Macrocyclic complexes of yttrium, the lanthanides and the actinides having peripheral coupling ...

28 Pages·2013·3.4 MB·English

USOO569624OA
United States Patent [19]
[11] Patent Number: 5,696,240
Vallarino et al. [45] Date of Patent: *Dec. 9, 1997
[54] MACROCYCLIC COMPLEXES OF Primary Examiner-Gary E. Hollinden
YTTRIUM, THE LANTHANIDES AND THE Assistant Examiner—Michael G. Hartley
ACTINIDES HAVING PERIPHERAL
Attorney Agent, or Fimn-Gerald R. Hibnick
COUPLING FUNCTIONALITIES
[57] ABSTRACT
[76] Inventors: Lidia M. Vallarino. 1009 West Ave.
Richmond. Va. 23220; Robert C. Leif. symmetrically di-functionalized water soluble macrocyclic
5648 Toyon Rd., San Diego, Calif. complexes of lanthanide. actinide and yttrium ions were
92115 obtained by metal templated. Schitf-base. cyclic condensa
tion of: (l) a functionalized 1,2-diaminoethane and a dicar
[* ] Notice: The term of this patent shall not extend
bonyl compound selected from the group consisting of
beyond the expiration date of Pat. No.
2.6-dicarbonylpyridine. 2.6-difcrmylpyridine. 2.5
5373.093.
dicarbonylfuran. 2.5-diformylfuran. 2.5-dicarbonyl
[21] Appl. No.: 351,827 thiophene and 2.5-diformylthiophene; or (2) 1.2
diaminoethane and a ring-substituted heterocyclic
[22] Filed: Dec. 8, 1994
dicarbonyl compound selected from a group consisting of
substituted 2.6-dicarbonylpyridine. substituted 2.6
Related US. Application Data
diformylpyridine. substituted 2,5-dicarbonylfuran. substi
tuted 2.5-diformy1furan; substituted 2.5-dicarbonyl
[63] Continuation-impart of Ser. No. 669,833, Mar. 15, 1991,
Pat No. 5,373,093. thiophene, and substituted 2,5-diformylthiophene. Asym
metrically functionalized water soluble macrocyclic com
[51] Im. c1.6 ...................... .. A61K 49/00; A61K 39/395;
A6113 5/055; C07F 5/00 plexes of the lanthanide. actinide and yttrium ions were
obtained by metal templated. Schiff-base. cyclic condensa
[52] US. Cl. ......................... .. 534/15; 534/10; 530/391.5;
530/402; 530/409; 540/465; 540/475; 424/9361; tion of appropriately substituted diamine and dicarbonyl
424/1.65 precursors, with such precursors contributing two heteroaro
[58] Field of Search ................................... .. 540/465. 474. matic moieties (pyridine. furan. thiophene. or a combination
540/477; 534/10. 14. 15. 16; 530/3915. thereof) to the resulting macrocyclic structure. The coordi
402, 409; 424/93. 1.65. 9.361 nation complexes thus formed are kinetically stable in dilute
aqueous solution. They are further reacted. or coupled.
[56] References Cited
through a substituent on the 1,2-diarninoethane or on the
U.S. PATENT DOCUMENTS pyridine, furan. or thiophene moieties, to one of the follow
ing: proteinaceous materials. polysaccharides,
4,678,667 7/1987 Meares et al. .......................... .. 424/85
polynucleotides. other biologically compatible macromol
OTHER PUBLICATIONS ecules or bridging molecules which, can be further reacted
Desreux et al. “Highly Stable Lanthanide Macrocyclic in or coupled to the above mentioned substrates. These mac
Search of New Constrast Agents for NMR Imaging". Nucl. rocyclic complexes are suitable in the preparation of reporter
Med. Biol. vol. 15, No. 1. pp. 9-15 (1988). molecules and for magnetic resonance, radiation imaging
DeCola et al., “Metal-templated Synthesis of Novel Mac and radiation therapy.
rocyclic Complexes of the Uranyl Ion”, Inorganics Chimica
Acta, 110. L1-L2, (1985). 16 Claims, No Drawings
5 ,696,240
1 2
MACROCYCLIC COMPLEXES OF linked oligonucleotide probes with multiple carboxy?uores
YTTRIUM, THE LANTHANIDES AND THE cein units. after deprotection of the primary amino groups of
ACTINIDES HAVING PERIPHERAL the lysine residues. However, the resulting oligonucleotides
COUPLING FUNCTIONALITIES “carrying multiple carboxy?uorescein labels gave low levels
of ?uorescence due to quenching” (1990B). These authors
This application is a Continuation-In-Part of Ser. No. reported that ‘The amount of ?uorescence per ?uorescein
moiety is 20 times less than that of carboxy?uorescein in the
07/669,833 ?led Mar. 15, 1991; issued as U.S. Pat. No.
