MACROPOLYCYCLIC RARE EARTH COMPLEXES AND APPLICATION AS FLUORESCENT TRACERS

COMPLEXES DE TERRES RARES MACROPOLYCYCLIQUES ET LEUR EMPLOI COMME TRACEURS FLUORESCENTS

English Abstract

A biological complex which consists of a biologically active molecule, associated by coupling
or adsorption to a macropolycyclic rare earth complex, said macropolycyclic rare earth
complex consisting of at least one rare earth salt complexed by a macropolycyclic compound
of the general formula:
(see fig.I)
in which Z is a trivalent or tetravalent atom; R is nothing when Z is trivalent or represents
hydrogen, a hydroxyl group, an amino group or a hydrocarbon radical when Z is tetravalent,
and the divalend radicals A, B and C independently of one another are hydrocarbon chains,
said hydrocarbon chains optionally contain one or more heteroatoms and said hydrocarbon
chains optionally interrupted by a heteromacrocycle, at least one of the radicals A, B or C also
containing a least one molecular unit or essentially consisting of a molecular unit, said
molecular unit possessing a greater triplet energy than the emission level of the complexed rare
earth ion. The invention also relates lo the macropolycyclic rare earth complexes as above
defined as well as to a process for detection or determination of an analyte and to a process for
enhancing the fluorescence of a rare earth.

French Abstract

Complexe biologique, composé d'une molécule active sur le plan biologique, associé par couplage ou adsorption à un complexe de terre rare macropolycyclique, ledit complexe de terre rare macropolycyclique se composant d'au moins un sel de terre rare complexé par un composé macropolycyclique de formule générale : (voir Fig. I), dans laquelle Z est un atome trivalent ou tétravalent ; R n'est rien lorsque Z est trivalent ou représente un hydrogène, un groupe hydroxyle, un groupe amino ou un radical d'hydrocarbure lorsque Z est tétravalent, et les radicaux divalents A, B et C indépendamment l'un de l'autre sont des chaînes d'hydrocarbure, lesdites chaînes d'hydrocarbure contiennent éventuellement un ou plusieurs hétéroatomes et lesdites chaînes d'hydrocarbure éventuellement interrompues par un hétéromacrocycle, au moins l'un des radicaux A, B ou C contenant également au moins une unité moléculaire ou étant essentiellement composé d'une unité moléculaire, ladite unité moléculaire possédant une énergie de triplet plus importante que le niveau d'émission de l'ion de terre rare complexé. L'invention concerne également les complexes de terres rares macropolycycliques définis ci-dessus, ainsi qu'un procédé de détection ou de détermination d'un analyte et un procédé d'amélioration de la fluorescence d'une terre rare.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. In a process for detection or determination of an analyte in a medium which
analyte is traced by associating said analyte or a receptor for said analyte with a fluorescent
label comprising reacting said label with said analyte or said receptor for said analyte to
form a labelled binding pair and detecting said analyte by measuring said label, wherein the
improvement comprises said label being a macropolycyclic rare earth complex consisting
of at least one rare earth salt complexed by a macropolycyclic compound of the general
formula:
<IMG> I,
in which Z is a trivalent or tetravalent atom; R is nothing when Z is trivalent or represents
hydrogen, a hydroxyl group, an amino group, or a hydrocarbon radical when Z is
tetravalent; and the divalent radicals A, B and C independently of one another are
hydrocarbon chains, said hydrocarbon chains optionally contain one or more heteroatoms
and said hydrocarbon chains optionally contain one or more heteroatoms and said
hydrocarbon chains optionally interrupted by a heteromacrocycle, provided that at least one
of the chain radicals A, B, and C contains a heterocycle, a heteromacrocycle, or a
polycyclic aromatic unit, and further provided that at least one of the chain radicals A, B
or C also contains at least one divalent energy donor radical that constitutes at least part of
said chain radical, said energy-donor radical possessing a greater triplet energy that the
emission level of the complexed rare earth ion.

56
2. In a process for detection or determination of an
analyte in a medium which analyte is traced by associating said
analyte or a receptor for said analyte with a fluorescent label
comprising reacting said label with said analyte or said receptor
for said analyte to form a selectively labeled binding pair and
detecting said analyte by measuring said label, wherein the
improvement comprises said label being a macropolycyclic rare
earth complex consisting of-at least one rare earth salt
selectively complexed by a macropolycyclic compound of the
general formula:
<IMG> I,
in which Z is a trivalent or tetravalent atom; R is nothing when
Z is a trivalent atom or represents hydrogen, a hydroxyl group,
an amino group or a hydrocarbon radical when Z is a tetravalent
atom; and the divalent radicals A, B and C independently of one
another are selected from the group consisting of hydrocarbon
chains, which hydrocarbon chains with one or more heteroatoms and
interrupted by a heteromacrocycle, wherein at least one of the
radicals A, B and C includes or

57
consists essentially of a radical selected from the groups consisting of:
<IMG> (1);
<IMG> (2);
<IMG> (3);
<IMG> (4);
<IMG> (5); and
<IMG> (6) .

58
3. A process of claim 2 in which at least one of the radicals A, B, and C includes
or consists essentially of radical 1, 2 or 5.
4. A process of claim 3, wherein Z is nitrogen and A and B are polyethoxylated
chains.
5. A process of claim 2 in which at least one of the radicals A, B, and C includes
or consists essentially of radical 3, 4 or 6.
6. A process of claim 5, wherein Z is nitrogen and A and R are polyethoxylated
chains.
7. A process of claims 2 wherein Z in nitrogen and A and B are polyethoxylated
chains.
8. A process of claim 2 wherein the macropolycyclic compound is a tris-bipyridine
macropolycycle of the formula:
<IMG>
or a phenanthroline-bis-bipyridine macropolycycle of the formula

59
<IMG>
9. A process of claim 2 wherein the macropolycyclic compound is a
tris-phenanthroline macropolycycle.
10. A process of claim 2 wherein the macropolycyclic compound is a bis-bipyridine
bi-isoquinoline macropolycycle of the formula

<IMG>
or a bis-bipyridine diphenylbipyridine macropolycycle of the formula
<IMG> .

61
11. A process of claim 2 wherein the macropolycyclic compound is a compound of
the formula
<IMG>
in which Z is a trivalent or tetravalent atom; R is nothing when
Z is trivalent or represents hydrogen, a hydroxyl group, an amino
group, or a hydrocarbon radical when Z is tetravalent; and A is a
divalent hydrocarbon chain that contains an energy-donor radical
that constitutes at least part of said chain, wherein said
energy-donor radical is anthracene, anthracenamide, naphthalene,
biphenyl, terphenyl, azobenzene, azopyridine, pyridine,
bipyridine, bisisoquinoline, diphenylbipyridine or a compound of
the formula
-C2H4-X1-C6H4-X2-C2H4-
or
-C2H4-X1-CH2-C6H4-CH2-X2-C2H4-
where X1 and X2, which can be identical or different, denote
oxygen, nitrogen, sulfur, or a group of the formula

62
<IMG>, <IMG>;
<IMG>; or
<IMG>
where X is oxygen or tuo hydrogen atoms.
12. The process of claim 2 wherein the macropolycyclic compound is a compound
of the formula
<IMG>
in which A is selected from the group consisting of

63
<IMG> ;
<IMG> ; <IMG> ;
<IMG> ; <IMG> ; and
<IMG>.

64
13. The process of claim 1 or 2 wherein said rare earth complex is substituted on one
or more atoms with a bonding arm selected from the group consisting of alkylamino,
arylamino, isothiocyano, cyano, isocyano, thiocyano, carboxyl, hydroxyl, mercapto, phenol,
imidazole, aldehyde, epoxide, thionyl halide, sulfonyl halide, nitrobenzoyl halide, carbonyl
halide, triazo, succinimide, anhydride, halogenoacetate, hydrazino, and dihalogenotriazinyl
radicals.

14. A macropolycyclic rare earth complex suitable as a fluorescent tracer consisting
of at least one rare earth salt complexed by a macropolycyclic compound of the formula:
<IMG>
in which Z is a trivalent or tetravalent atom; R is nothing when Z is trivlent or represents
hydrogen, a hydroxyl group, an amino group or a hydrocarbon radical when Z is
tetravalent; and the divalent radicals A, B and C independently of one another are
hydrocarbon chains which optionally contain one or more heteroatoms and are optionally
interrupted by a heteromacrocycle, provided that at least one of the chain radicals A, B and
C contains a heterocycle, a heteromacrocycle, or a polycyclic aromatic unit, and further
provided that at least one of the chain radicals A, B, and C contains at least one divalent
energy-donor radical that constitutes at least part of said chain radical, said energy-donor
radical possessing a greater triplet energy than the emission level of the complexed rare
earth ion.
15. A complex of claim 14 wherein the hydrocarbon chains are ethoxylated or
polythoxylated chains.
16. A macropolycyclic complex of claims 14 or 15 wherein the rare earth ion is
selected from the group consisting of europium, terbium, samarium, and dysprosium.
17. A macropolycyclic complex of claim 16 wherein the triplet energy of said
energy-donor radical undergoes radiant deactivation at a phosphorescence wavelength below
580 nm.
18. A macropolycyclic complex of claim 14 or 15 wherein the triplet energy of said
energy-donor radial undergoes radiant deactivation at a phosphorescence wavelength below
580 nm.

66
19. A macropolycyclic complex of claim 14 which consists of a terbium or
europium ion complexed by a macrocyclic compound selected from the group consisting
of (22)phenanthroline; (22)phenanthrolinamide, (22)anthracene; (22)anthracenamide;
(22)bisisoquinoline; (22)diphenylbipyridine; a tris-bipyridine macropolycycle; and a
phenanthroline-bisbipyridine macropolycycle.
20. A macropolycyclic complex of claim 14 wherein at least one of the chain
radicals A, B, and C contains a heterocycle or a heteromacrocycle.
21. A macropolycyclic complex of claim 14 wherein the energy-donor radical is
phenanthroline, azopyridine, pyridine, bipyndine, bisisoquinoline, diphenylbipyridine or a
radical of the formula
-C2H4-X1-C6H4-X2-C2H4-
or
-C2H4-X1-CH2-C6H4-CH2-X2-C2H4-
where X1 is oxygen, nitrogen, or sulfur, and X2 is a group of the formula
<IMG> ; <IMG> ; or
<IMG>.

67
22. The macropolycyclic rare earth complex of claim 14 wherein said divalent
energy-donor radical is selected from the group consisting of
<IMG> ;
<IMG> ;
<IMG> ;
<IMG> ;

68
<IMG> ; and
and
<IMG> .
23. The macropolycyclic rare earth complex of claim 14 wherein said
macropolycyclic compounds is a tris-bipyridine macropolycycle of the formula
<IMG>
or a phenanthroline-bis-bispyridine macropolycycle of the formula
<IMG> .

69
24. The macropolycyclic rare earth complex of claim 14 wherein said
macropolycyclic compound is a tris-phenanthroline macropolycycle.
25. The macropolycyclic rare earth complex of claim 14 wherein said
macropolycyclic compound is a bis-bipyridine biisoquinoline macropolycycle of the formula
<IMG>
or a bis-bipyridine diphenylbipyridine macropolycycle of the formula
<IMG> .

