A fluorescing lanthanide chelate of a lanthanide cation and a compound having the structure ##STR1## where X.sup.3, X.sup.4 and X.sup.5 that may be the same or different, each denotes a substituted ethynyl group, hydrogen, an alkyl group, an aryl group, a hydroxyl group, an alkoxyl group or an amino group, wherein at least one of X.sup.3, X.sup.4 and X.sup.5 denotes a substituted ethynyl group, and Z.sup.2 and Z.sup.6 that may be the same or different, each denotes a chelating group, hydrogen, an alkyl group or an amino group, wherein at least one of Z.sup.2 and Z.sup.6 denotes a chelating group.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention pertains to organic complexing agents capable of complexing 
with lanthanide cations and the formed lanthanide chelates useful as 
fluorescent compounds in the determination of physiologically active 
materials. 
The sensitivity of the analytical methods based on molecular fluorescence 
is limited by the background signal due to either Roman scattering or 
fluorescent impurities in the sample. Both of these interferences are 
generally short-lived phenomena, i.e. the radiative transition following 
the excitation of the molecule occurs within a microsecond time span. Thus 
any compound with a long-lived fluorescence can be effectively determined 
in the presence of short-lived background if a fluorometer with time 
resolution is at hand. In this fluorometer the sample is illuminated by an 
intermittent light source such thay the long-lived fluorescence is 
measurable during the dark period subsequent to the decay of the 
short-lived background. This invention relates to the synthesis and use of 
such compounds with the long-lived fluorescence. 
2. Description of the Prior Art 
The long-lived fluorescence (0.1-3 ms lifetime) of certain chelates of 
rare-earth metals has been known for some time. The use of these chelates 
in fluorometric immunoassay (FIA) with time resolution has been described 
in German OLS 2,628,158 and U.S. Pat. No. 4,374,120. In these publications 
the complexing agents are aromatic diketones. In German OLS 2,628,158 the 
fluorescent chelate is "conjugated", i.e. convalently bound to the antigen 
or antibody. The main shortcoming in this work is the aqueous instability 
of the chelates which hinders the use of the method at low concentrations. 
In Eur. Pat. Appl. 82850077.7 (publ. no. 64484) another ligand is attached 
either to the antigen or antibody. This ligand is non-fluorescent and 
serves only in carrying the lanthanide through the separation step of the 
antigen-antibody complex. After the separation the lanthanide cation is 
dissociated at low pH and another, fluorescent diketone chelate is formed 
and measured in aqueous micellar phase. This method gives a very good 
sensitivity but suffers from somewhat lengthy procedure. 
It would be very advantageous to have fluorescent probes with good aqueous 
stability which would allow shorter assay procedures and also the use of 
probes in other methods than immunoassay, e.g. in fluorescence microscopy. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The compound according to the invention, which compound is to be used as a 
complexing agent, has the following structure: 
##STR2## 
Each of X.sup.3, X.sup.4 and X.sup.5 that may be the same or different, 
denotes a substituted ethynyl group, hydrogen, an alkyl group, an aryl 
group a hydroxyl group, an alkoxyl group or an amino group, but at least 
one of X.sup.3, X.sup.4 and X.sup.5 denotes a substituted ethynyl group, 
and each of Z.sup.2 and Z.sup.6 that may be the same or different denotes 
a chelating group, hydrogen, an alkyl group, an aryl group, a hydroxyl 
group, an alkoxyl group or an amino group, but at least one of Z.sup.2 and 
Z.sup.6 denotes a chelating group. Fluorescing lanthanide chelates of such 
compounds can be used. The lanthanide used in such fluorescing lanthanide 
chelate is preferably terbium or europium. 
In the compounds according to the invention, the restrictions are: 
1. The carbon-carbon triple bond is conjugated with the pi-electron system 
of the pyridine ring. The selection of a triple bond instead of a single 
or double bond provides a rigidity to the molecule rendering it more 
difficult for absorbed energy to dissipate through rotational movement 
within the molecule. 
2. The chelating groups are so selected and substituted on the pyridine 
ring that energy transfer from the chromophoric structure (conjugated 
pi-electron system) to a chelated lanthanide ion is favored. The pyridine 
nitrogen participates in the chelation. 
3. Z.sup.2 and Z.sup.6 are both chelating groups. This gives quite an 
efficient protection against the transfer of absorbed energy to water 
molecules. 
