiation emitted from said excited complex.

[0199] The method follows conventional assay steps as will be evident for the skilled person. Preferred excitation wavelengths are in the range of from 320-370 nm. The skilled person is aware that the exact excitation wavelengths depend on the specific structure of the ligand. Further, the skilled person is aware that the emission wavelengths are specific for the used lanthanide (Tb.sup.3+, Eu.sup.3+, Dy.sup.3+, Sm.sup.3+), In case of Eu.sup.3+, which is preferred according to the invention, the preferred measured emission wavelength is in the range of from 610-620 nm, preferably about 615 nm.

[0200] In yet another aspect, the present invention relates to a method of labelling a biospecific binding reactant with a compound of the invention, comprising the steps of

a) providing a biospecific binding reactant; and
b) conjugating the biospecific binding reactant with the compound of formula (I) or formula (II).

[0201] The resulting compound may be a detection agent of the invention. The conjugation may occur via the reaction group Z of the compound of formula (I) or formula (II).

[0202] In another aspect, the present invention relates to the use of a compound of formula (I) or formula (II) of the invention for the in vitro detection of an analyte in a sample. The present invention thus also relates to the use of a detection agent of the invention in a specific bioaffinity based binding assay, e.g. utilizing time-resolved fluorometric determination of a specific luminescence. In one embodiment, the specific bioaffinity based binding assay is a heterogeneous immunoassay, a homogenous immunoassay, a DNA hybridization assay, a receptor binding assay, an immunocytochemical or an immunohistochemical assay.

[0203] In another aspect, the present invention relates to the use of a compound of formula (I) or formula (II) of the invention or the detection agent of the invention in bio-imaging applications. Such a use is particularly advantageous if the compound of formula (I) or formula (II) of the invention is a molecule with a neutral net charge or almost neutral net charge (i.e. the molecule comprises an overall net charge of from −3 to +5). This of course depends on the selection of the substituents. However, the compounds of the invention or the detection agent of the invention may for example be used as a contrasting agent. The contrasting agent may e.g. be used in MM or PET applications. The compounds of the invention or the detection agent of the invention may further be used for microscopy applications, e.g. in cell culture experiments, such as in confocal laser scanning microscopy and or hybridization experiments.

[0204] Still another aspect of the invention relates to a solid support material conjugated with a compound of formula (I) or formula (II) of the invention or the detection agent of the invention. The compound or the detection agent of the invention is typically immobilized to the solid support material either covalently or non-covalently.

[0205] In some embodiments, the solid support material is selected from a nanoparticle, a microparticle, a slide, a plate, and a solid phase synthesis resin.

[0206] The present invention is further illustrated by the following examples.

EXAMPLES

[0207] The following non-limiting examples are aimed to further demonstrate the invention.

[0208] The structures and synthetic routes employed are presented in Schemes 1-13 as provided at the end of the experimental part. Furthermore, reference is made to FIGS. 1-9. FIG. 10 provides the chemical structures of reference chelates Ref 1-3 used in this application.

[0209] In compounds 6 and 7 (Scheme 2), in compounds 65 and 66 (Scheme 13) as well as in compounds 45-48 (FIG. 6), both Eu(III) ions form equal coordination bonds and one of the two Eu(III) ions forms coordination bonds with two pyridine nitrogen atoms, three tertiary nitrogen atoms connected to the pyridine rings with CH.sub.2 bridges and four negatively charged oxygen atoms of the carboxylic groups. In compounds 26-29 (FIG. 2) and in compounds 35 and 36 (FIG. 4), both Eu(III) ions form equal coordination bonds and one of the two Eu(III) ions forms coordination bonds with three pyridine nitrogen atoms, two tertiary nitrogen atoms connected to the pyridine rings with CH.sub.2 bridges and four negatively charged oxygen atoms of the carboxylic groups (two carboxylic groups between the pyridine moieties and two carboxylic groups in pyridine rings). In compounds 56 and 57 (FIG. 8) and in compounds 58 and 59 (FIG. 9) one of the three Eu(III) ion forms coordination bonds with two pyridine nitrogen atoms, two tertiary nitrogen atoms connected to the pyridine rings with CH.sub.2 bridges, two negatively charged oxygen atoms of the carboxylic groups and two amide groups (—CONH—); the rest two Eu(III) ions form equal coordination bonds and one of the rest two Eu(III) ions forms coordination bonds with three pyridine nitrogen atoms, two tertiary nitrogen atoms connected with pyridine rings with CH.sub.2 bridges and four negatively charged oxygen atoms of the carboxylic groups (two carboxylic groups between the pyridine moieties and two carboxylic groups in pyridine rings).

[0210] .sup.1H-NMR spectra were recorded with Bruker AVANCE DRX 500 and 600 MHz. Tetramethyl silane was used as internal reference. Mass spectra were recorded PerSeptive Biosystems Voyager DE-PRO MALDI-TOF instrument using α-cyano-4-cinnamic acid matrix. UV-Vis spectra were recorded on Pharmacia Ultrospec 3300 pro. Fluorescence efficiencies were determined with Perkin-Elmer Wallac Victor plate fluorometer. Eu-content of Eu-chelates and labelled antibodies were measured by using ICP-MS instrument, PerkinElmer 6100 DRC Plus, in quantitative mode. The excitation, emission spectra and decay times were recorded on a Varian Cary Eclipse fluorescence spectrometer.

[0211] Conditions for HPLC purification runs: Reversed phase HPLC (RP-18 column). The solvents were A: triethyl ammonium acetate buffer (20 mM, pH7) and B: 50% acetonitrile in triethyl ammonium acetate buffer (20 mM, pH7). The gradient was started from 5% of solvent B and the amount of solvent B was linearly raised to 100% in 30 minutes.

[0212] Column chromatography was performed with columns packed with silica gel 60 (Merck). FC=Flash chromatography, RT=room temperature.

Example 1. Synthesis of Compound 2

[0213] A mixture of compound 1 (0.23 g, 0.51 mmol; Takalo, H., et al., Helv. Chim. Acta 79(1996)789)), tert-butyl (6-aminohexyl)carbamate (55 mg, 0.25 mmol), dry K.sub.2CO.sub.3 (0.28 g, 2.04 mmol) and dry MeCN (10 ml) under argon was stirred at RT overnight. After filtration and washes with MeCN, the product (0.24 g, 100%) was used for the next step without further purifications. .sup.1H NMR (CDCl.sub.3, δ ppm): 7.71 (2H, d, J=0.95 Hz), 7.59 (2H, d, J=0.95 Hz), 4.56 (2H, s), 4.15 (8H, q, J=7.15 Hz), 4.02 (4H, s), 3.78 (4H, s), 3.60 (8H, s), 3.14-3.01 (2H, m), 2.62-2.49 (2H, m), 1.59-1.49 (2H, m), 1.49-1.41 (6H, m), 1.27 (12H, t, J=7.15 Hz). .sup.13C NMR (CDCl.sub.3, δ ppm): 170.93, 160.44, 160.30, 160.10, 134.17, 123.49, 124.28, 78.90, 60.93, 59.74, 54.94, 54.43, 40.42, 29.95, 28.34, 26.85, 26.53, 14.15. MALDI TOF-MS mass: calculated (M+H.sup.+) 957.30, 959.30 961.30; found 957.15, 959.16, 961.07.