26. A macropolycyclic rare earth complex consisting of at least one rare earth salt
complexed by a macropolycyclic compound of the formula
<IMG>
in which Z is a trivalent or tetravalent atom; R is nothing when Z is trivalent or represents
hydrogen, a hydroxyl group, an amino group, or a hydrocarbon radical when Z is
tetravalent; and A is a divalent hydrocarbon chain that contains an energy-donor radical that
constitutes at least part of said chain, wherein said energy-donor radical is anthracene,
anthracenamide, naphthalene, biphenyl, terphenyl, azobenzene, azopyridine, pyridine,
bipyridine, bisisoquinoline, diphenylbipyridine or a compound of the formula
-C2H4-X1-C6H4-X2-C2H4-
or
-C2H4-X1-CH2-C6H4-CH2-X2-C2H4-
where X1 and X2 which can be identical or different, denote oxygen, nitrogen, sulfur, or a
group of the formula
<IMG> , <IMG>
<IMG>
<IMG>
where X is oxygen or two hydrogen atoms.

71
27. A biological complex which consists of a biologically active molecule,
which is associated by coupling or adsorption with a macropolycyclic rare earth complex,
said macropolycyclic rare earth complex consisting of at least one rare earth salt complexed
by a macropolycyclic compound of the general formula:
<IMG> I,
in which Z is a trivalent or tetravalent atom; R is nothing when Z is trivalent or represents
hydrogen, a hydroxyl group, an amino group or a hydrocarbon radical when Z is
tetravalent; and the divalent radicals A, B and C independently of one another are
hydrocarbon chains, said hydrocarbon chains optionally contain one or more heteroatoms
and said hydrocarbon chains optionally interrupted by a heteromacrocycle, provided that at
least one of the chain radicals A, B and C contains a heterocycle, a heteromacrocycle, or
a polycyclic aromatic unit, and further provided that at least one of the chain radicals A,
B and C also contains at least one divalent energy-donor radical that constitutes at least part
of said chain radical, said energy-donor radical possessing a greater triplet energy that the
emission level of the complexed rare earth ion.
28. A biological complex of claim 27 wherein the hydrocarbon chains of the
macropolycyclic compound are ethoxylated or polyethoxylated chains.
29. A biological complex of claim 27 wherein the rare earth ion is selected
from the group consisting of europium, terbium, samarium, and dysprosium.

72
A biological complex of claim 27 wherein triplet
energy of said energy-donor radical undergoes radiant
deactivation at a phosphorescence wavelength below 580 nm.
31. A biological complex of claim 27 wherein the rare
earth ion is terbium or europium and the macropolycyclic compound
is selected from the group consisting of (22)phenanthroline, (22)
phenanthrolinamide, (22) anthracene, (22) anthraceramide, (22)
bisisoquinoline, (22) diphenylbipyridine, the tris ipyridine
macropolycycle, and the phenanthroline-bisbipyridine
macropolycycle.
32. A biological complex of claim 27 wherein at least
one of the chains A, B, and C of the macropolycyclic compound
contains a hterocycle or a heteromacrocycle.
33. A biological complex of claim 27 wherein the
energy-donor radical of the macropoycyclic compound is
phenanthroline, azopyridine, pyridine, bipyridine,
bisisoquinoline, diphenylbipyridine or a radical of the formula
-C2H4-X1-C6H4-X2-C2H4-
or
-C2H4-X1-CH2-C6H4-CH2-X2-C2H4-

73
where X1 is oxygen, nitrogen, or sulfur, and X2 is a group of the
formula:
<IMG> , <IMG> , or
<IMG> .
34. A biological complex which consists of an analyte
or receptor thereof, which is associated by coupling or
adsorption with a macropolycyclic rare earth complex consisting
of at least one rare earth salt selectively complexed by a
macropolycyclic compound of the formula:
<IMG> I,

74
in which Z is a trivalent or tetravalent atom; R is nothing when
Z is trivalent or represents hydrogen, a hydroxyl group, an
amino group or a hydrocarbon radical when Z is tetravalent; and
the divalent radicals A, B and C independently of one another are
hydrocarbon chains, said hydrocarbon chains optionally contain
one or more heteroatoms and said hydrocarbon chains optionally
interrupted by a heteromacrocycle, provided that at least one of
the chain radicals A, B, and C contains a heterocycle, a
heteromacrocycle, or a polycyclic aromatic unit, and further
provided that at least one of the chain radicals A, B or C also
containing at least one divalent energy-donor radical, said
energy-donor radical possessing a greater triplet energy than the
emission level of the complexed rare earth ion, with the proviso
that when the rare earth salt is a europium or terbium salt, Z is
nitrogen, A is -(CH2)2-O-C6H2Q-O-(CH2)2- where Q is H or NH2, and
either B or C is the radical -(CH2)2-O-(CH2)2-, then the other of
B and C may not be the radical -(CH2)2-O-(CH2)2-O-(CH2)2-.

35. A biological complex which consists of an analyte
or a receptor thereof which is associated by coupling or
adsorption with a macropolycyclic rare earth complex, said
macropolycyclic rare earth complex consisting of at least one
rare earth salt selectively complexed by a macropolycyclic
compound of the general formula:
<IMG> I,
in which Z is a trivalent or tetravalent atom; R is nothing when
Z is a trivalent atom or represents hydrogen, a hydroxyl group,
an amino group or a hydrocarbon radical when Z is a tetravalent
atom; and the divalent radicals A, B and C independently of one
another are selected from the group consisting of hydrocarbon
chains, which hydrocarbon chains with one or more heteroatoms and
interrupted by a heteromacrocycle, wherein at least one of the
radicals A, B and C includes or consists essentially of a radical
selected from the group consisting of:
III
<IMG> ;

76
<IMG> (2) ;
<IMG> (3) ;
<IMG> (4) ;
<IMG> (5) ; and
<IMG> (6) .

77
36. A biological complex of claim 35 in which at least
one of the radicals A, B, and C includes or consists essentially
of radical 1, 2 or 5.
37. A biological complex of claim 35 wherein Z is
nitrogen and A and B are polyethoxylated chains.
38. A biological complex of claim 35 in which at least
one of the radicals A, B, and C includes or consists essentially
of radical 3, 4, or 6.
39. A biological complex of claim 38 wherein Z is
nitrogen and A and B are polyethoxylated chains.
40. A biological complex of claim 35 wherein Z is
nitrogen and A and B are polyethoxylated chains.
41. A biological complex of claim 35 wherein the
macropolycyclic compound is a tris-bipyridine macropolycycle of
the formula:
(IMG)

78
or a phenanthroline-bis-bipyridine macropolycycle of the formula
<IMG> .
42. A biological complex of claim 35 wherein the
macropolycyclic compound is a tris-phenanthroline macropolycycle.
43. A biological complex of claim 35 wherein the
macropolycyclic compound is a bis-bipyridine bi-isoquinoline
macropolycycle of the formula
<IMG>

79
or a bis-bipyridine diphenylbipyridine macropolycycle of the
formula
<IMG> .
44. A biological complex of claim 27 comprising a
compound of the formula
<IMG>
in which Z is a trivalent or tetravalent atom; R is nothing when
Z is trivalent or represents hydrogen, a hydroxyl group, an amino
group, or a hydrocarbon radical when Z is tetravalent; and A is
divalent hydrocarbon chain that contains an energy-donor radical

80
that constitutes at least part of said chain, wherein said
energy-donor radical is anthracene, anthracenamide, naphthalene,
biphenyl, terphenyl, azobenzene, azopyridine, pyridine,
bipyridine, bisisoquinoline, diphenylbipyridine, or a compound of
the formula
-C2H4-X1-C6H4-X2-C2H4-
or
C2H4-X1-CH2-C6H4-CH2-X2-C2H4-
where X1 and X2, which can be identical or different, denote
oxygen, nitrogen, sulfur, or a group of the formula
<IMG>
wherein X is oxygen or two hydrogen atoms.

81
45. A biological complex of claim 35 in which the
macropolycyclic compound is a compound formula
<IMG>
in which A is selected from the group consisting of
<IMG> <IMG>
<IMG> <IMG>
<IMG> <IMG>

82
46. A biological complex of claim 27 wherein the biologically active
molecule is selected from the group consisting of antigens, antibodies, monoclonal antibodies,
antibody fragments and antibody-antibody fragment combinations, drugs, receptors,
hormones, hormone receptors, bacteria, steroids, amino acids, peptides, viruses, vitamins,
nucleotides, polynucleotides, enzymes, enzyme substrates, lectins, nucleic acids, DNA, and
RNA.
47. A biological complex of claim 34 wherein the biologically active
molecule is selected from the group consisting of antigens, antibodies, monoclonal antibodies,
antibody fragments and antibody-antibody fragment combinations, drugs, receptors,
hormones, hormone receptors, bacteria, steroids, amino acids, peptides, viruses, vitamins,
nucleotides, polynucleotides, enzymes, enzyme substrates, lectins, nucleic acids, DNA, and
RNA.

83
48. A process for enhancing the fluorescence of a rare earth ion consisting
of
(a) complexing said ion with a macropolycyclic compound of the general formula:
<IMG>
in which Z is a trivalent or tetravalent atom; R is nothing when Z is trivalent or represents
hydrogen, an hydroxyl group, an amino group or a hydrocarbon radical when Z is
tetravalent; and the divalent radicals A, B and C independently of one another are
hydrocarbon chains which optionally contain one or more heteroatoms and are optionally
interrupted by a heteromacrocycle, provided that at least one of the chain radicals A, B and
C contains a heterocycle, a heteromacrocycle, or an aromatic unit, and further provided that
at least one of the chain radicals A, B and C contains at least one divalent energy-donor
radical that constitutes at least part of said chain radical, said energy-donor radical
possessing a greater triplet energy than the emission level of the complexed rare earth ion;
and
(b) exciting the complex so formed at the absorption wavelength of said energy-donor
radical which possesses said triplet energy.
49. A process of claim 48 wherein at least one of the chain radicals A, B and
C contains a heterocycle or a heteromacrocycle.