These three items are the heart of the invention. They define a group of 
compounds among which fluorescent lanthanide chelates can be found that 
very efficiently can be measured by time-delayed fluorescence 
spectrometry. Fluorescence can be measured even in aqueous media, a 
propety that has been rare for most known fluorescent lanthanide chelates. 
Compounds of the invention, accordingly, can be used as labelled reagents 
in favorable homogeneous immunoassays in which the time-resolved 
spectrofluorometric principle is employed. 
The pyridine nitrogen participates in the chelating (is a donor atom). 
The substituent in the ethynyl group may be either a substituted or 
unsubstituted aromatic group, e.g. a phenyl group or a naphthyl group, or 
a substituted or unsubstituted heteroaryl group, e.g. a pyridyl group, a 
quinolyl group, a thienyl group, a thiazolyl group, a benzthiazolyl group, 
a benzoxazolyl group or a benzimidazolyl group. 
The chelating group is a heteroatom-containing group, e.g. a 
N,N-bis(carboxymethyl)aminomethyl group, a 2,6-dicarboxypiperidinomethyl 
group or a carboxyl group. 
When both Z.sup.2 and Z.sup.6 denote the same heteroatom-containing groups, 
these groups may be joined by a bridge consisting of carbon and nitrogen 
atoms, so that the chelating group is a macrocycle. Preferred macrocyclic 
chelating compounds have the structure: 
##STR3## 
wherein n and m is 2 or 3 and Q is a group (such as amino or aminoalkyl) 
appended to the position 2', 3' or 4' of the phenyl group used to link up 
the physiologically active material such as an immunoreagent. 
In another preferred embodiment, the compound which complexes with the 
lanthanide metal has the following structure: 
##STR4## 
wherein the amino group may be appended to the positions 2', 3' or 4' and 
the other positions of the phenyl ring may be substituted by alkyl, such 
as methyl or ethyl, hydroxyl, alkoxyl, such as methoxyl or ethoxyl, amino, 
or halogen, such as fluorine or chlorine groups. 
A preferred fluorescently labeled binding reagent comprises a complex of a 
lanthanide metal having the structure: 
##STR5## 
wherein L is a linking group, such as a ureido, thioureido, an amide, such 
as --CONH--, -CONMe--; thioether, such as --S--, --S--S--; sulfonamide, 
such as --SO.sub.2 NH--, --SO.sub.2 NMe--; n is 1 or 2, and Y is a 
physiologically active material. Z.sup.2 and Z.sup.6 have been described 
above. 
The above compounds are also used as chelating ligands to replace the 
.beta.-diketones described in the European Pat. Appln 82850077.7. In such 
cases the compounds would not have any linking groups. 
Any fluorescent lanthanide metal can be used in the chelates, but the 
preferred lanthanide is europium.

EXAMPLE 1 
Synthesis of 
4-phenylethynyl-2,6-bis(N,N-bis(carboxymethyl)aminomethyl)pyridine 
The synthetic scheme for the preparation of the title compound is as 
follows: 
##STR6## 
Preparation of 
4iodo-2,6-bis(N,N-bis(t-butoxycarbonylmethyl)aminomethyl)pyridine (1) 
To a solution of 4-iodo-2,6-bisbromomethylpyridine (0.59 g, 1.5 mmol) and 
di-t-butyl iminodiacetate (0.74 g, 3.0 mmol) in 40 mL of dry acetonitrile 
was added 1.59 g of sodium carbonate (15 mmol) and the mixture was stirred 
for 24 h at room temperature. Filtration and evaporation of the filtrate 
left a yellow oil. The oil was taken into 20 mL of chloroform, washed 
twice with 10 mL of water and dried with Na.sub.2 SO.sub.4. Evaporation 
gave a yellowish oil which was purified by chromatographing on silica with 
toluene-ethanol (10:1). 
Preparation of 
4-phenylethynyl-2,6-bis(N,N-bis(carboxymethyl)aminomethyl)pyridine (2) 
A mixture of compound 1 (0.63 g, 0.875 mmol), 
bis(triphenylphosphine)palladium(II) chloride (12 mg, 0.0175 mmol), 
copper(I) iodide (7 mg, 0.0368 mmol), and phenylacetylene (83 mg, 0.875 
mmol) in 5 mL of triethylamine was deaerated with nitrogen and kept at 
40.degree. C. for 5 h. The mixture was diluted with 20 mL of chloroform 
and washed with water. The organic phase was dried with Na.sub.2 SO.sub.4, 
filtered and evaporated. The resulting yellowish oil was characterized by 
nmr spectrum. Oil was dissolved in 20 mL of trifluoroacetic acid and kept 
at room temperature for 16 h. Trifluoroacetic acid was evaporated in 
vacuo, the solid residue was triturated with 15 mL of ethyl ether and 
finaly recrystallized from ethanol. The yield was 0.24 g (59%). .sup.1 
H-NMR (DMSO): .delta. 3.20-3.60 (NH), 3.5 (8H), 3.98 (4H), 7.40-7.70 (7H), 
12.30-12.60 (OH). IR (KBr): 2210 cm.sup.-1 (C.tbd.C), 1730, 1630, 1390, 
1210 cm.sup.-1 (C.dbd.O and C--O). 