Example 2. Synthesis of Compound 3

[0214] A mixture of the compound 2 (0.23 g, 0.24 mmol) and N-(4-ethynylphenyl)-2,2,2-trifluoroacetamide (0.12 g, 0.58 mmol; Sund, H., et al. Molecules 22(2017)1807) in dry TEA (1 ml) and THF (2 ml) was de-aerated with argon. After addition of bis(triphenylphosphine)palladium(II) chloride (10 mg, 14 μmol) and CuI (6 mg, 28 μmol), the mixture was stirred overnight at 55° C. After evaporation to dryness, the product (0.27 g, 91%) was purified by FC (silica gel, 5% EtOH/CH.sub.2Cl.sub.2). .sup.1H NMR (CDCl.sub.3, δ ppm): 8.84 (2H, s), 7.65 (2H, s), 7.59 (2H, s), 7.56 (4H, d, J=8.70 Hz), 7.50 (4H, d, J=8.70 Hz), 4.17 (8H, q, J=7.10 Hz), 4.04 (4H, s), 3.76 (4H, s), 3.61 (8H, s), 3.08-3.01 (2H, m), 2.57-2.50 (2H, m), 1.55-1.47 (2H, m), 1.47-1.38 (6H, m), 1.26 (12H, t, J=7.1 Hz). .sup.13C NMR (CDCl.sub.3, δ ppm): 171.05, 160.31, 158.40, 155.90, 155.20, 154.93, 154.63, 154.38, 135.60, 132.67, 131.85, 123.08, 122.61, 121.11, 120.23, 118.98, 116.70, 114.41, 111.71, 92.03, 88.07, 78.92, 60.59, 60.51, 59.70, 54.89, 53.33, 40.45, 29.60, 28.30, 26.97, 26.55, 14.14. MALDI TOF-MS mass: calculated (M+H.sup.+) 1223.53; found 1223.90.

Example 3. Synthesis of Compound 4

[0215] The mixture of the compound 3 (0.25 g, 0.19 mmol) in TFA (2.8 ml) was stirred for 2 h at RT and evaporated to dryness. The residue was co-evaporated from diethyl ether (2×20 ml) and dissolved in dry MeCN (6 ml). After an addition of DIPEA (0.66 ml, 3.8 mmol) and a solution of compound 1 (0.17 g, 0.38 mmol) in dry MeCN (6 ml), the mixture was stirred for 67 h at RT. After evaporation to dryness, the residue was dissolved in CH.sub.2Cl.sub.2 (30 ml), washed with H.sub.2O (3×15 ml) and dried with Na.sub.2SO.sub.4. The product (0.31 g, 88%) was purified by (silica gel, 10% EtOH/CH.sub.2Cl.sub.2). .sup.1H NMR (CDCl.sub.3, δ ppm): 9.04 (2H, s), 7.66 (2H, s), 7.63 (2H, s), 7.58 (2H, s), 7.54 (2H, d. J=8.60 Hz), 7.49 (2H, s), 7.45 (2H, d, J=8.60 Hz), 4.15 (8H, q, J=7.2 Hz), 4.15 (8H, q, J=7.2 Hz), 4.04 (4H, s), 3.96 (4H, s), 3.75 (4H, s), 3.66 (4H, s), 3.61 (8H, s), 3.57 (8H, s), 2.58-2.51 (2H, m) 2.51-2.42 (2H, m), 1.57-1.46 (4H, m), 1.27 (12H, t, J=7.2 Hz), 1.25 (12H, t, J=7.2 Hz), 1.20-1.10 (4H, m). .sup.13C NMR (CDCl.sub.3, δ ppm): 171.07, 171.02, 160.84, 160.38, 159.87, 158.40, 155.82, 155.53, 155.22, 154.91, 135.72, 134.08, 132.58, 131.94, 124.26, 123.97, 123.07, 122.57, 121.12, 120.03, 119.04, 116.73, 114.44, 112.15, 92.09, 88.02, 60.67, 60.60, 60.54, 60.48, 59.82, 59.70, 59.46, 59.24, 55.40, 54.97, 54.88, 54.56, 29.59, 27.25, 14.15, 14.12. MALDI TOF-MS mass: calculated (M+H.sup.+) 1865.58, 1866.58; found 1864.34, 1866.53.

Example 4. Synthesis of Compound 5

[0216] A mixture of the compound 4 (0.29 g, 0.155 mmol), triethyl 2,2′,2″-{[4-(ethynyl)benzene-1,3,5-triyl]-tris(oxy)triacetate (0.15 g, 0.373 mmol; Sund, H., et al. Molecules 22(2017)1807) in dry TEA (1 ml) and THF (2 ml) was de-aerated with argon. After addition of bis(triphenylphosphine)palladium(II) chloride (10 mg, 14 μmol) and CuI (5 mg, 28 μmol), the mixture was stirred for 24 h at 55° C. After evaporation to dryness, the product (0.32 g, 82%) was purified by FC (silica gel, 5% EtOH/CH.sub.2Cl.sub.2). MALDI TOF-MS mass: calculated (M+2H.sup.+); 2522.59, found 2523.66.

Example 5. Synthesis of Compound 6

[0217] Compound 5 (0.30 g, 0.119 mmol) in 0.5 M KOH/EtOH (18.5 ml) was stirred for 30 min at RT. After an addition of H.sub.2O (9 ml), the mixture was further stirred at RT for 2 h. After EtOH evaporation and an additional overnight stirring at RT, the pH was adjusted to 6.5 by addition of 6 M HCl. EuCl.sub.3 (92 mg, 0.250 mmol) in water (1 ml) was added within 5 min and the pH was maintained at 6.0-6.5 with suitable additions of solid NaHCO.sub.3. After stirring overnight at RT, the pH was adjusted to 8.5 with 1M NaOH. The precipitate was removed by centrifugation and the supernatant evaporated to dryness. The product was purified by HPLC. Yield: 0.26 g (72%). R.sub.f(HPLC): 16.5 min. UV: 352 nm.

Example 6. Synthesis of Compound 7

[0218] Compound 6 (0.112 g, 37 μmol) in H.sub.2O (1.4 ml) was added within 5 min to a mixture of CSCl.sub.2 (29 μl, 0.52 mmol) and NaHCO.sub.3 (49 mg, 0.59 mmol) and CHCl.sub.3 (1.4 ml). After stirring for 20 min at RT, the aqueous phase was washed with CHCl.sub.3 (3×1.4 ml). The product was precipitated with acetone, centrifuged and washed with acetone. The product was used for the antibody labelling without any further purifications.

Example 7. Synthesis of Compound 8

[0219] A mixture of 4-nitrophenol (1.39 g, 10 mmol), 1,6-diaminohexane (1.54 ml, 10 mmol), dry Na.sub.2CO.sub.3 (4.24 g, 40 mmol) and dry DMF (25 ml) was for 2.5 h at 100° C. After evaporation to dryness and an addition of CH.sub.2Cl.sub.2 (50 ml), the mixture was washed with H.sub.2O (25 ml), 2M NaOH (25 ml), 5% NaHCO.sub.3 (25 ml), H.sub.2O (25 ml) and dried with Na.sub.2SO.sub.4 (25 ml). The product (0.84 g, 28%) was purified by FC (silica gel, 10% EtOAc/petroleum ether). .sup.1H NMR (D.sub.6-DMSO, δ ppm): 8.20 (2H, d, J=9.3 Hz), 7.13 (2H, d, J=9.3 Hz), 4.12 (2H, t, J=6.5 Hz), 3.54 (2H, t, J=6.7 Hz), 1.86-1.79 (2H, m), 1.79-1.72 (2H, m), 1.47-1.43 (4H, m). .sup.13C NMR (D.sub.6-DMSO, δ ppm): 164.49, 141.17, 126.34, 115.44, 68.99. 35.52, 32.62, 28.69, 27.69, 23.99. MALDI TOF-MS mass: calculated (M+H.sup.+); 302.04, 304.04, found 301.65, 303.65.

Example 8. Synthesis of Compound 9

[0220] After stirring of a mixture of N,N′-(hexane-1,6-diyl)bis(2,2,2-trifluoroacetamide) (0.98 g, 3.18 mmol) and NaH (0.27 g, 6.68 mmol; 60% in oil) in dry DMF (10 ml) for 30 min at RT, 3-nitrobenzylbromide (1.44 g, 6.67 mmol) was added. The reaction mixture was stirred for overnight at RT, and evaporated to dryness. After an addition of H.sub.2O (50 ml), the solid crude product was filtrated and washed with H.sub.2O. A dried solid material was suspended in 30% EtOAc/petroleum ether, filtrated and washed with EtOAc/petroleum ether. Yield: 1.67 g (91%). MALDI TOF-MS mass: calculated (M+H.sup.+) 579.17; found 579.02.