Description

13~3?~
-- 1 --
Macropolycyclic rare earth complexes and application
as fluorescent tracers
The present invention, which is the result of
work carried out in collaboration with Professor J.M.
LEHN and his research team at the Université Louis
Pasteur in Strasbourg, relates to the field of
fluorescence and more particularly of fluorescent macro-
polycyclic complexes which are especially suitable as
tracers in immunological determinations.
It is known that detection methods using
fluorescence are intrinsically very sensitive and could
make it possible to obtain lower detection limits than
those reached by means of radioactivity measurements,
in particular through the use of laser sources (I.
Wieder, Immunofluorescence and related staining tech-
niques, 1978, Elsevier).
In practice, however, these detection limits are
not reached because the measurement is made in a matrix
which does not have the properties required to achieve
this optimum. In general, the measurement medium is
turbid and favors diffusion, and other molecules
fluorescing at the same wavelength can be present in the
measurement medium.
In some cases, the improvements made to the
measuring equipment are not sufficient to substantially
improve this detection limit or are very expensive
(laser, monochromators, etc.).
This state of things is even more troublesome
in the field of biochemistry and immunology, where very
small quantities of active molecules have to be measured
in biological media which can be turbid or can contain
proteins or other molecules which are themselves
fluorescent (turbidity and intrinsic fluorescence of the
serum).
In immunological determinations using a

- 2 - 1339200
fluorescent tracer, when the antigen or antibody is
labeled with the fluorescent molecule, the latter must
possess the following properties:
- it must possess a chemical function which permits
coupling with the biological molecule without de-
naturing it or modifying its immunological properties;
- the molar absorption coefficient of the fluorescent
molecule must be as high as possible;
- the quantum yield of fluorescence must be the
highest;
- the Stokes shift must be as large as possible;
- the emission wa~elength must be greater than 500 nm
if possible; and
- it must be soluble in water or buffer solutions.
These conditions are specified for example in the
article by E. SOINI in Clin. Chem. 25, 353 (1979). Now,
the molecules known to date are far from possessing all
these properties.
The fluorescence of certain rare earth chelates
has been known for many years through the work relating
to their application in the field of lasers (A.P.B.
SINHA, Spectroscop. Inorg. Chem. 2, 255 (1971)).
These complexes are formed of:
- firstly a chelating molecule possessing an
electron system capable of populating the triplet state
Tl by inter-system crossing after excitation of the
singlet state Sl following the absorption of luminous
energy; and
- secondly a rare earth ion which, in the case
of fluorescence applications, possesses a strong ion
fluorescence intensity (Eu, Tb) or a moderate intensity
(Sm, Dy), and whose resonance le~el is populated by the
transfer of non-radiant energy from the triplet state of
the complexing agent.
In order to ~btain a strong fluorescence of the

- 1~3920~ -
rare earth chelate, it is necessary for the triplet
energy of the complexing molecule to be greater than
that of the resonance level of the ion, and for it to have
a sufficient life-time (preponderance of this type of
deactivation compared with other processes such as
phosphorescence and non-radiant deactivation, for example
thermal deactivation).
In this case, after excitation in the ab-
sorption band of the chelate, a fluorescence characteris-
tic of the rare earth ion is observed.
These compounds have great advantages inthe field of detection by the measurement of fluorescence;
for example, they are characterized by :
- a large Stokes shift,
- a high molar absorption coefficient,E, relative to
that of the rare earth,
- the possibility of simultaneous detection of several
chelates by changing the rare earth,
- an emission spectrum characteristic of the rare earth
(line spectrum,~ of maximum emission ~ 500 nm),
and
- a long life-time ~from a microsecond to a millisecond).
U.S. Patent 4 058 732 describes a method of
analytical spectroscopy by fluorescence which uses
fluorescent molecules (rare earth chelates) having a
relatively long life-time. In view of the fact that the dif-
fusion due to the particles of the matrix, and the fluores-
cence of the majority of the organic molecules which make
up the matrix, are short-lived phenomena (generally lasting
less than 1 ~sec.), the saia patent recommends a pulsed
excitation and a functionalized rare earth chelate
for labeling the biological molecules to be identified,
detection taking place between each pulsation
and after a sufficiently long time for the
undesired phenomena to have substantially decreased.

133921~0
-- 4 --
According to the prior art cited below, rare
earth chelates theoretically satisfy all the conditions
for forming a class of ideal fluorescent molecules, in
particular as tracers for biological molecules in
immunological determinations, in cytology and for cell
classifiers: -
- U.S. Patent 4 058 732
- SOINI, Clin. Chem. 25, 353 (1979)
- L.M. VALLARI~'O et al. in Automation of
Uterine Cancer Cytology 2, Proceedings of 2nd Int. Conf.,
G.L. WIED, G.F. GAHR and P.H. BARTEL, Editors, Tutorials
of Cytology, Chicago LL 1977.
Now, in practice, it is found that the chelates
used hitherto do not have all the following properties
required by these applications, these being mainly:
- a high quantum yield of fluorescence and a high molar
absorption coefficient,
- a suitable triplet energy of the chelating agent,
- non-inhibition by the solvent (water or other solvent)
or by molecules present in the medium in which the
measurement is made,
- ease of functionalization for the purpose of coupling
with molecules of biological interest or other molecules,
- selectivity of the chelation in favor of the rare
earth and at the expense of other cations which may be
present in large quantities in the measurement medium,
- solubility in water under the conditions of applica-
tion of the immunological determinations, and
- a high stability, in particular at low dilution.
The ~-diketone chelates described in U.S. Patent
4 048 732 are only sparingly soluble in water and bio-
logical media, if at all, and their fluorescence is
largely inhibited by water.
Other chelates hsve been proposed as tracers in
immunological determinations; in particular, there may

1339200
-- 5 --
be mentioned the chelates derived fro~ EDTA, HEDTA and
DTPA, imidodiacetate and the like, and described for
exa~ple in: Proc. ~at. Acad. Sci. USA 72, 4764 (1975),
V.S. Patent 4 374 120, European Patent Application
A-0068875 published January 5, 1983 andGerman Offenlegungsschrift
3 033 691 published March 19, 1981. Their efficacy is assumed to
originate from the high value of their stability constant, which
is generally greater then 1ol0
A criterion of this kind, based solely on thermo-
]0 dynamic considerations, is certainly not c~fficie~t
since it nas been sho~n that the life-ti~e of d Tb(~II)
~DTA ccm~lex is increased by the ~ddition of apotrans-
ferrin ~Biochem. l9, 5~57, 198~), w~ich is characteristic
c~ a protein-chelate bond.
It has recentlg been recommended, ~hen using
tracers ~f the Eu(III) EDTA type such as those described
in U.S. Patent 4 374 120, to use a buffer containing
DTP~ in order to re~ove the free europium vhich has
dissociated from the chelate, clearly pro~ing the in-
s~abilitg of such chelates (Clin. Chem. 29, 60, 1983).
This stEte of the--prior srt sho~s that the rare
earth chel~tes employed hitherto cannot be used st lov
dilution in aqueous media containing other cations or
in biological mEdia, and in particular in i0munological
deter~inations, and that criteria such as the kinetic
stability (rate of dissociation of the complex) and t~e
selecti~itg of ~or~ticn of the rare earth comple~ should be
taken into account.
It ~ay be noted moreover that, by ~irtue of
their high formation constant (in general ~ 10l~), thD
majority of the chelates of the prior art have found sn
application in technical r imagery, vhich uses r-
e~itting hesvg ions of relati~elg short life-ti0e,0bt~ined
- in dilute ~queous solutions. The fixatlon of the ion
~ust then take place rapidl~ on the ~olecule of biological

- 133~200
-- 6 --
interest which carries the chelating agent;- in par-
ticular, the rste of formation of the chelate must be
high:
.
L + n EDTA ~ ~ (EDTA)
. ~ n
kl and k-1 bein8 the rate constants of formation and
dissociation respecti~ely; at equilibrium, we have:
kl L (EDTA)n
k-l ~L] [~TA]
For the complexes used hitherto, and for the
reasons referred to above, the chelates which have
~lways been used make it possible to obtain a high
value of kl 1~1.9 1022 M 1 min. 1 for Eu3+ EDTA - see
J. inorg. nucl. Chem. 1971, 33, p. 127]. Consequently,
for a given value of K, the dissociation rate constant
is relatively large, which means that the rate of ion
exchange between the chelate and the solution is
relatively rapid.
Macropolycyclic rare earth complexes haYe now
been found which possess excellent properties of
selecti~ity and stability, especially of kinetic
stability in aqueous media and biological media. These
macropolycyclic complexes are particularly suitable as
fluorescent tracers for biological substances in
im~unological detection or determination techniques
using fluorescence. They are also suitable as reagents
in luminescence reactions.
It has actually been found that it is possible
to enhance the fluorescence of a rare earth ion in-
aqueous solution by complexing the ion with a macropoly-

- 1339200
-- 7 --
cyclic compound possessing a donor unit with a greater
triplet energy than the emission level of the rare
earth ion, and by exciting the donor unit.
Whereas the excitation of a rare earth ion
produces a very weak fluorescence because rare earths
generally have low molar absorption coefficients ~,
excitation of the donor unit of the macropolycyclic
compound defined below makes it possible to enhance
the fluorescence characteristic of the rare earth ion.
The rare earth complexes thus formed are excellent
fluorescent tracers; furthermore, these are compounds
which are stable in aqueous media and possess a very
high selectivity in respect of rare earth ions.
In contrast to the chelates of the prior art,
which are characterized by a high formation constant,
the rare earth complexes according to the invention
have as essential characteristic a high kinetic
stability (low rate of dissociation). This characteris-
tic is all the more important because the biological
solutions used in immunological detection and determina-
tion methods generally contain proteins which themselves
are also capable of fixing the rare earth ion.. On the
other hand, the rate of formation of the complexes of
the invention is not critical. The formation of the
rare earth ion complex, on the one hand, and the coupling
of the resulting complex with the biological molecule,
on the other, can be carried out separately. Conse-
quently, the formation of the rare earth ion complex
according to the invention can be carried out under non-
biological conditions (use of organic solvents,possibility of supplying energy, time, etc.).
According to a first feature, the present in~en-
tion therefore relates to the use of macropolyc~clic
rare earth complexes as fluorescent tracers, especially
in immunological detection and determination methods,

8 13392QO
the said complexes consisting of at least one rare
earth salt complexed by a macropolycyclic compound of
the general formula:
~,,
R Z / ~ \ Z - R I,
~ ~3,/
in which Z is a trivalent or tetravalent atom such as
nitrogen, carbon or phosphorus, R is nothing or repre-
sents hydrogen, the hydroxyl group, an amino group or a
hydrocarbon radical, snd the divalent radicals ~ ,
~ and ~ independently of one another are hydrocarbon
chains which optionally contain one or more heteroatoms
and are optionally interrupted by a heteromacrocycle,
at least one of the radicals ~ , ~ or ~ also
containing at least one molecular unit or essentially
consisting of a molecular unit, the said molecular unit
possessing a greater triplet energy than the emission
level of the complexed rare earth ion.
According to another feature, the invention
also relates to a process for enhancing the fluorescence
of a rare earth ion, which consists in complexing at
least one rare earth ion with a macropolycyclic compound,
such as defined above, possessing a molecular unit with a
greater triplet energy than the emission level of the
rare earth ion, and in exciting the complex thus formed at
the absorption w.~velength of the said molecular unit.
The rare earth complexes defined above are new
compounds except for the europium and terbium complexes
obtained with the macropolycyclic compound of the
formula below:

-9- 133s2ao
\
N ~ R=H or NH2
\~/ ~/ ~//
O
/ \ '
Thus, the present invention also relates, by
way of new compounds, to the rare earth complexes
consisting of at least one rare earth salt complexed
by a macropolycyclic compound of the formula I above,
with the proviso that, if the rare earth salt is a
europium or terbium salt, Z is nitrogen, ~ is-(CH )2-
0-c6H3R-O-(cH2)2_ and with R=B or NH2, ~ and ~ are
not simultaneously the radical -(cH2)2-o-(cs2)2-o-(cH2)2-
in one case and the radical -(CH2)2-O-~C~2~2- in the other.
The hydrocarbon chains which form the radicals
O , ~ and ~ can contain 2 or more carbon atoms and
can optionally be interrupted by one or more hetero-
atoms chosen from the group consisting of oxygen, sulfur
or nitrogen atoms.
The preferred hydrocarbon chains for the pur-
poses of the invention are polyether chains, in par-
ticular ethoxylated or polyethoxylated chains.
The molecular unit which constitutes an essential
component of the macropolycyclic compound according to
the invention is a triplet energy donor unit composed of mo-
lecules or groups of molecules possessing a greater triplet
energy than the emission level of the complexed rare earth ion.
The energy transfer takes place from the triplet
level of the donor unit to one of t~e emission levels

-l33~2ao
-- 10 --
of the complexed rare earth. For example, europium
possesses the emission levels 5Do at 17270 cm 1, 5Dl at
19030 cm 1 and possibly 5D2, and terbium possesses the
emission level 5D4 at 20480 cm 1.
The triplet energy donor units suitable for the
purposes of the invention must possess a triplet energy
which is greater than or equal to that of the emission
levels of the rare earth ion. For example, in the case
of the europium and terbium complexes according to the
invention, the triplet level of the donor unit must be
greater than 17270 cm 1.
As the phenomenon of phosphorescence is due to
the radiant deactivation of a triplet state, a preferred
criterion for the donor units can be the phosphorescence
emission wavelength of these units. For example, the
chosen units will emit a phosphorescence at lower wave-
lengths (higher energies) than those corresponding to
the population of the emission levels of the rare earth.
In the present case, the phosphorescence wavelength of
the donor unit will have to be below 580 nm.
It is pointed out that the complexes according
to the invention can have one or more donor units, the
said donor units constituting either all or part of the
radicals ~ , O and O .
Without implying a limitation, molecular units
which can be used are any triplet sensitizers having the
requisite triplet energy, for example those described in
European Patent Application A-0068875 or in Spectroscop.
Inorg. Chem. 2, 255, 1971, these documents being cited
in the present description by way of reference.
Particularly preferred molecular units for the
purposes of the invention are phenanthroline, anthracene,
benzene, naphthalene, biphenyl and terphenyl, bipyridines,
biquinolines such as the bisisoquinolines, for example 2,2'_
bipyridine, azobenzene, azopyridine, pyridine or 2,2'-
bisisoquinoline.

133~200
11
The chains below may be mentioned in particular
as examples of radicals ~ , ~ and ~ containing an
energy donor unit:
-C2H4-Xl-C6H4-x2 2 4
-C2H4-xl-cH2-c6H4-cH2 X2 2 4
Xl snd X2, which can be identical or different, denote
o~ygen, nitrogen or sulfur;
-Ic~
- CH21~L CH2--
X X
.. ~-
-C-CH2-0 ~ ~0 CH2 C
X being oxygen or hydrogen.
The macropolycyclic rare esrth complexes accor-
ding to the invention can be obtained by the conventional
processes for the preparation of metal complexes, which
consist in reacting the complexing compound with a com-
pound donating the cation to be complexed. Processes of
this type are described especially in U.S. Patents 3 888
377, 3 966 766 and 4 156 683, which are cited in the
present description by way of reference.
For example, the macropolycyclic complexes can

133~200
- 12 -
be obtained by reacting a rare earth cation donor com-
pound with the macropolycyclic compound having the
characteristics defined above, each compound advan-
tageously being in solution, preferably in the same
solvent or in compatible solvents which are inert to-
wards complex formation. In general, acetonitrile,
DMS0 or ethanol is used as the solvent.
The rare earth cation donor compound which can
be used is any rare earth salt, advantageously a
chloride an a~etylacetonate or a nitrate.
The reaction is advantageously carried out at
the boiling point of the solven't.
If the macropolycyclic complex formed is
soluble in the reaction solvent, it is isolated from
the solution by evaporation to dryness. If the macro-
polycyclic complex formed crystallizes from the reaction
solvent, it is separated off by filtration or any other
appropriate conventional means. The complexes thus
obtained can be purified by crystallization.
The above reaction can also be carried out using
a solution of the macropolycyclic compound and,the cation
donor compound in the crystalline form. A synergistic
agent for protecting against deactivation can also be
introduced into the coordination sphere of the cation,
as described in J. Chem. Phys. 40, 2790 (1964) and 41,
157 (1964).
In the remainder of the present description, the
macropolycyclic complexes according to the invention
are also called "cryptates" and the macropolycyclic com-
pound by itself is also called "cryptand". The nomen-
clature as defined by LEHN will be adopted below for
denoting the cryptates and cryptands. For this purpose,
reference may be made especially to the a-rticles by
J.M. LEHN in Struct. 80nding (Berlin) 16, 1, 1973 and
Acc. Chem. Res. 11, 49 (1978).

1339200
- 13 -
All the rare earth ions are suitable for the
purposes of the present invention. However, preference
will be given to those which exhibit the most intense
ion fluorescence, i.e. terbium and europium and, to a
lesser extent, samarium and dysprosium.
As macropolycyclic compounds suitable for the
purposes of the invention, it is possible to use the
known cryptands, for example:
1) The benzo-cryptands of the general formula:
N \ ~ / N
in which ~ , ~ and ~ independently of one another
represent the groups 2B~ 2 and 1, which have the
following meanings:
2B = -C2H4-Xl-C6H4-X2 C2 4
-C2H4-xl-cH2-c6H4-cH2 X2 2 4
2 = -C2H4-Yl-c2H4-y2 C2 4
= -C2H4-Z-C2H4
in which Xl 2' Yl 2 and Z each represent a heteroatom
chosen from the group consisting of oxygen, sulfur and
nitrogen, it being possible for Xl and X2, and Yl and
Y2, to be identical or different.
Examples of such cryptands which may be mentioned
are those in which the units ~ , ~ and ~ are
respectively composed as follows:

13392~0
- 14 -
~ ~ ~ = (2Bll); (2B21); (2B22); (2B2B2); and
the compound of the formula:
~ ~N ~
0~ \ ~0 0~/ ~~
~,o~oJ
2) The cryptands containing nitrogen heterocycles,
such as those described in JACS 99, 4583 (1977), of the
formula I in which:
- ~ and ~ , which are identical, represent
the polyethoxylated chain of the formula
( 2)2 ~ (CH2)2-o-(cH2)2-~ and
- ~ is a hydrocarbon chain containing a
nitrogen heterocycle as the energy donor unit.
Examples of such compounds which may be mentioned in
particular are the compounds below:
- (22)pyridinamide ~
. .
15 - (22)pyridinamine N ~ J ~ N
'~'
- t22)bipyridine IC 2 72

-15- 1 3392 f~ o
'/~
o=c C-O
- (22) bipyridinamide 1 ~

1333200
- 16 -
~ ~Y ~
- (22)azopyridine o~c\ c
/O
3) The cryptands containing several nitrogen hetero-
cycles as donor units, such as the compound of the
formula below:
0~~0
N~ O O~N
\ /\o /~)\o
described in J. Am. Chem. Soc. 1979, 101, p. 1047.
4) The polycyclic cryptands containing aromatic units,
for example the compounds corresponding to the formula
below:
~CH2- C N
- ,~~ ~~
~H2 ~ Q~
~C. ~ X . o,
or the compounds of the formula I in which the hydro-
carbon chain carrying the donor unit or units is

133~2~0
- 17 -
interrupted by a hetero~acroc~cle, such as the compounds
of the formulae below:
O/YZ ~A ~N~
2 ~ ~N
X ~ 0, N
/N o~ o /~\ ~
~ ~ R = ~
.~oAo~
- N h~
\_~o /o~
Such compounds are described especially in :
Angew. Chemie 86, 443 (1974); Tet. letters 21, 941 (1980)
and Chem. Comm. 833, 1981.
Other cryptands which can be used are the
macropolycyclic compounds of the formula I abo~e in which
the molecular unit or units possessing a triplet energy
greater than the energy of the rare earth to be cryptated
are chosen from the group consisting of phenanthroline,
anthracene, bipyridines and the bisquinolines, possibly

133~700
~ t8 ~
substituted in appropriate positions and by groups
capable of increasing the efficiency of the transfer
of energy or to modify the excitation spectrum of the rare
earth cryptate LJ. Phys. Chem. 1964, vol. 68, p. 3324~.
One example of such groups is the phenyl group.
In these new macropolycyclic compounds,
at least one of the radicals ~ , ~ or ~ preferably
corresponds to one of_the formulae_ below :
~ c,_o U C~lCH2
--C--CH2 ~CH2--C-- ;--CH2--CH2~ CH2--CH2--
H~ O ~ C=O
lS ~ or
~ particular class of these macro-
polycyclic compounds consists of the compounds of the

1~3~2~0
formula I in which Z is nitrogen, ~ and ~ are
two mono- or polyethoxylated chains, preferably diethoxy-
lated, and ~ corresponds to one of the formulae above.
These new macropolycyclic compounds also
include the compounds of the formula I in which ~ and
~ each represent the group :
~ N N
f 2 CH~
and ~ is one of the following groups :
~ ~ or
H2C CH2 H2 CH~
/
~ ~ or
10~ N N ~ N N ~
H21C C\2 H~C C~2
Another particular class of these new
macropolycyclic compounds is constituted by the compounds
of the formula I in which Z is nitrogen and
and G are identical and represent one of the above
indicated heterocycles, namely the 2, 2'-bipyridine
and phenanthroline.

133~0~
_ 20 -
The macropolycyclic compounds of the for-
mula I in which the molecular moiety or moieties are
phenanthroline, anthracene, bipyridines or ~uinolines
are new compounds with the exception of the compounds of
formula I in which ~ and ~ are each a chain of
the formula -(C~2)2-~-~cH2)2 ~ (CH2)2
qroup of the formula
N ~ ~
~ C CH2
or the group of formula
~ N N
OjC C=O
which are described in Chen. Ber. 111, ~aqes 200-204 (1978~.
The macropolycyclic compounds of the for-
mula I can be obtained by known chemical processes invol-
ving mainly condensation and/or addition reactions.
The processes described in French Patent
70 21079 (2052947) and U.S. Patents 3 888 877, 3 966 766
and 4 1 56 683 may be mentioned in particular as exampies
of such processes. These processes are especially suitable
for the preparation of compounds of the formula I in which
Z is nitroqen or phosphorus.
To obtain compounds of the formula I in
~hich Z is carbon and ~ is as defined above, analogous
condensation processes will be used.
In all cases, it suffices to use, as
startlng materials, chemical compounds each containlng
one of the radicals ~, ~ or ~ and ends groups
capable of being substituted or containing radicals which
can easily be removed.