Fluorescence of the europium chelate of compound 2 
The relative fluorescence yield .PHI..sub.rel of the europium chelate of 
compound 2 was measured in an equimolar 10.sup.-5 M solution of compound 2 
and europium chloride in pH 8.5 borate buffer. Fluorescence measurements 
were done on a Perkin-Elmer LS5 (trade mark) spectrofluorimeter using the 
phosphorescence mode which allowed the decay curves of the lanthanide 
fluorescence to be measured. The fluorescence yield is reported relative 
to the fluorescence of the uncomplexed europium cation using the equation: 
##EQU1## 
where I.sub.che and I.sub.Eu are the preexponential terms of the emission 
decay curves measured at 615 nm for the chelated and uncomplexed europium, 
respectively. The excitation wavelength for the uncomplexed europium was 
395 nm and for the chelate of compound 2 294 nm. C.sub.Eu and C.sub.che 
are the concentrations of free and complexed europium, respectively, and 
k.sub.Eu and k.sub.che the corresponding decay constants. For compound 2 
the relative fluorescence yield becomes 7.8.times.10.sup.5. This parameter 
is relatively independent on the instrument used for the measurement. 
EXAMPLE II 
Synthesis of 3,5-bis(phenylethynyl)-4-hydroxy-2,6-pyridinedicarboxylic acid 
(3) 
##STR7## 
Dimethyl ester of 3,5-diiodo-4-hydroxy-2,6-pyridinedicarboxylic acid (1.0 
g, 2.2 mmol), phenylacetylene (0.7 g, 6.9 mmol), palladium(II) acetate (19 
mg, 0.085 mmol), triphenylphosphine (45 mg, 0.170 mmol) and copper(I) 
iodide (8 mg, 0.043 mmol) were dissolved in the mixture of triethylamine 
(15 mL) and dimethylformamide (5 mL). The mixture was stirred under 
nitrogen atmosphere for 24 h at room temperature and 7 h at 40.degree. C. 
Triethylammonium iodide was filtered off and filtrate evaporated in vacuo. 
The residue was dissolved in chloroform, washed with water, dried with 
sodium sulfate and evaporated. The residue was dissolved in the mixture of 
petroleum ether and ethyl acetate (10:7) and chromatographed on silica. 
The yield of the methyl ester of the title compound was 0.3 g (33%). 
.sup.1 H-NMR (CDCl.sub.3): 67 3.90 (6H), 7.80-7.15 (10H, arom.). 
IR (KBr): 1735, 1210 cm.sup.-1 (C.dbd.O and C--O), 2208 cm.sup.-1 
(C.tbd.C). 
Methyl ester was hydrolyzed by stirring at room temperature in 0.5M 
solution of KOH in ethanol for 4 h. The mixture was diluted with water and 
acidified to pH 1.5 with 6M hydrochloric acid. The mixture was stirred for 
1 h, the precipitate filtered off and washed with water. 
IR (KBr): 1720, 1330-1190 cm.sup.-1 (C.dbd.O and C-O), 2206 cm.sup.-1 
(C.tbd.C). 
Relative fluorescence yield of the europium complex: .PHI..sub.rel 
=1.4.times.10.sup.5 at .lambda..sub.ex 330 nm. 
EXAMPLE III 
##STR8## 
Preparation of 
4-(4-aminophenylethynyl)-2,6-bis(N,N-bis(t-butoxycarbonylmethyl)aminomethy 
l)pyridine (4) 
A mixture of compound 1 (1.08 g, 1.5 mmol), 
bis(triphenylphosphine)palladium(II) chloride (21 mg, 0.03 mmol), 
copper(I) iodide (11 mg, 0.06 mmol) and p-aminophenylacetylene (176 mg, 
1.5 mmol) in 10 mL of triethylamine was deaerated with nitrogen and kept 
at 40.degree. C. for 1.5 h. The mixture was diluted with 40 mL of 
chloroform, washed with water and dried with sodium sulfate. The solution 
was evaporated in vacuo and the product was obtained as a yellow oil. 