Example 9. Synthesis of Compound 10

[0221] After stirring of a mixture of N,N′-(hexane-1,6-diyl)bis(2,2,2-trifluoroacetamide) (0.42 g, 1.36 mmol) and NaH (0.11 g, 2.86 mmol; 60% in oil) in dry DMF (5 ml) for 30 min at RT, a solution of the compound 8 (0.82 g, 2.72 mmol) in dry DMF (6 ml) was added. The reaction mixture was stirred for 22 h at 75° C., and evaporated to dryness. The residue was dissolved in CH.sub.2Cl.sub.2 (40 ml), was washed with H.sub.2O (2×20 ml) and dried with Na.sub.2SO.sub.4. The product (0.51 g, 50%) was purified by FC (silica gel, from 30% to 40% EtOAc/petroleum ether). .sup.1H NMR (D.sub.6-DMSO, δ ppm): 9.38 (2H, s), 8.20 (4H, d, J=9.1 Hz), 7.13 (4H, d, J=9.1 Hz), 4.12 (4H, t, J=5.8 Hz), 3.38-3.30 (4H, m), 3.17 (4H, q, J=6.5 Hz), 1.80-1.70 (4H, m), 1.64-1.50 (8H, m), 1.50-1.40 (8H, m), 1.35-1.20 (12H, m). .sup.13C NMR (D.sub.6-DMSO, δ ppm): 154.49, 156.75, 156.47, 155.89, 155.61, 141.18, 126.35, 120.37, 118.96, 115.78, 115.44, 113.51, 68.99, 68.96, 47. 42, 46.83, 28.70, 28.56, 28.53, 28.51, 26.66, 26.64, 26.30, 26.26, 26.19, 26.15, 26.07, 25.91, 25.49, 25.43, MALDI TOF-MS mass: calculated (M+H.sup.+) 751.32; found 751.33.

Example 10. Synthesis of Compound 11

[0222] After stirring of a mixture of the compound 9 (1.46 g, 2.52 mmol) in 0.5M KOH (20 ml) and CH.sub.2Cl.sub.2 (30 ml) overnight at RT, a second set of CH.sub.2Cl.sub.2 (30 ml) was added, and the mixture was washed with H.sub.2O (2×20 ml) and dried with Na.sub.2SO.sub.4. Yield: 0.97 g (100%). .sup.1H NMR (D.sub.6-DMSO, δ ppm): 8.20 (2H, s), 8.07 (2H, dd, J=7.9 and 1.6 Hz), 7.77 (2H, d, J=7.9 Hz), 7.59 (2H, t, J=7.9 Hz), 3.80 (4H, s), 2.46 (4H, t, J=7.0 Hz), 1.48-1.37 (4H, m), 1.32-1.24 (4H, m). .sup.13C NMR (D.sub.6-DMSO, δ ppm): 148.20, 144.32, 134.91, 129.83, 122.53, 121.76, 52.38, 48.98, 29.91, 27.16. MALDI TOF-MS mass: calculated (M+H.sup.+) 387.21; found 386.81.

Example 11. Synthesis of Compound 12

[0223] Compound 12 was synthesized from compound 10 using a method analogous to the synthesis described in example 10. Yield: 89%. .sup.1H NMR (D.sub.6-DMSO, δ ppm): 8.19 (4H, d, J=9.3 Hz), 7.13 (4H, d, J=9.3 Hz), 4.14-4.08 (4H, m), 2.48-2.43 (8H, m), 1.78-1.70 (4H, m), 1.46-1.30 (16H, m), 1.29-1.22 (4H, m). .sup.13C NMR (D.sub.6-DMSO, δ ppm): 164.53, 141.20, 126.31, 115.45, 69.11, 49.87, 49.78, 30.05, 29.94, 28.86, 27.34, 27.00, 26.81, 25.79. MALDI TOF-MS mass: calculated (M+H.sup.+) 559.35; found 559.18.

Example 12. Synthesis of Compound 13

[0224] N-hydroxysuccinimide (0.19 g, 1.69 mmol) and N,N-dicyclohexylcarbodiimide (0.35 g, 1.69 mmol) was added to a solution of 2-(4-iodophenoxy)acetic acid (0.47 g, 1.69 mmol) in dry 1,4-dioxane (5 ml). After stirring for 2.5 h at RT, a solution of the compound 11 (0.33 g, 0.85 mmol) in dry 1,4-dioxane (2.5 ml) was added, and the mixture was stirred for 24 h at RT. The mixture was filtrated, the solid material washed with 1,4-dioxane (4×5 ml) and the filtrate was evaporated to dryness. The residue was dissolved in CH.sub.2Cl.sub.2 (40 ml), washed with 10% NaOH (20 ml), 5% NaHCO.sub.3 (20 ml), H.sub.2O (20 ml) and dried with Na.sub.2SO.sub.4. The product (0.42 g, 55%) was purified by FC (silica gel, from 0% to 1% EtOH/CH.sub.2Cl.sub.2). MALDI TOF-MS mass: calculated (M+H.sup.+) 907.08; found 906.94.

Example 13. Synthesis of Compound 14

[0225] After an addition of DIPEA (0.24 ml, 1.36 mmol) and PyAOP (0.35 g, 0.75 mmol) into a solution of compound 12 (0.19 g, 0.34 mmol) and 2-(4-iodophenoxy)acetic acid (0.19 g, 0.68 mmol) in dry DMF (7.5 ml), the mixture was stirred for 2 h at RT and evaporated to dryness. The product (0.31 g, 84%) was purified by FC (silica gel, from 1% to 2% EtOH/CH.sub.2Cl.sub.2). MALDI TOF-MS mass: calculated (M+Na.sup.+): 1101.77; found 1101.35.

Example 14. Synthesis of Compound 15

[0226] A mixture of 4-bromo-6-hydroxymethyl-2-carboxyethylpyridine (1.56 g, 6.0 mmol; Takalo, H., et al., Helv. Chim. Acta 79(1996)789)), bis(triphenylphosphine)palladium(II) chloride (84 mg, 0.12 mmol) and CuI (46 mg, 0.24 mmol) in dry TEA (5 ml) and THF (10 ml) was de-aerated with argon. After an addition of trimethylsilylacetylene (1.2 ml, 8.4 mmol), the mixture was stirred overnight at RT. After evaporation to dryness, the product (1.66 g, 100%) was purified by FC (silica gel, from 2% to 5% EtOH/CH.sub.2Cl.sub.2).

Example 15. Synthesis of Compound 16

[0227] A mixture of the compound 15 (0.277 g, 1.0 mmol) and PBr.sub.3 (113 μmol) in dry CHCl.sub.3 (10 ml) was stirred for 3 h at RT. After an addition of CHCl.sub.3 (20 ml), the mixture was neutralized with 5% NaHCO.sub.3 (20 ml), the aqueous phase was extracted with CHCl.sub.3 (10 ml), the combined organic phases were dried with Na.sub.2SO.sub.4. The product (0.27 g, 79%) was purified by FC (silica gel, 5% EtOH/CH.sub.2Cl.sub.2). .sup.1H NMR (D.sub.6-DMSO, δ ppm): 7.90 (1H, s), 7.86 (1H, s), 4.74 (2H, s), 4.37 (2H, q, J=7.1 Hz), 1.34 (3H, t, J=7.1 Hz), 0.27 (9H, s). .sup.13C NMR (D.sub.6-DMSO, δ ppm): 164.03, 158.48, 148.59, 132.59, 129.02, 125.90, 102.05, 101.27, 61.99, 33.74, 14.45, −0.16. MALDI TOF-MS mass: calculated (M+H.sup.+) 340.04, 342.04; found 339.62, 341.62.