conjugates with ten lysines”, even when the lysine residues
5.373.093 on Dec. 13. 1994.
were separated by two or four spacers. This concentration
BACKGROUND OF THE INVENTION quenching of conventional organic ?uorophores in multiple
10
label polymers is a well known phenomenon. as reported in
1. Field of the Invention
Shapiro (1995). Practical Flow Q'tometry. Third Edition
This invention concerns a process for the synthesis of page 91. Wiley-Liss, New York. NY.
functionalized, water-soluble. macrocyclic complexes of the
The luminescence of the complexes of certain lanthanide
lanthanides having atomic numbers 57-71. of the actinides
(HI) ions. for example europium (111) and terbium (III). has
having atomic numbers 89-103. and of yttrium(]Il) having
the beneficial features of narrow band emission, very large
atomic number 39.
Stokes shifts and long lifetimes; these features allow the
This invention also includes processes for the coupling of lanthanide emissions to be very effectively separated from
such complexes to biologically active or biologically com the luminescence of the background. The luminescent
patible molecules. or to biological cells through peripheral lanthanide(llI) complexes also undergo less concentration
20
pendant substituents having one or more reactive sites. quenching than conventional organic ?uorophores. For these
These complexes are useful particularly as reporter mol reasons, the complexes of the luminescent lanthanides have
ecules in irnmunoassays. analytical cytology, histological
found application as biological labels.
staining, and imaging processes (Leif et al., 1977, Clinical
The complexation of lanthanide ions with organic as well
Chemistry 23. 1492. Development of Instrumentation and
as inorganic compounds and the subsequent use of the
Fluorochromes for Automated Multiparameter Analysis of
resultant complexes in both industrial and biological envi
Cells; Leif et al.. 1976. Markers for Instrumental Evaluation
ronments has been previously reported. Such complexes.
of Cells of the Female Reproductive Tract: Existing and
also referred to as “coordination compounds”. are typically
New Markers, in The Automation of Uterine Cancer Cytol
formed by the union of a lanthanide ion with appropriate
ogy. Ed. Wied. Bahr and Bartels pages 313-345). The
ligands, which can be negatively charged or ionically neu
macrocyclic complexes with a single coupling functionality
tral.
can be attached to small molecules such as nucleic acid
The complexes of the lanthanide(lll) ions with unidentate
bases. These modi?ed bases after incorporation into nucleic
ligands or bidentate chelating ligands are known to be
acids can serve as reporter molecules for molecular biology
extremely labile, achieving complexation and ligand
and analytical cytometry.
35 exchange equilibrium almost instantaneously. Thus. even
2. Description of the Prior Art
very stable complexes of the lanthanide(llI) ions and biden
The sensitivity of ?uorescence measurements for the tate chelating ligands, e.g. tris(acetylacetonato)-lanthanum
analysis of biological samples is often limited by back
(HI) exist in solution as a mixture of species. the uncom
ground signal due to auto?uorescence or Raman scattering. plexed (solvated) metal ion being present in an appreciable
For instance, a multilaboratory survey found the average amount. Lanthanide complexes of fused polychelating non
auto?uorescence of human lymphocytes to equal that of 657 cyclic ligands are somewhat less labile; whereas, those of
?uorescein molecules (Schwartz et al., 1993, Annals N.Y. cyclic ligands are relatively inert.
Academy of Science 677 pages 28-39). The stability and lability of the lanthanide(llI)-liga.nd
Concentration quenching has also limited the number of complex can be critical. depending upon the particular use
conventional organic ?uorophores that can be attached to a 45 and/or environment. More speci?cally. the time frame of
molecular species. For instance. Haralambidis et al., metal-exchange and ligand-exchange kinetics is a major
(1990A) "The synthesis of polyamide-oligonucleotide con consideration when such complexes are used as probes in
jugate molecules”. Nucleic Acids Research 18 pages biological systems. In the dilute aqueous solutions or
493-499 described the synthesis of both peptide aqueous-organic solutions required for these systems. often
oligodeoxyribonucleotide and polyamide—oligonucleotide involving contact with potentially competing ligands. such
carboxy?uorescein conjugates employing an Applied Bio complexes. if labile. can dissociate. Where such dissociation
systems Inc. automated DNA synthesizer. The peptide or does occur. the value of the probe is diminished or lost.