- 21 - -1339200
By way of example, it may be indiczted
that the compound of the formula I in which Z is carbon,
R is the hydroxyi sroup and ~l ~ and ~ each
represent 2,2'-bipyridine can be obtained by condensing
6,6'-dilithiobipyridine with 6,6'-dicyano-2,2'-bipyri-
dine, hydrolyzing the cyano group of the macrocycle thus
obtained and condensing the resulting product with 6,6'-
dilithiobipyridine.
In a preferred procedure, the new macro-
polycyclic compour.ds of the formula I in which Z is
nitroc,en and ~ and ~ are polyethoxylated chains
can be obtained by reacting :
- the nitrogen mac-ocycle consisting of the two poly-
ethoxylated chains, with
-the hydrocarbon chain containing-the said molecular
unit, the said chain having cleavable end groups such
as, for example, halogeno groups.
This cou?ling reaction is advantageously
carried out in an anhydrous solvent, for example di-
methyl sulfoxide (DMS0) or acetonitrile, if approp~iate
in the presence of a reducing agent, such as sodium hydri-
de or sodium car~onate. The macropolycyclic compound is
then obtained ir. the form of a sodium salt.
The reaction is preferably carried ou. at a
temperature below the boiling point of the solvent, for
example at between 60 and 100~C.
It is possible, in order to obtain the
free macropolycyclic compound to react said sodium salt
witp silver ni~rate to form the macropolycyclic silver
complex, which is there.after treated with a stream of
H2S. The formed precipitate is then neutralized with a
solution of (N(CH3)40~ and extraced with methylene chlori-
de.
It will be noted that the macropolycyclic
rare earth complex according to the invention may be
obtained from the free complex or from a salt thereof,
X

1339200
- 22 -
such as the sodium salt for example.
In the above process, any one of the
nitrogen-containing monocyclic macrocycles described in U.S.
Patent 3 966 766 can advantageously be used as the
macrocycle, the preferred compounds being : 1,7,10,16-
tetraoxa-4,13-diazacyclooctadecane of the general formula :
~ 0/ \0~
~ \ / ~
or 1,7,16-trioxa-4,13-diazacyclodecane of the general
formula :
H ~ ~ ~H
It is pointed out that the preferred
complexes according to the invention, obtained from the
above macrocycles, can be considered as derivatives of
cryptates of the type 222 or 221, in which the ether
units in at least one of the hydrocarbon chains have been
substituted by energy donor units (in general aromatics or
polyaromatics possibly containing heteroatoms)-
The europium cryptates of type 222 or 221
have very low rates of dissociation in aqueous solutions,
altho~gh their formation constant is relatively low
(K = 10 and 105 o~ the cryptates 221 and 222 res-
pectively - Inorganic Chem. 1981, 20, p. 616 and J.A.C.S.
1980, 102, p. 2278. Su~h cryptates do not satisfy the
stability criteria def~ned in the prior art. On the other
hand, substitution of the ether units by energy donor units,
which provides the preferred complexes according to the
invention, does not modify the dissociation characteristic
of these cryptates but makes it possible, by excitation
of the energy donor unit in its absorption band, to obtain
a greatly enhanced fluorescence characteristic of the
cryptated r,are earth.

1339200
_ 23 -
If the rare earth cryptates forming the
subject of the present invention are used specifically
to label biologically active molecules with the aid of
a covalent bond, they can be substituted on one or more
S of their constituent atoms by one or more sufficiently
accessible substituents possessing one or more molecular
units which permit covalent coupling with the biological
molecule under operating conditions compatible with its
biological integrity.
Non-limiting examples of these molecular
units which may be mentioned are alkylamino, arylamino,
isothiocyano, cyano, isocyano, thiocyano, carboxyl,
hydroxyl, mercapto, phenol, imidazole, aldehyde, epoxide,
thionyl halide, sulfonyl halide, nitrobenzoyl halide,
1; carbonyl halide, triazo, succinimido, anhydride, halo-
genoacetate, hydrazino, dihalogenotriazinyl and other
radicals (8iol. Chem.245, 3059 (1970). The length
of the arm bonding the macrocyclic complex to the
molecule of biological interest can vary from 1 to 20
atoms, for example, and can contain carbon atoms as
well as heteroatoms such as N, 0, S and P. The invention
therefore also relates to the biological complexes
consisting of a biologlcal molecule which is associated
by coupling or adsorption with a macropolycyclic complex
according to the invention.
The coupling can be carried out using any
of the reagents described for this purpose in the litera-
tu~e and the labeled molec~le can be separated from the
unreacted macropolycyclic complex by any suitable means
of separation (for exa~ple gel filtration).
Among the molecules of b~ological lnterest
which can advantageously be labeled with the rare earth
cryptates forming the subject of the present invention,
there may be mentioned, without implying a limitation,
antigens, antibodies; monoclonal antibodies, fragments

13392~0
- 24 -
and antibody-fragment combinations, drugs, receptors, hor-
mones, hormone receptors, bacteria, steroids, amino
acids, peptides, vitamins, viruses, nucleotides or poly-
nucleotides in hybridization methods, enzymes and their
substrates, lectins, nucleic acids, DNA and RNA.
The macropolycyclic rare earth complexes
according to the present invention find an important
application as fluorescent tracers in immunological
determinations, either in the so-called methods of
determination by competition or in the so-called methods
of determination by excess, in the homogeneous or hetero-
geneous phase, the said determinations being des-
cribed in the prior art (LANDO~, Ann. Clin. Biochem. 1981,
18, p. 253 and SOINI, Clin. Chem. 25, 353, 1979).
In the heterogeneous methods, it is
possible advantageously to use :

13392~0
-
_ 25 _
- tubes coated with sntibodies specific for the sub-
stance to be determined, and to read off the fluores-
cence by the methods described above, directly through
the tube (Clin. Chem. 29, 60, 1983),
- or a different solid phase, in particular a narrow
strip or a gelatinous film on which a medium containing
a specific antibody has been deposited beforehand, the
fluorescence being read off at a different angle from
the excitation and the reflection of the exciting wave
or directly through the support, if this is transparent.
Because of the line spectrum of these tracers,
it is also possible to detect several antigens simul-
taneously either by using cryptates of different rare
earths whose fluorescence lines do not overlap (for
example Tb and Eu), or by using conventional fluorescent
tracers (fluorescein or rhodamine) and tracers according
to the present invention.
Another application relates to immunochemistry,
where the fluorescence of the labeled cell is detected
by microscopy in the manner described by R.C. NAIRN in
"Fluorescent Protein Tracing, Longman Group Ltd.", 1976,
which also has the possibility of carrying out multi-
detection.
Another application of the rare earth complexes
according to the invention relates to cytology and cell
classifiers, where the use of a tracer having a line
spectrum at high wavelength, coupled with the use of
conventional tracers, makes it possible to perform multi-
parameter analyses.
Furthermore, with a given cryptand and different
rare earth ions, it is possible, with a single exciting
wavelength, which is that of the molecular UDit trans-
ferring the energy and which can be 8enerated with a
single source (for example a laser), to obtain two
fluorescence line spèctra characteristic of the two

-26- 1339200
cryptated ions (for example Tb and Eu) and thus disclosing the respective biological
molecules to which the cryptates are fixed.
Another application relates to the use of the rare earth cryptates according to
the present invention in the field of genetic engineering, for example as indicators in
hybridization reactions such as those described in European Patent Applications A-0
070 685 and A-0 070 687, both published January 26, 1983.
The invention will now be described in greater detail by means of the non-
limiting illustrative examples below.
Example 1
A- Preparation of (22) phenanthroline (hereafter called: (22)phen)
~ ~o~
~O ~ SO > N O ~'
Br 8: 60-~OO-C ~
di-CH2Br-phen N2O4 macrocycle (22)phen
The solvent used in this synthesis is DMSO, which was dried for several days
over a 4 ~ molecular sieve and, if appropriate, distilled in vacuo.
1 mmol (366 mg) of dry and freshly chromatographed
dibromomethylphenanthroline compound (di-CH2Br-phen) was dissolved in 10 to 20
ml of DMSO. It was transferred to a dry dropping funnel; the volume was made up to
100 ml with dry DMSO.

13392~0
- 27 -
100 to 200 mg of NaH in oil (FLUKA) were placed
in a dry round-bottomed flask; 5 ml of dry toluene
(or hexane) were added to the mixture, which was left
to settle. The solution was evaporated in vacuo
(1 torr) for 1 hour. The NaH was redissolved in 20 ~l
of dry DMS0 (evolution of H2), with heating to 50~C if
appropriate.
1 mmol of the N204 macrocycle (262 mg) ~as
dried and 10 to 20 ml of dry DMS0 were added.
The solutions of NaH and ~2~4 macrocycle were
each introduced into a dropping funnel and the volume
was made up to ]00 ml with dry DMS0.
100 ml of dry DMS0 were placed in a three-
necked round-bottomed flask. This flask was equipped
with the two dropping funnels, a reflux condenser and
a drying system (silica gel). The flask was heated to
60-100~C,with magnetic stirring.
The contents of the two dropping funnels were
simultaneously added dropwise over a period of 1 to 2
hours.
The reaction mixture was heated for 1 hour
after the reactants had been added. The mixture was
then distilled in vacuo to remove the solvent.
It is possible to add a small quantity of water
in order to remove the hydrides and the bases.
The reaction mixture was reduced to dryness to
give a dry or pasty red residue.
100 to 200 ml of CH2Cl2 were added; the paste
was triturated at room temperature for at least 30
minutes. The residue ~as filtered off and washed. The
organic liquid phase was retained and evaporated in
vacuo. The residue obtained was dried in vacuo (1 torr,
at least 30 minutes).
100 ml of ethyl ether were addçd to the residue,
the components were mixed and the mixture was left to

- 28 - 13392QO
settle; this operation was repeated 3 to 4 times.
To remove the remainder of the monocyclic N204
macrocycle more completely, 50 ml of hexane and 1 ml of
CH30H were added to the residue; the solution was
evaporated in vacuo (gentle heating) until turbidity
appeared; the solution was decanted and the operation
~as repeated several times.
The red residue was tested by slab chromatography
(Polygram Alox N/UV254 from Macherey-Nagel); migration
in the medium CH2C12/CH30H 9/1 gave the following
values:
(22)phen Rf = 0.4 - 0.8
Monocyclic N204 macrocycle Rf = 0.2 - 0.5
The residue was chromatographed on a column
(20 cm, 0 15 cm) packed with neutral activated aluminum
oxide 90 of 70-230 mesh (Merck), the eluent consisting
of the following mixtures:
CH2C12/CH30H (5~), CHC13/C2H50H (5~), CH30H/hexane 9/1.
Yields from 12 to 35% according to the operating
conditions.
This gave the sodium salt of (22)phen, of the
formula [Na+C(22)phen-Br.H20], the ele~ental analysis
of which was as follows:
found: C 53.00 H 6.21 N 9.44
calculated: C 53.16 H 6.18 N 9.55
B - Formation of the complex [Eu3+C(22)phen]
0.07 mol (19.4 mg) of anh~drous EuC13 was
dissolved in 4 ml of dry CH3CN and the solution was
heated under reflux for 1 hour 30 minutes. 35 mg of
the previously obtained sodium salt of ~22)phen in 2.5
ml of CH3CN were added. The mixture was heated under
reflux for 2 hours 30 minutes. After the mixture had
been left to stand overnight at 4~C, the yellow pre-
cipitate was filtered off.
The precipitate collected (11 mg) exhibits a
* Trade Mark