Example 16. Synthesis of Compound 17

[0228] A mixture of 4-bromo-2,6-dibromomethylpyridine (3.50 g, 10 mmol; Takalo, H., et al. Acta Chem, Scand. Ser. B 248(1988).sub.373), glycine ethyl ester hydrochloride (14.0 g, 0.10 mmol) and di-isopropylethylamine (35 ml) in dry MeCN (130 ml) was stirred overnight at RT. After evaporation to dryness, the residue was dissolved in CH.sub.2Cl.sub.2 (100 ml), washed with H.sub.2O (3×50 ml), dried with Na.sub.2SO.sub.4, and the product was purified by FC (silica gel from 10:20:70 to 15:30:55 TEA/EtOAc/petroleum ether. Yield 2.48 g (64%). .sup.1H NMR (CDCl.sub.3, δ ppm): 7.43 (2H, s), 4.20 (4H, q, J=7.2 Hz), 3.91 (4H, s), 3.46 (4H, s), 2.25-2.15 (2H, bs), 1.28 (6H, t, J=7.2 Hz). .sup.13C NMR (CDCl.sub.3, δ ppm): 172.00, 160.40, 133.83, 60.75, 54.00, 50.34, 14.18. MALDI TOF-MS mass: calculated (M+H.sup.+): 388.09, 390.09, found 388.75, 390.75.

Example 17. Synthesis of Compound 18

[0229] 4-Bromo-6-bromomethyl-2-carboxyethylpyridine (2.06 g, 6.39 mmol; Takalo, H., et al. Helv. Chim. Acta 79(1996).sub.789) was added in small portions to a mixture of the compound 17 (2.49 h, 6.39 mmol), dry K.sub.2CO.sub.3 (3.53 g, 25.6 mmol) in dry MeCN (215 ml) within 2 h at RT. After stirring for 22 h at RT, the mixture was filtrated and the filtrate evaporated to dryness. The product was purified by FC (silica gel 10:25:65 TEA/EtOAc/petroleum ether. Yield 2.03 g (50%). .sup.1H NMR (CDCl3, δ ppm): 8.12 (1H, d, J=1.7 Hz), 8.07 (1H, d, J=1.7 Hz), 7.55 (1H, d, J=1.5 Hz), 7.43 (1H, d, J=1.5 Hz), 4.46 (2H, q, J=7.2 Hz), 4.19 (4H, q, J=7.2 Hz), 4.09 (2H, s), 3.95 (2H, s), 3.90 (2H, s), 2.3-2.1 (1H, bs), 1.43 (3H, t, J=7.2 Hz), 1.29 (3H, t, J=7.2 Hz), 1.29 (3H, t, J=7.2 Hz). .sup.13C NMR (CDCl3, δ ppm): 171.98, 170.81, 164.03, 161.57, 160.41, 159.70, 148.54, 134.26, 133.90, 129.13, 126.95, 124.60, 123.77, 62.16, 60.75, 60.63, 59.61, 59.48, 55.38, 54.00, 50.36, 14.19, 14.17, 14.14. MALDI TOF-MS mass: calculated for (M+H.sup.+): 629.06, 631.05. 633.06, found 629.54, 631.56, 633.54.

Example 18. Synthesis of Compound 19

[0230] A mixture of compound 18 (0.75 g, 1.19 mmol) and triethyl 2,2′,2″-{[4-(ethynyl)benzene-1,3,5-triyl]-tris(oxy)triacetate (1.17 g, 2.86 mmol) in dry TEA (10 ml) and DMF (20 ml) was de-aerated with argon. After an addition of bis(triphenylphosphine)palladium(II) chloride (32 mg, 48 μmol) and CuI (18 mg, 95 μmol), the mixture was stirred overnight at 55° C. After evaporation to dryness, the product (1.23 g, 80%) was purified by FC (silica gel, from 10:90:0 to 10:88:2 EtOH/CH.sub.2Cl.sub.2/TEA). .sup.1H NMR (D.sub.6-DMSO, δ ppm): 7.86 (1H, s), 7.78 (1H, s), 7.35 (1H, s), 7.31 (1H, s), 6.25 (2H, s), 6.22 (2H, s), 4.88 (4H, s), 4.86 (4H, s), 4.82 (2H, s) 4.81 (2H, s), 4.34 (2H, q, J=7.1 Hz), 4.20-4.13 (12H, m), 4.09-4.02 (4H, m), 3.96 (2H, s), 3.80 (2H, s), 3.54 (2H, s), 3.35 (2H, s), 1.32 (3H, t, J=7.1 Hz), 1.24-1.14 (24H, m). .sup.13C NMR (D.sub.6-DMSO, δ ppm): 172.27, 171.12, 168.55, 168.54, 168.50, 164.66, 161.01, 160.85, 160.68, 160.47, 160.18, 158.43, 147.80, 133.34, 132.55, 127.05, 124.50, 122.53, 121.56, 94.61, 94.26, 93.88, 93.77, 93.41, 93.38, 89.00, 87.20, 65.77, 65.72, 65.38, 61.67, 61.08, 60.26, 59.25, 59.19, 54.93, 53.96, 50.18, 46.04, 14.43, 14.41, 14.38, 14.36. MALDI TOF-MS mass: calculated (M+H.sup.+) 1285.49; found 1285.26.

Example 19. Synthesis of Compound 20

[0231] A mixture of compound 19 (0.64 g, 0.50 mmol), 16 (0.20 g, 0.60 mmol) and dry K.sub.2CO.sub.3 (0.28 g, 2.0 mmol) in dry MeCN (10 ml) was stirred overnight at RT. The mixture was filtrated, inorganic salt was washed with MeCN and the filtrate was evaporated to dryness. The product (0.54 g, 70%) was purified by FC (silica gel, from 5% to 10% EtOH/CH.sub.2Cl.sub.2). .sup.1H NMR (D.sub.6-DMSO, δ ppm): 7.85 (1H, d, J=1.0 Hz), 7.78 (1H, d. J=1.0 Hz), 7.77 (1H, s), 7.37 (1H, s), 7.34 (1H, s), 6.24 (2H, s), 6.23 (2H, s), 4.87 (4H, s), 4.85 (4H, s), 4.82 (2H, s), 4.81 (2H, s), 4.36-4.28 (4H, m), 4.20-4.12 (12H, m), 4.07-4.01 (6H, m), 4.01 (2H, s), 3.95 (2H, s), 3.92 (2H, s), 3.53 (2H, s), 3.50 (2H, s), 1.31 (3H, t, J=7.2 Hz), 1.29 (3H, t, J=7.1 Hz), 1.24-1.13 (26H, m), 0.21 (9H, s). .sup.13C NMR (D.sub.6-DMSO, δ ppm): 171.13, 171.08, 168.53, 168.49, 164.65, 164.39, 161.05, 160.99, 160.85, 160.62, 160.47, 158.61, 158.52, 147.77, 133.38, 132.65, 131.70, 127.94, 127.06, 124.98, 124.52, 123.03, 122.96, 101.92, 100.89, 94.50, 94.28, 93.89, 93.78, 93.41, 88.99, 87.42, 65.77, 65.73, 65.39, 61.72, 61.65, 61.07, 61.03, 60.27, 60.24, 59.40, 59.37, 59.20, 55.27, 55.21, 54.89, 14.37, −0.24. MALDI TOF-MS mass: calculated (M+H.sup.+) 1544.60; found 1544.55.