.sup.1 H-NMR (CDCl.sub.3): .delta. 1.47 (36H), 3.49 (8H), 4.02 (4H), 
6.50-7.65 (6H). MS: parent peak at 708. 
Preparation of 
4-(4-(p-aminobenzamido)phenylethynyl)-2,6-bis(N,N-bis(t-butoxycarbonylmeth 
yl)aminomethyl)pyridine (5) and the corresponding tetracarboxylic acid (6) 
Compound 4 (60 mg, 0.084 mmol), 39 mg (0.168 mmol) of 
N-trifluoroacetyl-4-aminobenzoic acid and 2mL of dry pyridine were mixed 
and the solution was evaporated. The residue was dissolved in 1 mL of dry 
pyridine, and 200 mg (0.674 mmol) of 
1-(2-mesitylenesulfonyl)-3-nitro-1,2,4-triazole was added. The solution 
was stirred for 45 min, 2 mL of concentrated sodium bicarbonate solution 
was added and the mixture was stirred for 10 min. The mixture was 
extracted with 5 mL of chloroform, the organic phase was separated and 
evaporated. The residue (compound 5) was purified with flash 
chromatography on a silica column with 5% of methanol in chloform as an 
eluent. 
TLC (silica): R.sub.f =0.33 in methanol-chloroform (1:9), R.sub.f =0.78 in 
acetonitrile-water (4:1). 
UV (ethanol): .lambda..sub.max 306 nm. 
The product (compound 5) from the previous step (30 mg) was dissolved in 
2.5 mL of trifluoroacetic acid and the solution was stirred overnight at 
room temperature. The solution was evaporated, the residue triturated with 
5 mL of diethyl ether and the light yellow powder (compound 6) was 
filtered off. 
TLC (silica): R.sub.f =0.35 in acetonitrile-water (4:1). 
UV (water, pH 9): .lambda..sub.max 298 nm. 
Europium complex of compound 6 was prepared by dissolving 6 in water and 
adding equimolar amount of europium(III) chloride. The pH was adjusted to 
8 and the solution was evaporated almost dry. A few milliliters of acetone 
was added and the product was filtered off. This compound was not further 
characterized but converted directly to the corresponding isothiocyanato 
compound for coupling purposes. The aforementioned europium complex (40 
mg) was dissolved in 1 mL of water and 30 mg of sodium bicarbonate was 
added. Thiophosgene (25 .mu.l) was dissolved in 1 mL of chloroform and 
this solution was added dropwise into the aqueous solution of europium 
chelate. The heterogeneous mixture was stirred for 1 h, some water and 
chloroform were added, and the phases were separated. The water phase was 
evaporated to almost dry, some acetone was added, and the precipitate 
(compound 7) was filtered off. 
UV (water): .lambda..sub.max 333 nm. 
EXAMPLE IV 
##STR9## 
Synthesis of 
15-bromo-3,7,11-tritosyl-3,7,11,17-tetraazabicyclo[11.3.1]-heptadeca-1(17) 
,13,15-triene (8) 
Disodium salt of N,N',N"-tritosyl-4-aza-1,7-heptanediamine (2.55 g, 4.0 
mmol) was dissolved in 30 mL of DMF, and 1.37 g (4.0 mmol) of 
4-bromo-2,6-bisbromomethylpyridine in 25 mL of DMF was added at 75.degree. 
C. during 1.5 h. The mixture was stirred at 75.degree. C. for 2 h, 100 mL 
of water was added and the precipitate was filtered off and washed with 
water. After recrystallization from ethanol the yield of compound 8 was 
2.66 g (86%), m.p. 187.degree.-9.degree. C. 
.sup.1 H-NMR (CDCl.sub.3): .delta. 1.62 (4H), 2.42 (3H), 2.45 (6H), 2.83 
(4H), 3.24 (4H), 4.24 (4H), 7.29 (2H), 7.35 (4H), 7.59 (2H), 7.71 (2H), 
7.72 (4H). 
IR (KBr): 1595 cm.sup.-1 (pyrid.), 1340, 1155 cm.sup.-1 (SO.sub.2). 