133~2QO
- 29 -
strong fluorescence and this also applies when it is
in solution in H20 and CH30H (UV 254 nm).
No change in the excitation and emission spectra
was observed after three months in aqueous solution at
a concentration below 10 4 mol/liter.
Example 2
A - Preparation of (22)phenanthrolinamide (hereafter
called (22)phenamide)
u ~N ~ ~~ =~
Cl C~= ~ \~ ~Q~
~o~,
di-COCl-phen (22) (22)phenamide
The solvent used in this synthesis ïs aceto-
nitrile distilled over P205. The process was carried
out in a dry glass apparatus.
305 mg of dry recrystallized di-COCl-phen
(1 mmol) were dissolved in 50 ml of CH3CN; after one
night, the solution was filtered and transferred to a
dropping funnel; the volume was made up to 90 ml with
CH3CN.
524 mg of (22) (2 mmol) were dried in vacuo
(< 1 torr; overnight). The product was dissolved in
90 ml of CH3CN and transferred to a dropping funnel.
The two dropping funnels were fitted to a 2 to
3 liter round-bottomed flask containing 1 liter of
CH3CN. The two reactants were added simultaneously
over a period of 2 hours to 2 hours 30 minutes, with
rapid stirring (magnetic). Stirring was continued for
30 minutes after the addition. The solvent was removed

_ 30 _ 133~200
in vacuo at 50~C. The residue was dried for at least
1 hour at P ~ 1 torr. The residue was mixed for 30
minutes in 300 ml of CH2C12 and filtered off. The
filtrate was evaporated to dryness. The residue was
chromatographed on neutral activated Alox 90 (column
30 cm, 0 1.5 cm), the eluent being CH2C12 containing
170 of CH30H. The first product eluted was recovered.
310 mg of (22)phenamide were recovered with a
yield of 63~. The product was recrystallized from a
mixture of toluene/hexane 1/1.
Melting point: 300 - 302~C
IR amide band at 1630 cm 1
Mass spectrum: M+ at 494 for ~W = 494.55
Elemental analysis: C26H30~1~06 C H N
Calculated: 63.15 6.11 11.33
round: ~ 59.8 5.7 10.5
~ 59.9 5.9 10.6
B - Formation of the complex [Eu C(22)phenamide]
4j mg (0.17 mmol) of anhydrous EuC13 were
dissolved in 10 ml of dry CH3CN. A solution of 80 mg
(0.16 mmol) of (22)phenamide in 2 ml of CH2C12 was
heated under reflux for 3 hours, 50 ml of CH3CN were
added and the mixture was heated to the boiling point
of the CH3CN (CH2C12 is removed by eYaporation).
Then the solution of Eu was added.
The mixture was heated under reflux for 2 hours
and then left to stand at room temperature. The ~hite
p~ecipitate W8S recovered by filtration (50 mg). It
exhibited a strong red fluorescence ( ~ excitation-
254 nm) and the same applied in solution in H20, CH30H
and DMS0.
Elemental analysis for EuC(22)phenamide(0H)C12.3H20
X

1339200
- 31 -
C26H37~4010Cl2Eu ~l~' = 788.47 C H ~'
Calculated: 39.61 4.73 7.11
Found: ~ 40.1 4.9 7.2
~ 40.3 4.8 7.1
No changes in the excitation and emission
spectra of this complex were observed after three
months in aqueous solution at a concentration below
10 4 mol/liter.
ExamDle 3 - Pre?aration of (22)anthracenamide
COCl
I
CH2
IY.2
COCl
1 2
' 11
O=~C ~ . 11=o
CH2 ~~~ CH2
~,
In this example, the method of high dilution is
used.

1339200
- 32 -
- 0.564 g of the acid dichloride 1 (1.7 mmol) was
dissolved in 70 ml of anhydrous CH2C12 and the solution
was introduced into a 100 ml dropping funnel;
- 0.446 g of the macrocycle (22), i.e. 1.7 mmol, and
0.47 ml of triethylamine (2 x 1.7 mmol) were also
dissolved in 70 ml of anhydrous CH2C12 and the solution
was introduced into a 100 ml dropping funnel.
0.4 ml of toluene and 0.15 liter of anhydrous
methylene chloride were introduced into a 3 liter three-
necked flask. The two reactants prepared above in the
dropping funnels were introduced simultaneously into
the flask over a period of 5 hours (14 ml/hour).
The organic phase was collected, concentrated
to a few milliliters and then transferred to a column
of SiO2 under pressure (column diameter 3.5 cm; height
17 cm; eluent CH2C12/1% of methanol).
This gave a pale yellow, crystalline fluorescent
product.
Yield: 45%
Thin layer chromatography (TLC): solvent CH2C12/2% of
methanol; SiO2; Rf = 0.6
H NMR: solvent CDC13/CD2C12 5/5, at 200 MHz
ppm 2.53 (m)~
2.93 (m)
3-18 (m)~, 24 H, HCH2 + OCH2 crown
3.42 (m)
4.02 (d)J
A AB 16 Hz 4H OCCH2N
~B = 5-03
7.6 (m) 4H'
8.43 (d) 2H ~ aromatic
8.82 (d) 2H,
Microanalysis: C30H3606N2
Calculated: C 69.20 H 6.97 N 5.38
Found: C 69;07 H 7.00 N 5.22

33 ~ 1339200
~lolecular weight 520.55
The acid dichloride 1 used as the starting
material in this example can be obtained by the processes
described by M.T~ iller et al. in R.~'. Amidon and P.O.
Tawney, J.A.C.S. 77, 2845 (1955) or by B.M. ~likhailov,
Izvest. Akad. ~ank. SS. Osdel Khim. Nank. 1948, 420-6;
CA 42, 6350.
Example 4 - Preparation of (22)anthracene
c ~ C~2 ~ C~2
o = C C=o CH2 CH
~ ~ O ~ \ ~ ~ ~ o ~ ~ I
~ /o~ o ~N ~o ~o~
380 mg of the diamide 3 (0.73 mmol) were
partially dissolved in 20 ml of anhydrous THF contained
in a round-bottomed flask.
The flask, fitted with a reflux condenser, was
placed under an argon atmosphere and 8 ml of 1.1 M
B2H6 in anhydrous THF were added at room temperature.
The whole reaction mixture was kept under reflux
overnight and the excess diborane was then destroyed at 0~C
with a few drops of distilled water.
The solvents were evaporated off and 40 ml of a
6 ~ solution of HCl were added to the residual white

- 34 - 133~200
solid.
The solution was heated under reflux for 30
hours under an argon atmosphere and became dark green.
It was left to cool, the water was then
evaporated off and the resulting solid was pumped dry
with a vane pump for 1 hour and was then dissolved in
50 ml of distilled water; 50 ml of CH2C12 ~ere added,
the two phases were shaken and the aqueous phase ~as
then separated off and rendered basic at 0~C with an
aqueous solution of LiOH to pH 13; a solid precipitated.
A further 50 ml of CH2C12 were added and the two phases
were shaken and left to settle. The organic phase was
recovered and dried over MgS04.
The crude product was transferred to a column
of alumina and eluted with CH2C12/1% of methanol. A
pure crystalline product was recovered on a TLC plate.
Yield: 62 - 70~
TLC: A1203; eluent CH2C12/6~ of MeOH; Rf: 0.33
Melting point: > 260~C
13C NMR: solvent CDC13
ppm 25.2 (aromatic CH2)
54.3
56 3 ~ NCH2 macrocycle + branch
69.3
0 0 ~ OCH2 macrocycle
123.9 (1)'
125.5 (2)
129 9 (3) t
3~ lH NMR: solvent CDC13
ppm 2.39 (t) 8H NCH2 macrocycle
2.55 and 2.76 (0) - 2 x 4H OCH2 N ~ O

1339200
- 35 -
2.89 and 3.19 (m) - 2 x 4H OCH2 0 ~ 0
3.03 and 3.82 (t) - 2 x 4H -CH2-CH2
7.47 and 8.31 (AB) - 4H
) aromatic H
7.50 and 8.36 (AB) - 4H
~icroanalysis: C30H4004N2 (molecular weight 492.6)
Calculated: C 73.13 H 8.18 N 5.6
Found: C 73.06 H 8.04 N 5.2
The complex [Eu3+C(22)anthracene] was prepared
by following the procedure described in Example 1.
Example 5 - Preparation of the macropolycyclic compound
of the formula (7) and the correspondin~ europium
complex
~--N N
N
~ N N
6 7

- 36 - 1339200
A - Preparation of 6,6'-bis-bromomethyl-2,2'-bipyridine
~ ~ NBS
CH3 CH3
Br Br
molecular ~eight 184molecular weight 342
12 12 2 12 10 2 2
A mixture of 6,6'-dimethyl-2,2'-bipyridine
(2.76 g, 15 mmol) and N-bromosuccinimide (5.10 g, 28.6
mmol) in CC14 (150 ml) is heated under reflux for 30
minutes. Benzoyl peroxide (30 mg) was subsequently
added to the mixture, this was heated under reflux
again for 2 hours and the succinimide was then filtered
off. Ihe solution was cooled to 0~C and the solid was
filtered off and washed with methanol to gi~e the bis-
dibromide in the form of a white crystalline solid
(1.65 g).
Yield 32%; melting point 180-181~C.
The CC14 solution was concentrated and chromato-
graphed on a column (silica gel) by elution with a
mixture of methylene chloride/methanol 98/1 to give:
- 6,6'-bis-dibromomethyl-2,2'-bipyridine 0.9 g, 12%
- 6,6'-bis-bromomethyl-2,2'-bipyridine 1.38 g, 27%
- 6-meth~1-6'-bromomethyl-2,2'-bipyridine 0.55 g, 14%
B - Preparation of the macropolycyclic compound
A mixture of the bis-bipyridine macrocycle of
the formula (5) (0.15 g, 0.38 mmol) (described in J. Org.
Chem. 1983, 48, 4848) and Na2C03 (0.4 g) in acetonitrile
(200 ml) was heated to the reflux temperature and a
solution of 6,6'-bis-bromomethyl-2,2'-bipyridine (0.12 g,