Example 20. Synthesis of Compound 21

[0232] A mixture of compound 20 (1.43 g, 0.922 mmol) and tetrabutylammonium fluoride (0.29 mg, 1.11 mmol) in CH.sub.2Cl.sub.2 (30 ml) was stirred for 70 min at RT. After an addition of CH.sub.2Cl.sub.2 (30 ml), the mixture was washed with 10% aqueous citric acid solution (30 ml), H.sub.2O (30 ml) and dried with Na.sub.2SO.sub.4. The product (1.36 g, 100%) was used for the next step without any further purifications. .sup.1H NMR (D.sub.6-DMSO, δ ppm): 7.85 (1H, d, J=1.1 Hz), 7.84 (1H, s), 7.83 (1H, s), 7.79 (1H, d, J=1.1 Hz), 7.37 (1H, s), 7.36 (1H, s), 6.24 (2H, s), 6.22 (2H, s), 4.87 (4H, s), 4.86 (4H, s), 4.82 (2H, s), 4.81 (2H, s), 4.59 (1H, s), 4.36 (4H, m), 4.21-4.12 (12H, m), 4.07-4.00 (6H, m), 4.02 (2H, s), 3.95 (2H, s), 3.92 (2H, s), 3.52 (2H, s), 3.49 (2H, s), 1.34-1.26 (6H, m), 1.26-1.12 (26H, m). .sup.13C NMR (D.sub.6-DMSO, δ ppm): 171.10, 168.54, 168.52, 168.50, 168.48, 164.66, 164.43, 161.09, 160.99, 160.85, 160.66, 160.47, 158.68, 158.58, 147.84, 144.79, 133.35, 132.63, 131.50, 128.22, 127.09, 125.20, 124.53, 122.96, 122.88, 94.55, 94.27, 93.89, 93.79, 93.40, 88.97, 87.38, 86.48, 80.81, 65.77, 65.73, 65.39, 61.73, 61.65, 61.08, 61.04, 60.26, 60.25, 59.42, 59.34, 59.25, 57.95, 55.10, 54.95, 14.38, 14.35. MALDI TOF-MS mass: calculated (M+H.sup.+) 1472.56; found 1472.45.

Example 21. Synthesis of Compound 22

[0233] A mixture of compound 13 (0.45 g, 0.50 mmol) and SnCl.sub.2x2H.sub.2O (1.12 g, 5.0 mmol) in abs EtOH (50 ml) was stirred for 6 h at 85° C. The mixture was evaporated to ca. half volume, H.sub.2O (20 ml) was added. After neutralization with solid NaHCO.sub.3, the mixture was extracted with CH.sub.2Cl.sub.2 (40 ml) and 25% EtOH/CH.sub.2Cl.sub.2 (3×20 ml) and the combined organic fraction were dried with Na.sub.2SO.sub.4. The product (0.38 g, 90%) was used for the next step without any further purifications. MALDI TOF-MS mass: calculated (M+H.sup.+) 847.55; found 847.11.

Example 22. Synthesis of Compound 23

[0234] Compound 23 was synthesized from compound 14 using a method analogous to the synthesis described in example 21. The product (yield 40%) was purified by FC (silica gel, from 2% to 5% EtOH/CH.sub.2Cl.sub.2). MALDI TOF-MS mass: calculated (M+H.sup.+) 1019.81; found 1019.34.

Example 23. Synthesis of Ligand Ester 24

[0235] A mixture of compound 21 (0.215 g, 0.146 mmol) and 22 (59 mg, 70 μmol) in dry TEA (1 ml) and DMF (2 ml) was de-aerated with argon. After an addition of bis(triphenylphosphine)palladium(II) chloride (10 mg, 14 μmol) and CuI (6 mg, 28 μmol), the mixture was stirred 24 h at RT. After evaporation to dryness, the product (101 mg, 40%) was purified by FC (silica gel, from 5% to 10% EtOH/CH.sub.2Cl.sub.2). MALDI TOF-MS mass: calculated (M+H.sup.+) 3536.40; found 3536.79.

Example 24. Synthesis of Ligand Ester 25

[0236] Compound 25 was synthesized from compounds 23 and 21 using a method analogous to the synthesis described in example 23 stirring for 22 h at 55° C. The product (yield 49%) was purified by FC (silica gel, 5:89:1 EtOH/CH.sub.2Cl.sub.2/TEA). MALDI TOF-MS mass: calculated (M+H.sup.+) 3706.53; found 3706.67.

Example 25. Synthesis of Eu Chelate 26

[0237] A mixture of ligand ester 24 (48 mg, 13.6 μmol), 0.5 M KOH in EtOH (10 ml) and CH.sub.2Cl.sub.2 (5 ml) was stirred for 2 h at RT and evaporated to ca. half volume. After an addition of H.sub.2O (5 ml), the mixture was stirred for 30 min at RT. The rest of EtOH was evaporated and the aqueous solution was stirred overnight at RT. The pH was adjusted to 6.5 by addition of 6 M HCl. EuCl.sub.3 (11 mg, 30 μmol) in water (0.2 ml) was added within 5 min and the pH was maintained at 6.0-6.5 with suitable additions of solid NaHCO.sub.3. After stirring for overnight at RT, the pH was adjusted to 8.5 with 1 M NaOH. The precipitate was removed by centrifugation and the supernatant evaporated to dryness. The product was purified by HPLC. Two isomers were found. Yield: 29 mg (45%). R.sub.f(HPLC): 18.2 and 20.4 min. MALDI TOF-MS mass: calculated (M+H.sup.+) 3273.53; found 3273.46.

Example 26. Synthesis of Eu Chelate 27

[0238] Eu chelate 27 was synthesized from the ligand ester 25 using a method analogous to the synthesis described in example 25. The product (yield 45%) was purified by HPLC. Two isomers were found R.sub.f(HPLC): 22.2 and 24.2 min. MALDI TOF-MS mass: calculated (M+H.sup.+) 3443.70; found 3441.79.

Example 27. Synthesis of Eu Chelate 28

[0239] Eu chelate 28 was synthesized from the Eu chelate 26 using a method analogous to the synthesis described in example 6. The product was used for the antibody labelling without any further purifications. R.sub.f(HPLC): 21.1 and 25.8 min.

Example 28. Synthesis of Eu Chelate 29

[0240] Eu chelate 29 was synthesized from the Eu chelate 27 using a method analogous to the synthesis described in example 6. The product was used for the antibody labelling without any further purifications. R.sub.f(HPLC): 25.5 min.

Example 29. Synthesis of Compound 30

[0241] A mixture of compound 11 (1.08 g, 2.80 mmol), BrCH.sub.2COO.sup.tBu (0.87 ml, 5.88 mmol), dry K.sub.2CO.sub.3 (1.55 g, 11.2 mmol) in dry MeCN (50 ml) was stirred for 24 h at RT. The mixture was evaporated to dryness and the product (1.54 g, 90%) was purified by FC (silica gel, from CH.sub.2Cl.sub.2 to 2% MeOH/CH.sub.2Cl.sub.2). MALDI TOF-MS mass: calculated (M+H.sup.+) 615.73; found 615.12.

Example 30. Synthesis of Compound 31

[0242] A mixture of compound 31 (1.52 g, 2.47 mmol) in trifluoroacetic acid (10 ml) was stirred for 2.5 h at RT, and evaporated to dryness. The product was precipitated by Et.sub.2O (100 ml), filtrated, washed with Et.sub.2O and dried in vacuum desiccator. The product as TFA salt was dissolved in 10% NaOH (30 ml), the mixture was made acid (pH ca. 2.0) with 1M HCl, and the product was separated and washed with H.sub.2O. Yield: 1.34 g (98%). MALDI TOF-MS mass: calculated (M+H.sup.+) 503.22; found 502.89.

Example 31. Synthesis of Compound 32

[0243] Compound 32 was synthesized from compound 31 and 4-jodoaniline using a method analogous to the synthesis described in example 13. The product (0.45 g, 100%) was purified by FC (silica gel, EtOAc).

Example 32. Synthesis of Compound 33

[0244] Compound 33 was synthesized from the compound 32 using a method analogous to the synthesis described in example 2 stirring for 22 h at 80° C. The product (48%) was purified by FC (silica gel, EtOAc).