Detosylation of compound 8 
Compound 8 (2.65 g, 3.4 mmol) was dissolved in 18 mL of conc. sulfuric acid 
(95-98%) and stirred for 8 h at 105.degree.-110.degree. C. Sodium 
hydroxide solution (15%) was added to the cooled solution until its pH was 
ca. 10. The mixture was extracted with dichloromethane (8.times.60 mL). 
The extract was dried and evaporated on a rotary evaporator. The residue 
was yellow oil, which partly crystallized (0.65 g, 69%). 
.sup.1 H-NMR (CDCl.sub.3): .delta. 1.79 (4H), 2.66 (4H), 2.81 (4H), 3.26 
(3H), 3.86 (4H), 7.33 (2H). 
Synthesis of 
15-phenylethynyl-3,7,11-tris(carboxymethyl)-3,7,11,17-tetraazabicyclo[11.3 
.1]heptadeca-1(17),13,15-triene (10) 
Detosylated compound 8 (compound 9) (0.31 g, 1 mmol), 1.38 g (10 mmol) of 
potassium carbonate and 0.59 g (3 mmol) of t-butyl bromoacetate in 25 mL 
of dry acetonitrile was stirred for 24 h at room temperature. The mixture 
was filtered and the filtrate evaporated in vacuo. The resulting yellowish 
oil, bis(triphenylphosphine)palladium(II) chloride (14 mg, 0.02 mmol), 
copper(I) iodide (8 mg, 0.04 mmol) and phenylacetylene (102 mg, 1 mmol) 
were dissolved in the mixture of triethylamine (10 mL) and tetrahydrofuran 
(6 mL), the solution was deaerated with nitrogen and kept at 45.degree. C. 
for 24 h. The mixture was filtered and the filtrate evaporated in vacuo. 
The resulting dark oil was dissolved in trifluoroacetic acid (20 mL) and 
kept at room temperature for 24 h. After evaporation the residue was 
triturated with ether, the solid residue was filtered off and washed with 
ether. The yield of white microcrystalline product was 0.6 g. IR (KBr): 
2215 cm.sup.- (C.dbd.C), 1730, 1680, 1200 cm.sup.-1. 
Relative fluorescence yield of the europium complex: 
.PHI..sub.rel =1.6.times.10.sup.4 at 80 .sub.ex =294 nm. 
EXAMPLE V 
Competitive assay of human IgG (HIgG) on solid phase by measuring the 
change of signal in solution 
Human IgG (1 mg) was labelled with the isothiocyanato derivative of Eu 
chelate (compound 7) by incubating it with 25-fold excess of the reagent 
in buffer solution (pH 9.5) at 4.degree. C. overnight, and thereafter 
separating the unreacted fluorescent label from the conjugated HIgG by gel 
filtration on Sepharose 6B (Pharmacia Fine Chemicals). 
10 ng of the labelled HIgG was then incubated in microtitration strip wells 
(Elfab, Finland) coated with rabbit-anti-human IgG together with HIgG 
standards (0-4000 ng/mL) in 0.25 mL of buffer containing BSA (DELFIA.TM. 
Assay Buffer, LKB-Wallac, Finland) at room temperature for 2 h, whereafter 
the fluorescence of solution in the strip wells was measured on a 
fluorometer with time resolution (1230 ARCUS,.TM.LKB-Wallac, Finland). The 
extent of binding of the labelled HIgG to the solid-phase bound antibodies 
was measured by reading the fluorescence intensity in solution. The 
labelled HIgG competes with the added standards. An increased standard 
concentration is seen as an increase in the measured signal as less of the 
labelled HIgG is bound to the rabbit-anti-human IgG on the solid phase 
(FIG. 1). The assay requires no actual separation as the result can be 
read from the incubation mixture. 
EXAMPLE VI 
Sandwich fluorescence assay of mouse IgG (MIgG) by measuring the change of 
signal in solution 
Anti-mouse-IgG was labelled with the Eu-complex (7) as described in Example 
IV. A sandwich assay of mouse IgG was performed by incubating standards of 
MIgG (0-4000 ng/mL) in anti-mouse IgG coated strip wells for 2 h, 
whereafter (after washing) 200 ng/250 .mu.L of the labelled anti-mouse-IgG 
in DELFIA.TM. Assay buffer (LKB-Wallac) was added to the strips and 
incubated further 2 h at room temperature. After incubation the 
fluorescence of solution in the strip wells was measured on a fluorometer 
with time resolution. The resulting standard curve is presented in FIG. 2. 
As the MIgG concentration increases, more of the labelled anti-mouse-IgG 
is bound to the solid phase and the fluorescence signal in the incubation 
solution decreases.