1339200
- 37 -
0.38 mmol) was then added over a period of 3 hours.
The mixture was subsequently heated under reflux for
20 hours. The Na2C03 was filtered off and the filtrate
was evaporated. The residue was filtered on a short
column of alumina by elution with CH2C12/MeOH 98/2
to give the sodium salt of the tris-bipyridine macro-
bicyclic compound in the form of a white solid (0.16 g :
73%); melting point above 270~C).
Microanalysis : C36 H30 8'
calculated : C 63.81 H 4.46 N 16.53
found : C 63.79 H 4.48 N 16.49
NMR H : solvent CDC13
3.85 (s, 6CH2) ;
7.33 (dd, J=7,2 ; 1,2; 6H ; H-C(5); H-Ct5'))
7.82 (t, J=7.2; 6H ; H-C(4) ; H-C(4'))
7.90 (dd ; J=7.2; 1.2; 6B; H-C(3); H-C(3'))
A mixture of silver nitrate AgN03(15 mg,
0.08 mmol ) and of the sodium salt obtained above (20 mg,
0.03 mmol ) was heated with 5 ml of CH30H for 30 minutes.
The methanol was evaporated and the resulting complex
was purified over a column of silica gel with CH2C12/CH30H
as eluent (96/4).
The resulting silver complex (20 mg) was
dissolved in a water-methanol mixture (1:1 - 5 ml) and
treated with a stream of H2S for 15 minutes. The formed
precipitate was centrifuged and the solution neutralized
with N(CH3)40H O.lN and extracted with methylene chloride
(3.5 ml). The solution was drled on MgS04 and evaporated;
the resulting solid waS filtered on silica gel CH2C12:
CH30H (96:4) to give the free complex (13 mg; 86~) with
the following characteristics :
NMR H : solvent CDC13
3.82 (s, 12H, CH2)
7.44
7.78 ABX System J = 8; 7.5 and 0.9 Rz
7.83 L

1339~00
- 38 -
Microanalysis : 36 30 8 (S74.7)
Found : 75.37 H 4.98 N 19.41
calculated : C 75.24 H 5.26 N 19.50
The macropolycyclic compound obtained
was used to prepare the complex ~u C(tris-bipyridine
macropolycycle)~ by following the procedure described in
Example 1.
Example 6 - Preparation of the bis-bipyridinephen-
anthroline macropolycyclic compound of the formula (9)
and the corresponding europium complex
5 ~ ~ ~ CN~CNreflux
Br r ~ Na2C03
14 10 2 2 \ ~ \ ~ /
C24H22N6
C38H30N8
?
0.158 mmol of the bis-pyridine macrocycle (5) was weighed
into a 500 ml two-necked round-bottomed flask. 0.75 mmol
of Na2C03 (approx. 5-fold excess) and 105 ml of freshly
distilled CH3CN were added. The mlxture was heated under
reflux for 30 minutes, with magnetlc stirring.
An equimolecular dibromophenanthrollne
solution in 100 ml of acetonitrile was then added dropwise.
- Refluxing and magnetic stirrlng we~e conti-
nued throughout the rather slow addition t2 hours 10
minutes)-

~39~ 1339200
The mixture was then heated for a further
18 hours under the same conditions. The solution obtained
was evaporated to dryness. The crude product, dissolved
in CH2C12, was washed with water. (Thin layer chromato-
graphy on alumina : eluent CH2C12/MeOH 90/10.)
This crude product was purified on a
column of standardized alumina (activity II-III), the
eluent being CH2C12/MeOH 98/2.
MiCrOana1YSiS C38 ~30 8 ~ 2
calculated : C 63.42 H 4.45 N 15.57
found : C 62.77 H 4.46 N 15.31
RMN H solvent - CDC13
3.91 ( S, 8H, CH2 - bPY)
4.07 (S, 4H, CH2-Phen~
7.36 (dd, J=7.2; 1,3; 4H; H-C(5) ; H - C(5') of
the bipyridine)
7.64 (d, J = 8.2; 2H; H-C(3), H-C(8) of phenanthroline,
7.78 (S, 2H, H-C(5), H-C(6) of phenanthroline
7. 83 (t, J=7.2; 4H; H-C(4); H-C(4') of bipyridine
7.91 (dd, J=7.2; 1,3; 4H; ~-C(3); R-C(3') of ~i'pyridine
.27 (d, J=8.2 ; 24 ; H-C (4);
4-C (7) of phenanthroline.
The macropolycyclic compound thus obtained
was used to prepare the complex ~Eu C(bis-bipyridinephen-
25 anthroline macropolycycle)~ by following the procedure
described in Example 1.
The excitation and emission wavelengths
characteristic of this complex are shown in the Table
below.
30 EXamP1e 7
The macropolycycllc complexes below were
obtained by following the procedure of Example 1 using the
macropolycyclic compounds (22) pyridinamide and (2228) res-
pectively : - -
LEU C (22)PYridinamid ~
rTb C ~222B)7

1339200
- 40 -
Example 8 - Preparation of the (22)bisisoquinoline macro-
polycyclic compound of the formula tl2) and of the
corresponding europium complex.
s ~ ~J ~ 3
Br Br
12 26 2 4
20 l4 2 2 macrocycle N204
~ 10 11
>.~
- ~ N N ~
~ Na ~ Br
/~\
\~~\ /~~/ '
32 38 4 4
a~ preparation of l,l'-bis(bromomethyl)-3,3' biisoquino-
line of the formula lO
This compound was prepared from l-methyl-3-
hydroxyisoquinoline of formula " ~~ ~ OH
~1
CH3
The l-methyl-3-hydroxyisoquinoline was prepared by
cyclization of N-(-)-phenyl ethyl-diethoxyacetamide. 72 g

1339200
- 41 -
of this starting compound, prepared according to the
method of preparation described by H. FUKUMI et al in
"HETEROCYC$ES" 9(9) 1197 (1978) and purified by distil-
lation under reduced pressure (140~C- 0.1 mmHg), were
poured dropwise under stirring for one hour into 400 ml
of concentrated H2S04 cooled down to 10~C. The reaction
mixture was cooled during the addition,so that its
temperature does not exceed 10~C. The reaction mixture
was then stirred for ten hours at room temperature,
then poured into 600g of ice. After filtration, the clear
solution was neutralized with 20% aqueous medium under
efficient cooling. The yellow precipitate was filtered
off, washed with water and dried in vacuo. 41.5g of
l-methyl-3-hydroxyisoquinoline were obtained (yield 90%).
Said compound was then tosylated. A suspension of 41.5g
of l-methyl-3-hydroxyisoquinoline was then stirred with
pyridine (250 ml) under cooling in an ice-water bath.
P-toluenesulfonyl chloride (70g, 1.5 equivalent) was then
gradu~ly added over a period of 30 minutes. The yellow
starting compound has then disappeared. The reaction was
monitored by TLC. As soon as the reaction was complete,
water (50 ml) was added, and the stirring was continued
for 1 hour. It was then diluted with 700 ml of water and
neutralized with solid Na2C03. The resulting precipitate
was filtered off, carefully washed with water and dried.
70.5g of a non-hydroscopic product, stable in air was
obtained (yield : 86%).
The resulting l-methyl-3-p-toluenesulfonyl-
oxyisoquinoline was then coupled according to the method
of preparation described by M. Tiecco et al in SYNTHESIS
736, 1984.
- In a three-necked flask equipped with
a magnetic stirrer, flushed with argon, 1 1 of dimethyl-
formamide (DM~), 236 3 g of triphenylphosphine and 53.5 g
of NiC12, 6H20 were introduced, forming a deep blue-gree

- 42 - 133~200
solution. When the temperature of the oil bath reached
50~C, 14.64 g of zinc powder were added and the colour
of the solution changed to a brown-red colo~r. After one
hour, the solution of the starting product (70.5 g in
200 ml of DMF), namely the l-methyl-3-p-toluenesulfonyl-
oxyisoquinoline, was quickly added dropwise with stirring,
under heating at 50~C, said temperature was maintained
for additional 6 hours. After cooling down to room temper-
ature, the reaction mixture was poured into 4 1 of
diluted ammonia (1.51 of H20 and 500 ml of 20% N~3).A
vigOurous air stream was bubbled through the suspensionto oxidize the Ni(Ph3P)4 (Ph=phenyl). The end of oxydation
was indicated by the disappearance of the brown colour.
The suspension was filtered off, washed with water and
placed in 400 ml of 20% HCl to change the biisoquinoline
into its water-insoluble hydrochloride. The suspension
was then shaked with two portions of 400 ml of ethyl ether
to remove the triphenylphosphine and the acid layer was
filtered off, washed with 100 mlof water and acetone
to remove traces of Ph3P and Ph3P0. The hydrochloride
was placed in a round-bottomed flask with 200 ml of
20% ammonia and stirred overnight to release the base.
The white product thus obtained (26 g), namely the 1,1-
dimethyl-3,3'-biisoquinoline was filtered off, carefully
washed with water and dried in vacuo (yield : 81~1. This
product was poorly sol~ble in most of the solvents and
sparingly soluble in CH C13 and THF. The 1,1-dimethyl-3,3'-
biisoquinoline thus obtained was then brominated to form
the l,l'-bis (bromomethyl)-3,3'-biisoqulnoline.
1.20 g of 1,1'-dimethyl-3,3'-bllsoquinoline
was dissolved in 500 ~1 of CC14 under reflux and N-bromo-
succinimide (2.26 g, 3 equivalents) was added. After ten
minutes, 10 g of 2,2'-azobis (2-methylpropionitrile) were
added. The reaction was monitored by thin layer chromato-
graphy. Two other parts of initiator (10 mg each) were

- 43 _ 133~2~0
added over a period of one hour. After three hours, the
solution was evaporated to dryness and the residue was
treated with 150 ml of methanol, stirred for 30 minutes
and filteredoff. The resulting solid was washed with 100 ml
of methanol. The filtration cake was dried in vacuo,
dissolved in boiling toluene (100 ml) and quickly filtered.
The product precipitated during the night in the refri-
gerator (1.28g-yield:68%).
b) Preparation of the macropolycyclic compound of the
formula 12
To a mixture under stirring of 1.416g of
N204 macrocycle, and 3.39 g of Na2C03 in 150 ml of CH3CN,
a suspension of l,l'-bis (bromomethyl)-3,3'-biisoquino-
line in CH3CN (100 ml) was added over a period of 3 hours.
Stirring was continued for 20 hours. After filtration
and washing with C~3CN and evaporation, a crude product
was obtained and chromatographed twice, first over alumina
(eluents : 3~ CH30H in CHC13 then 10% CH30H in CHC13 V/V)
then over silica gel (eluent 10% of CH30H in CHC13). The
yield after two purifications was of 0.289 g, namely 14~.
Another purification was carrled out by cristallizationin a mixture ethanol-ether by vapour diffusion.
c) Preparation of the complex CEU Ct22)biisoquinoline7
24.5 mg (1 equivalent) of anhydrous
europium nitrate were dissolved in 0.5 ml of anhydrous
CH3CN. The sodium complex of (22) biisoquinoline (37.0 mg)
in 0.3 ml of CH3CN was added and the resulting mixture
was treated by following the method disclosed in example
2b. The pale yellow precipitate thus obtained was diluted
in 3ml of acetonitrile and heated for 2 hours. The solution
was allowed to stand at room temperature for several days.
A yellow crystallized product of the formula 12 was
obtained.