Example 33. Synthesis of Ligand Ester 34

[0245] The ligand ester 34 was synthesized from compounds 33 and 21 using a method analogous to the synthesis described in example 23. The product (61%) was purified by FC (silica gel, from 7.5:91.5:1 to 10:89:1 EtOH/CH.sub.2Cl.sub.2/TEA). MALDI TOF-MS mass: calculated (M+H.sup.+) 3534.74; found 3534.17.

Example 34. Synthesis of Eu Chelate 35

[0246] Eu chelate 35 was synthesized from the ligand ester 34 using a method analogous to the synthesis described in example 25. The product (yield 24%) was purified by HPLC. R.sub.f(HPLC): 21.3 min.

Example 35. Synthesis of Eu Chelate 36

[0247] Eu chelate 36 was synthesized from the Eu chelate 35 using a method analogous to the synthesis described in example 6. The product was used for the antibody labelling without any further purifications.

Example 36. Synthesis of Compound 37

[0248] 2-(Boc-oxyimino)-2-phenylacetonitrile (4.93 g, 20 mmol) was added in small partitions within 20 min into a solution of diethylenetriamine (1.08 ml, 10 mmol), H.sub.2O (10 ml), 1,4-dioxane (10 ml) and TEA (4.18 ml, 30 mmol). After stirring for overnight at RT, H.sub.2O (20 ml) was added and the mixture was extracted with EtOAc (40 ml+20 ml). The combined organic phases were washed with 10% NaOH (2×15 ml), H.sub.2O (2×15 ml) and dried with Na.sub.2SO.sub.4. The product (2.58 g, 78%) was purified by FC (silica gel, from 5:95:0 to 20:79:1 MeOH/CH.sub.2Cl.sub.2/TEA). .sup.1H NMR (CDCl.sub.3, δ ppm): 4.94 (2H, bs), 3.21 (4H, q, J=5.8 Hz), 2.73 (4H, t, J=5.8 Hz), 1.45 (18H, s). .sup.13C NMR (CDCl.sub.3, δ ppm): 79.14, 48.74, 40.23, 28.33.

Example 37. Synthesis of Compound 38

[0249] Trifluoroacetic acid anhydride (11.1 ml, 80 mmol) was added within 15 min into ice-cold solution of 4-aminophenylacetic acid (3.02 g, 20 mmol) in trifluoroacetic acid (30 ml). After stirring for 15 min on ice-bath, the mixture was stirred for 2 h at RT and H.sub.2O (50 ml) was added. A cooled mixture was filtrated and the product (3.93 g, 80%) was washed with H.sub.2O and dried. .sup.1H NMR (D.sub.6-DMSO, δ ppm): 12.33 (1H, s), 11.22 (1H, s), 7.59 (2H, d, J=8.6 Hz), 7.29 (2H, d, J=8.6 Hz), 3.57 (2H, s). .sup.13C NMR (D.sub.6-DMSO, δ ppm): 171.50, 155.23, 154.94, 154.64, 154.35, 135.16, 132.86, 130.30, 121.39, 119.63, 117.34, 115.04, 112.75, 40.43.

Example 38. Synthesis of Compound 39

[0250] N-hydroxysuccinimide (0.575 g, 5.0 mmol) and N,N-dicyclohexylcarbodiimide (1.03 g, 5.0 mmol) was added to a solution of compound 38 (1.23 g, 5.0 mmol) in dry 1,4-dioxane (20 ml). After stirring for 4 h at RT, the mixture was filtrated and the solid material was washed with 1.4-dioxane (3×5 ml) and the filtrate was evaporated to dryness. The residue was dissolved in dry DMF (15 ml) and the 6-aminohexanoic acid (0.655 g, 5.0 mmol) was added. The mixture was stirred for one week at RT. The mixture was evaporated to dryness, and after an addition of H.sub.2O (25 ml), the cooled mixture was filtrated, the product (1.80 g, 100%) was washed with H.sub.2O (3×10 ml) and dried. .sup.1H NMR (D.sub.6-DMSO, δ ppm): 11.80 (1H, bs), 11.23 (1H, bs), 7.99 (1H, t, J=5.5 Hz), 7.57 (2H, d, J=8.6 Hz), 7.27 (2H, d, J=8.6 Hz), 3.38 (2H, s), 3.02 (2H, q, J=6.9 Hz), 2.18 (2H, t, J=7.4 Hz), 1.53-1.43 (2H, m), 1.43-1.33 (2H, m), 1.30-1.20 (2H, m). .sup.13C NMR (D.sub.6-DMSO, δ ppm): 174.79, 170.09, 155.19, 154.90, 154.61, 154.32, 134.90, 134.43, 129.80, 121.39, 117.35, 115.05, 112.75, 42.19, 38.84, 33.97, 29.16, 26.31, 24.56. MALDI TOF-MS mass: calculated (M+H.sup.+) 361.34; found 363.96.

Example 39. Synthesis of Compound 40

[0251] N-hydroxysuccinimide (0.12 g, 1.0 mmol) and N,N-dicyclohexylcarbodiimide (0.21 g, 1.0 mmol) was added to a solution of compound 39 (0.36 g, 1.9 mmol) in dry 1,4-dioxane (30 ml) and DMF (5 ml). After stirring for 3 h at RT, a solution of compound 37 (0.30 g, 1.0 mmol) in 1.4-dioxane (3 ml) was added and the mixture was stirred for 2 days at RT. After evaporation to dryness, the residue was dissolved in 1,4-dioxane (10 ml), filtrated and the solid material was washed with 1,4-dioxane and the filtrate was evaporated to dryness. The residue was dissolved in CH.sub.2Cl.sub.2 (30 ml), washed with H.sub.2O (3×10 ml) and dried with Na.sub.2SO.sub.4. The product (0.37 g, 57%) was purified with FC (silica gel, from 5% to 7.5% MeOH/CH.sub.2Cl.sub.2. .sup.1H NMR (D.sub.6-DMSO, δ ppm): 11.21 (1H, s), 7.80 (1H, t, J=5.5 Hz), 7.57 (2H, d, J=8.5 Hz), 7.27 (2H, t, J=8.5 Hz), 6.98 (1H, t, J=5.9 Hz), 6.80 (1H, t, J=5.4 Hz), 3.58 (2H, s), 3.27 (2H, t, J=6.5 Hz), 3.23 (2H, t, J=6.5 Hz), 3.05-2.97 (4H, m), 2.23 (2H, t, J=7.3 Hz), 1.50-1.44 (2H, m), 1.44-1.34 (2H, m), 1.36 (18H, s), 1.28-1.21 (2H, m). .sup.13C NMR (D.sub.6-DMSO, δ ppm): 172.70, 170.15, 156.13, 156.08, 155.21, 154.96, 154.72, 154.47, 135.00, 134.51, 129.91, 121.46, 119.16, 117.25, 115.34, 113.43, 78.25, 78.03, 55.38, 47.72, 45.64, 42.28, 39.03, 32.44, 29.47, 28.69, 28.65, 26.67, 25.08. MALDI TOF-MS mass: calculated (M+H.sup.+) 646.34; found 647.36.

[0252] Beside of the wanted product 40, 0.18 g of NHS-activated form of 39 was obtained from which an additional amount of the compound 40 (0.164 g, 65%) was prepared in DMF and TEA.

Example 40. Synthesis of Compound 41

[0253] A mixture of compound 40 (0.52 g, 0.812 mmol) in CH.sub.2Cl.sub.2 (20 ml) and trifluoroacetic acid (5 ml) was stirred for 4.5 h at RT, and evaporated to dryness. After an addition of Et.sub.2O (30 ml), the product (0.532 g, 97%) was removed by centrifugation, washed with Et.sub.2O (2×15 ml) was dried in vacuum desiccator. .sup.1H NMR (D.sub.6-DMSO, δ ppm): 11.25 (1H, s), 8.06 (1H, t, J=5.5 Hz), 7.99 (2H, bs), 7.79 (2H, bs), 7.57 (2H, d, J=8.5 Hz), 7.27 (2H, d, J=8.5 Hz), 3.53.3.45 (4H, m), 3.05-2.96 (4H, m), 2.96-2.90 (2H, m), 2.30 (2H, t, J=7.6 Hz), 1.53-1.45 (2H, m), 1.45-1.37 (2H, m), 1.31-1.25 (2H, m). .sup.13C NMR (D.sub.6-DMSO, δ ppm): 174.01, 170.22, 159.17, 158.95, 158.72, 158.50, 155.25, 155.00, 154.76, 154.52, 135.02, 134.50, 129.92, 121.50, 118.17, 117.90, 117.25, 115.94, 115.33, 113.99, 44.99, 43.13, 42.27, 39.03, 37.58, 37.36, 32.48, 29.50, 26.53, 24.68. MALDI TOF-MS mass: calculated (M+2H.sup.+) 447.50; found 447.16.

Example 41. Synthesis of Compound 42

[0254] A mixture of compound 1 (180 mg, 0.40 mmol), 41 (67 mg, 0.10 mol) and DIPEA (209 μl, 1.2 mmol) in dry MeCN (5 ml) was stirred overnight at 52° C. After evaporation to dryness, the product (163 mg, 84%) was purified by FC (silica gel, 5% MeOH/CH.sub.2Cl.sub.2). MALDI TOF-MS mass: calculated (M+H.sup.+) 1932.45, 1930.45; found 1932.59, 1930.69.

Example 42. Synthesis of Ligand Ester 43

[0255] A mixture of compound 42 (160 mg, 84.0 μmol) and diethyl 2,2′-{{4-[(trimethylsilyl)ethynyl]-1,3-phenylene}bis(oxy)}diacetate (129 mg, 0.422 mmol; Sund, H., et al. Molecules 22(2017)1807) in dry TEA (1 ml) and THF (2 ml) was de-aerated with argon. After an addition of bis(triphenylphosphine)-palladium(II) chloride (10 mg, 14 μmol) and CuI (6 mg, 28 μmol), the mixture was stirred 24 h at 55° C. After evaporation to dryness, the product (148 mg, 62%) was purified by FC (silica gel, from 5% to 10% EtOH/CH.sub.2Cl.sub.2). MALDI TOF-MS mass: calculated (M+H.sup.+) 2832.19; found 2831.19.

Example 43. Synthesis of Ligand Ester 44

[0256] This ligand ester 44 was synthesized from compound 42 and ethyl 2-(4-ethynyl-3-methoxyphenoxy)acetate using a method analogous to the synthesis described in example 41. The product (60%) was purified by FC (silica gel, from 5% to 10% EtOH/CH.sub.2Cl.sub.2). MALDI TOF-MS mass: calculated (M+2H.sup.+) 2545.12; found 2545.17.

Example 44. Synthesis of Eu Chelate 45

[0257] Eu chelate 45 was synthesized from the ligand ester 43 using a method analogous to the synthesis described in example 25. The product (46%) was purified by HPLC. R.sub.f(HPLC): 12.8 min. UV: 348 nm.

Example 45. Synthesis of Eu Chelate 46

[0258] Eu chelate 46 was synthesized from the ligand ester 44 using a method analogous to the synthesis described in example 25. The product (yield 73%) was purified by HPLC. R.sub.f(HPLC): 16.2 min. UV: 348 nm.

Example 46. Synthesis of Eu Chelate 47

[0259] Eu chelate 47 was synthesized from the Eu chelate 45 using a method analogous to the synthesis described in example 6. The product was used for the antibody labelling without any further purifications.

Example 47. Synthesis of Eu Chelate 48

[0260] Eu chelate 48 was synthesized from the Eu chelate 46 using a method analogous to the synthesis described in example 6. The product was used for the antibody labelling without any further purifications.

Example 48. Synthesis of Compound 49

[0261] A mixture of Boc-Gly-OSu (0.27 g, 1.0 mmol) and 4-iodoaniline (0.22 g, 1.0 mmol) in dry DMF (2.0 ml) was stirred for one week at RT. After evaporation to dryness, the product (0.32 g, 85%) was purified by FC (silica gel, 50% EtOAc/petroleum ether). .sup.1H NMR (D.sub.6-DMSO, δ ppm): 10.3 (1H, s), 7.64 (2H, d, J=8.8 Hz), 7.43 (2H, d, J=8.8 Hz), 7.06 (1H, t, J=6.1 Hz), 3.70 (2H, d, J=6.1 HZ), 1.39 (9H, s). .sup.13C NMR (D.sub.6-DMSO, δ ppm): 168.91, 156.40, 139.26, 137.86, 121.71, 87.04, 78.54, 44.29, 28.67. MALDI TOF-MS mass: calculated (M+2H.sup.+) 378.05; found 378.71.

Example 49. Synthesis of Compound 50

[0262] A mixture of compound 49 (0.65 g, 1.73 mmol) and TFA (10 ml) was stirred for 5 h at RT and was evaporated to dryness. After and addition of Et.sub.2O (50 ml), the mixture was stirred for ca. 30 min, the product (0.63 g, 93%) was filtered, washed with Et.sub.2O (50 ml) and dried in in vacuum desiccator. .sup.1H NMR (D.sub.6-DMSO, δ ppm): 10.59 (1H, s), 8.15 (2H, s), 7.70 (2H, d, J=8.8 Hz), 7.35 (2H, d, J=8.8 Hz), 3.79 (2H, s). .sup.13C NMR (D.sub.6-DMSO, δ ppm): 165.49, 158.79, 158.58, 158.38, 158.17, 138.48, 138.16, 121.76, 120.75, 118.77, 116.78, 88.08, 41.57, 31.18. MALDI TOF-MS mass: calculated (M+H.sup.+) 276.99; found 276.53.

Example 50. Synthesis of Compound 51

[0263] A mixture of compound 50 (0.62 g, 1.59 mmol) and DIPEA (1.38 ml, 7.95 mmol) in dry MeCN (20 ml) was stirred at RT until a clear mixture was obtained and BrCH.sub.2COOEt (176 μl, 1.59 mmol) was added. After stirring overnight at 55° C., the mixture was evaporated to dryness and the product (1.12 g, 67%) was purified by FC (silica gel, from 50:49:1 to 75:24:1 EtOAc/petroleum ether/TEA). .sup.1H NMR (D.sub.6-DMSO, δ ppm): 9.98 (1H, s), 7.63 (2H, d, J=8.8 Hz), 7.47 (2H, d, J=8.8 Hz), 4.11 (2H, q, J=7.1 Hz), 3.42 (2H, s), 1.19 (3H, t, J=7.1 Hz). .sup.13C NMR (D.sub.6-DMSO, δ ppm): 172.63, 170.65, 139.02, 137.82, 121.82, 87.17, 60.55, 52.66, 50.48, 14.60. MALDI TOF-MS mass: calculated (M+H.sup.+) 363.02; found 362.63.

Example 51. Synthesis of Compound 53

[0264] A mixture of compound 52 (0.43 g, 0.90 mmol; Sund, H., et al. Molecules 22(2017).sub.1807), NH.sub.2CH.sub.2COOEt x HCl (31 mg, 0.22 mmol), DIPEA (160 μl, 0.90 mmol) in dry MeCN (15 ml) was stirred for 2 h at RT. After evaporation to dryness, the product (130 mg, 66%) was purified by FC (silica gel, 2% MeOH/CH.sub.2Cl.sub.2).

Example 52. Synthesis of Compound 54

[0265] A mixture of compound 51 (151 mg, 0.20 mmol), 53 (89 mg, 0.10 mmol) and dry K.sub.2CO.sub.3 (55 mg, 0.40 mmol) in dry MeCN (5 ml) was stirred for 3-4 days at RT. The mixture was filtrated, the precipitate was washed with MeCN and the filtrate was evaporated to dryness. The product (101 mg, 69%) was purified by FC (silica gel, from 2 to 5% MeOH/CH.sub.2Cl.sub.2). MALDI TOF-MS mass: calculated (M+H.sup.+) 1456.23; found 1456.35.