133~200
- 44 -
Example 9 : Preparation of the (22) diphenylblpyridine
macropolycyclic compound of the formula 18
~ t ~o~3
Br 16 Br
~0)
N N
~ ~ Na
18
a) Preparation of 6,6'-bis (bromomethyl)-4,4'-diphenyl-
2,2'-bipyridine (16)
This compound was prepared from the 4,4-
diphenyl 2,2'-bipyridine available on the market, according
to the following method.
6,6'-dimethyl-4,4'-diphenyl-2,2'-bipyridine
To a suspension under stirring of 4,4'-
diphenyl-2,2'-bipyridine (4 g, 13 mmol) in-anhydrous
tetrahydrofuran (TRF), cooled with an ice-bath, was added
- 20 dropwise 1,5M methyllithium (3 equivalents) under nitro-
gen atmosphere. After this addition, the solution was
stirred for additional 30 minutes under the same conditions.
The dark red mixture thus obtained was then heated and

1339200
- 45 -
stirred at 40~C for 3 hours under nitrogen.
An excess of water was slowly added at
0~C under nitrogen. The organic phase was separated and
the aqueous phase was extracted three times with dichloro-
methane. Manganese dioxide was then added t20 to 40 timesthe weight of the starting compound) to the orange organic
solution. The mixture was stirred at room temperature
for 30 to 50 minutes. The reaction was followed by thin
layer chromatography (A1203- eluent:toluene).
Magnesium sulfate was then added to the reaction medium,
which was stirred for 30 additional minutes. The filtrate
was then filtered off and evaporated. The residue was
chromatographed over alumina (eluent : toluene/hexane 1:1)
and two products were obtained :
the 6,6'-dimethyl-4,4'-diphenyl-2,2'-bipyridine (0.85 g;l9~)
the 6-monomethyl-4,4'-diphenyl-2,2'-bipyridine(0.45 g:10.8~)
6,6'-dimethyl-4,4'-diphenyl-2,2'-bipyridine-N,N'-dioxide
In a 250 ml flask containing an ice-cooled
stirred solution of the above compound (0.28~, 0.83 mmol)
in chloroform (60 ml), it was gradually added a solution
of m-chloroperbenzoic acid (0.57 g 3.3 mmol) in chloro-
form (60 ml). Stirring was continued for 3 hours, during
which the mixture was left to return to room temperature.
The reaction mixture was treated with an aqueous solution
of sodium bicarbonate (3.3 mmol) stirred for 30 minutes.
The organic phase was separated and
evaporated in vacuo. The residue was re-dissolved in di-
chloromethane (CH2C12) and passed over a column of basic
alumina. A second chromatography was carried out on
standard alumina to completely purify the product (0.21 g;
68~). -
6,6'-bis(acetoxymethyl)-4,4-diphenyl-2,2'-bipyridine
0.21 g (0.57 mmol) of the above compound
was heated for one hour under reflux in acetic anhydride
(1.3 ml). The solution was concentrated in vacuo and tolu-

133~200
- 46 -
ene was added until the formation of azeotrope of the
mixture.
The resulting solid was re-dissolved
in dichloromethane, washed witha 10~ aqueous solution of
sodium bicarbonate, dried and the solvent was evaporated.
The crude product was passed over a column of silica gel
and eluted with dichloromethane to give 0.126 g (yield :
49~) of the desired compound.
6,6'-bis(bromomethyl)-4,4l-diphenyl-2,2'-bipyridine(16)
A solution under stirring of the above
compound (0.053 g; 0.117 mmol) and of 47% hydrobromic
acid (0.9 ml) was heated for 4 hours at 130~C.
The solution was then cooled in a
methanol/ice-bath . 15 ml of water, 40 ml of
chloroform, then gradually a saturated solution of sodium
carbonate, were added thereto, until the pH of the solution
became alkaline.
The organic phase was separated and the
aqueous phase was extracted with chloroform (2 x 10 ml).
All the organic fractions were combined and evaporated.
The residue was chromatographed over an alumina column,
with the chloroform as eluent. 40 mg (yield:69~) of the
dibromide of the formula 16 were obtained.
b) Preparation of salt ~Na C(22) diphenyl-bipyridine Br7
of the formula 18
The method described in example 5b was
followed by using :
21.2 mg (0.081 mmol) of N204 macrocycle of the formula 17
43 mg (0.40 mmol) of sodium carbonate
40 mg (0.081 mmol) of dibromide of the formula 16
29.8 mg (yield:53~) of the complex of the formula 18 were
obtained.

1339200
- 47 -
Example 10. Preparation of the diphenylbipyridine-bisbi-
pyridine macropolycyclic complex of the formula 19
~ ~
/ 19
N Na Br
The method of the above example was repea-
ted by using the bis-pyridine macrocycle of the formula
(5) (example 5) instead of the N204 macrocycle of the
formula 17~ There were used;
24.7 mg (0.063 mmol) of 5 bis-pyridine macrocycle
31.1 mg (0.063 mmol) of dibromide of the formula 16
o.l mg (0.94 mmol) of sodium carbonate
The reaction lasted for 23 hours and the compound of the
formula 19 was obtained with a yield of 20%.

- 48 - 1339200
Example 11 : Preparation of the biisoquinoline bis-bipy-
ridine macropolycyclic compound of the formula 20
< ~ 20
5 N Na Br
The method of the example 8 was repeated
by using the bis-bipyridine macrocycle of the formula 5
instead of the N204 macrocycle of the formula 11. Equi-
molecular quantities of the compound of the formula S
and of dibromide of the formula 10, were used, and
the compound of the formula 20 was obtained (31.7 g ;
yield : 20%)
Example 12 : Preparation of the (22) bipyridine macropoly-
cyclic compound
Following the method of example S and usingthe N204 macrocycle of the formula 11 instead of bis-
bipyridine of the formula 5 and the macropolycyclic compound
of the formula 21 was obtained with a yield of 12~.

1339200
- 49 _
N N ~
21
\ Na ~ Br~
/1 \\
G~~,~~
Example 12
Also using the method of operation of
Example 1 with the macropolycyclic compounds obtained
according to examples 9 to 11, the following macrocyclic
complexes were respectively obtained :
/Eu C(22) bipyridine~
~Eu C (bpy;bpy.biisoq.)7
~Eu C (22) diph.bpy~
rTb t22) bipyridine~
Likewise, by operating according to
the method of example 5, the complex fEu C(macropoly-
cycle tris-phenanthroline)~ was prepared by using the
6,6'-bis-bromomethyl-phenanthroline and the diamine bis
(phenanthrolinediyl) macrocycle. The corresponding
macropolycyclic compound had the following characteristics :
y 42 38 8' ' 2 ( ~ )
calculated : C 65.71 H 4.17 N 14.6
found : C 65.23 H 4.26 N 13.1
RMN lH: solvent CDC13
4.03 4.45 (very broad; AB, 12H, CH2)
7.66 (d, J=8.1; 6~; H-C(3) ; H-C(8))
7.78 (s, 6H, H-C(5); H-C(6))
8.27 (d, J=8.1, 6H; H-C(4); H-C(7))
.

~ 50 - 1339200
fluorescent characteristics of the cryptates of the
invention
a) excitation peaks for a given emission wavelength
For each complex was determined, for
a given emission wavelength, characteristic of the rare
earth ion of the complex, the excitation wavelengths ;
the recorded excitation peaks do not correspond to those
of the rare earth ion alone. In addition, it has been
noted that by exciting the complex at the wavelength
corresponding to the excitation peak determined above,
the maximum of fluorescence was characteristic of the rare
earth ion.
The above determinations were made with a
spectrometer PERKIN-ELMER LS 5 in the operating conditions
indicated in table I hereunder.
b) Measurement of the life-time~ of europium and terbium
cryptates.
On a spectrometer LS5, the emiss$on spec-
trum of the peak situated between 610 and 620 nm for
20 europium, and between 540 and 550 for terbium, was recorded,
the solution being excited in one of the adsorption peaks.It was operated in phosphorescence method, the value of
the slit being fixed to 1 ms=tg.
Recordings were made for several values of
25 td (time limit) namely 0.1 ; 0.2; 0.3 ; 0.4 and 0.5ms.
The intensity of the peak It was measured
and ~was determined by the formula :
t o
0.434t
30 ~; e
log I -log I
by tracing the curve log It
I may be determined and qZ may be calcula-
ted as a function of td. The obtained results are indicated
in table II hereunder.
* Trade mark

T.~BLE I
EXCITATION AND EMISSION CHARACTERISTJCS OF THE COMPLEXES 0~ TIIE IN~'ENTION
(Perkin-Elmer LS 5)
. . . EXCITATION EMISSION
Complex Concentratlon Wldth of Expallslon
(solvent) the fnc~or
slits ~iven ~ rccorded 1 ~iven ~ fluorescence
emission excitation exci~ation maximum
obser~cd at:
[Eu3+C(22)phen] 3.7-10 6 mol/l2.5/20 nm 10 61S nm ~290 nm (peak) 290 nm {615 nm (peak)
(water) 312 nm 593 nm
(shoulder) (second
330 nm peak)
~ (shoulder)
[Eu3+C(22)phenamide] 2.5-10 4 mol/l2.5/10 nm ~ 613 nm 290 nm (peak) 290 nm 613 nm
(water) 323 nm (peak)
333 nm
(shoulder) 590 nm
345 nm
(shoulder)
[Eu3+C(tris-bi- 7-10 mol/l 2.5/10 nm 15 6I7nm maximum at 300 nmmaximum at
pyridine macro- (0.1 M phos- 300 nm 6I7l~m
cycle)] phate buffer,
pH 7.5) .
[Eu3+C(phenanthroline_ 2.8-10 5 mol/l 2.5/10 nm 2 ~80 nm 590 nm ~~~
bis-bipyridine)] (water) - C~
613 nm C~
o

1339200
-- 52 --
E E E E E E E E
o ~- J ~
n ~ .0 _J
Z
E E E E
Ll a.) ~ O ~ ~ ~
_~ 01 G
E-~ . .'' X X
, _ c~ ~ Y
O ~ 4
X Z ~ a, C C ~ C
o ~ ~ ~ o
~ ~ o u~
o
. c~ ~
- E E c
-- L "~ c u
o ~ u
cn
, CU
O
~ L I ~ O
O
.
CU ~L ~
C ~ cE C ~ , C
~r S Cl JJ-- ~ ~ ~
J J~ .C r~ _ _ _
L 3 ~ ~
a
z~
o ~ u ~) o
C E _ ,~
O O -- , O -- O ~ ~ ~
cn _ --~0 --~ ~ ~ 3
t~ O
.
.1 1~ I
~, u ~ C'J ~ O
C ~,~ u ~
~U ~ ~ . _ ~ I
D
E -- ~ C'J,
c~+ ~ cu +C~ 'D ~ C
C
C~

133~200
-- 53 --
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a5
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o _ J O t~ ~ O ~ ~5 ~D
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c e EC cE e
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H ~--1 X ~ ~ ~5
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X ~ '5'3 5~ e 5~
o t~ ~ o ~
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H -- ~1~ ~ ~1 ~ ~) ~1
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m ~ 0 a5
O O ~) u~ ,c5 rc5
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E ~ ~ ~
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r ~ ~ ~A
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O O O U~ O O ~¦ . 0
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1_¦ 5A OA _ OA5~ OA
~, ~ 5~ r ~ D
3 , ,_ , D