Example 53. Synthesis of Ligand Ester 55

[0266] A mixture of compound 21 (255 mg, 173 μmol) and 54 (101 mg, 70 μmol) in dry TEA (1 ml) and DMF (2 ml) was de-aerated with argon. After an addition of bis(triphenylphosphine)palladium(II) chloride (10 mg, 14 μmol) and CuI (6 mg, 28 μmol), the mixture was stirred 24 h at RT. After evaporation to dryness, the product (193 mg, 67%) was purified by FC (silica gel, from 5% to 10% EtOH/CH.sub.2Cl.sub.2). MALDI TOF-MS mass: calculated (M+Na.sup.+) 4166.50; found 4166.02.

Example 54. Synthesis of Eu Chelate 56

[0267] Eu chelate 56 was synthesized from the ligand ester 55 using a method analogous to the synthesis described in example 25. The product (yield 18%) was purified by HPLC. R.sub.f(HPLC): 14.3 min. MALDI TOF-MS mass: calculated (M+H.sup.+) 3756.52; found 3756.51.

Example 55. Synthesis of Eu Chelate 57

[0268] Eu chelate 57 was synthesized from the Eu chelate 56 using a method analogous to the synthesis described in example 6. The product was used for the antibody labelling without any further purifications.

Example 56. Synthesis of Eu Chelate 58

[0269] Eu chelate 58 was synthesized from ethyl 2-{{2-{{2-[2-(4-iodophenoxy)acetamido]ethyl}amino}-2-oxoethyl}amino}acetate and 53 using methods analogous to the synthesis steps described for the Eu chelate 56 in examples from 52 to 55.

Example 57. Synthesis of Eu Chelate 59

[0270] This Eu chelate 59 was synthesized from the Eu chelate 58 using a method analogous to the synthesis described in example 6. The product was used for the antibody labelling without any further purifications.

Example 58. Synthesis of Compound 60

[0271] A mixture of compound 52 (0.24 g, 0.50 mmol; Sund, H., et al. Molecules 22(2017)1807), NH(CH.sub.2COOEt).sub.2 (95 mg, 0.50 mmol), dry K.sub.2CO.sub.3 (0.35 g, 2.5 mmol) in dry MeCN (10 ml) under argon was stirred for 5.5 h at RT. After filtration and evaporation to dryness, the product (97 mg, 33%) was purified by FC (silica gel, 30% EtOAc/petroleum ether). The product was used directly in the next step as it does not tolerate storage.

Example 59. Synthesis of Compound 61

[0272] A mixture of compound 60 (90 mg, 0.154 mmol), BocNH(CH.sub.2).sub.6NH.sub.2 (0.17 g, 0.77 mmol), dry K.sub.2CO.sub.3 (0.21 g, 1.54 mmol) in dry MeCN (5 ml) under argon was stirred for 3 h at RT. After evaporation to dryness, the product (85 mg, 77%) was purified by FC (silica gel, 10% MeOH/CH.sub.2Cl.sub.2). MALDI TOF-MS mass: calculated (M+H.sup.+) 720.36; found 720.43.

Example 60. Synthesis of Compound 62

[0273] A mixture of compound 61 (106 mg, 0.15 mmol), compound 1 (68 g, 0.15 mmol), dry K.sub.2CO.sub.3 (41 mg, 0.30 mmol) in dry MeCN (5 ml) under argon was stirred for 7 h at RT. After filtration and evaporation to dryness, the product (97 mg, 60%) was purified by FC (silica gel, 5% MeOH/CH.sub.2Cl.sub.2). MALDI TOF-MS mass: calculated (M+H.sup.+) 1092.41, 1090.41; found 1092.00, 1089.95.

Example 61. Synthesis of Compound 63

[0274] A solution of compound 62 (97 mg, 89 μmol) in trifluoroacetic acid (1.3 ml) was stirred for 2 h at RT. The solution was evaporated to dryness and further evaporated twice from diethyl ether (2×20 ml). The residue was dissolved in dry MeCN (3 ml), and after an addition of DIPEA (0.32 ml, 1.8 mmol) and compound 1 (81 mg, 180 μmol), the mixture was stirred for 4 d at RT under argon. After evaporation to dryness, the product (125 mg, 81%) was purified by FC (silica gel, 5% MeOH/CH.sub.2Cl.sub.2). MALDI TOF-MS mass: calculated (M+H.sup.+) 1734.46, 1732.46; found 1734.64, 1731.73.

Example 62. Synthesis of Ligand Ester 64

[0275] The ligand ester 64 was synthesized from compound 63 and triethyl 2,2′,2″-{[4-(ethynyl)benzene-1,3,5-triyl]-tris(oxy)triacetate in dry DMF instead of THF using a method analogous to the synthesis described in example 4. The product (yield 33%) was purified by FC (silica gel, from 5 to 10% MeOH/CH.sub.2Cl.sub.2). MALDI TOF-MS mass: calculated (M+H.sup.+) 2716.12; found 2715.52.

Example 63. Synthesis of Eu Chelate 65

[0276] Eu chelate 65 was synthesized from the ligand ester 64 using a method analogous to the synthesis described in example 5. The product (yield 47%) was purified by HPLC. R.sub.f(HPLC): 14.1 min. UV: 347 nm.

Example 64. Synthesis of Eu Chelate 66

[0277] Eu chelate 66 was synthesized from the Eu chelate 65 using a method analogous to the synthesis described in example 6. The product was used for the antibody labelling without any further purifications.

Example 65. Labeling of Antibody with Labelling Reagents 7, 28, 47, 57, 59 and 66

[0278] Labeling of a TnI antibody was performed similarly as described in Sund, H., et al., et al. Molecules 22(2017)1807 by using 350 mM Na.sub.2CO.sub.3 buffer (pH 9.8) as reaction buffer and 300 fold excess of the labelling reagents 7, 28, 47, 57, 59 or 66. The reactions were carried out overnight at RT. The labeled antibody was separated from the excess of chelates on Superdex 200 GL 10/30 gel filtration column (GE healthcare) by using TRIS-saline-azide buffer (50 mM TRIS, 0.9% NaCl, pH 7.75) as an eluent. The fractions containing the antibody were pooled and the Eu concentration was measured by UV.

Example 66. Troponin I Immunoassay

[0279] The TnI antibody labeled with the chelate 7, 28, 47, 57, 59 and 66 was tested in sandwich immunoassay for cardiac troponin I. As a reference compound a TnI antibody labelled with Ref 1 (Von Lode, P., et al., Anal. Chem. 74(2003)3193), Ref 2 (Sund, H., et al., Molecules 22(2017).sub.180) and Ref 3 in FIG. 10 was used. 10 μl of diluted tracer antibody (3 ng/μl or 5 ng/μl) and 35 μl of TnI standard solution were pipetted to a pre-coated assay well (single wells in 96 well plate format, wells coated with streptavidin and a biotinylated capture antibody against TnI, Radiometer Turku Oy). The reaction mixtures were incubated for 20 min at 36° C. with shaking. The wells were washed 6 times and dried prior to measurement with Victor™ Plate fluorometer. The brightness of the novel labeling reagents were estimated from the measured signals of novel chelates and Ref 1-3, and by using the brightness values of Ref 1-3 which were 8000, 37200 M.sup.−1 cm.sup.−1 (Sund, H., et al. Molecules 22(2017)180) and 22800 M.sup.−1 cm.sup.−1 respectively. The preliminary results are summarized in Table 1.

TABLE-US-00001 TABLE 1 Chelate label εϕ (M.sup.−1cm.sup.−1) 7 38000 28 58000 47 44000 57 55000 59 75000 66 41000

[0280] As shown from the values of Table 1, the measured brightnesses are really high and based on the observations the labels with two binding groups seem to have unexpected high luminescence.

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