polyarnide was ?rst assembled on a solid support. The Alternatively, vibrational quenching of the ?uorescence by
tenninal amino group was converted to an amide by reaction interaction with water molecules may occur. For instance as
with an mm-hydroxycarboxylic acid derivative. The free 55 reported by Evangelista. R. A.. A New Europium Chelate for
hydroxyl group was then esteri?ed with a phosphoramidate Protein Labelling and Time-Resolved Fluorometric
and the peptide- or polyamide- substituted polynuclectide Applications, Clinical Biochemistry 1988, 21. 173-178, the
was subsequently assembled by sequential reaction with dinegative anionic ligand 4.7-bis(chlorosulfophenyl)-1.10
methyl N. N -diisopropyl nucleoside phosphorarnidates. Pro phenanthroline-2,9-dicarboxilic acid (BCPDA) forms a
tected lysine residues were included in both the peptide and ?uorescent complex with europium?ll). However, this com
the polyamide to provide primary amino functionalities plex is not detected at concentrations required to detect
suitable for dye conjugatation. In a following paper. many analytes of interest which are present in picogram or
Haralambidis et al. (1990B)[Haralambidis J.. Angus K.. lower concentrations. Since BCPDA complex of europium
Pownall 8., Duncan L.. Chai M.. and Tregear G. W. ‘The (111) ion binds su?icient water to quench the ?uorescence.
preparation of polyamide-oligonucleotide probes containing 65 the added inconvenient step of drying the samples must be
multiple non-radioactive labels”, Nucleic Acids Research. performed prior to measurement. Furthermore. the quantum
18 pp 501-505 1990.] reported labeling the polyamide e?iciency of the BCPDA europium(lII) complex is relatively
5,696,240
3 4
low, so that as reported by Khosravi, M. J. and Diamandis, 4,4"-diphenyl-2.2,40;6'2"-terpyridine were excited at 340
E. P., Time-Resolved Immuno?uorometry of Follitropin in nm and emitted respectively at 614 nm and 544 nm.
Serum Clinical Chemisny, 35, No. 1, 181 (1989), it was It should be pointed out that the poly-chelating ligands
necessary to increase the sensitivity for analytes which are described in US. Pat. Nos. 5,216.134 and 5.324.825 are
present in low concentration, by binding the chelate to organic compounds capable of existence as metal-free spe
thyroglobulin as an intermediate step to build a multilayer cies. Their lanthanide?ll) complexes are formed by com
system. plexation of the metal ions with the pre-synthesized ligands
Soini and Lovg'en, Time-Resolved Fluorescence of Lan and, if complexes containing different lanthanides are
thanide Probes and Applications in Biotechnology, CRC mixed, metal scrambling can occur resulting in loss of
Critical Reviews in Analytical Chemistry Vol. 18., Issue 2 labeling speci?city. The complexes of these ligands have
10
another limitation. The absorption of light by the heteroaro
(1987) pages 105-154; Soini and Hemmila, Fluorescence
Spectroscopy Assay Means. US. Pat. No. 4,374,120, (1983) matic ligands, which is the ?rst step in the energy pathway
leading to lanthanide emission. occurs at low wavelengths
have reported on the use of a ?uorescent chelate of a
lanthanide. The ligand was DTPA (W0 Patent 03698. 1984). (272-342 nm). In this spectral region conventional ?uores
which forms a strong but non?uorescent complex with cence microscope objectives absorb a considerable portion
europium?ll). This complex was dissociated with acid. of the exciting radiation, with resulting lower emission
intensity by the lanthanide centers.
Hemmila et al. Europium as a label in Time-Resolved
Irnrnuno?uorometric Assays, Anal. Biochem. 137, 335-343 Xu, US. Pat. No. 5,316,909 (1994), has utilized the
(1984); and the solubilized europium??) was complexed interaction between B-diketonate complexes of luminescent
with a beta-diketone in a micellar phase. These dissociation lanthanide(ll1') and yttrium (111). in the presence of a syn
complexation steps resulted in an increase in complexity of ergistic compounds. to provide a co?uorescence effect
the procedure; in a decrease in the concentration of the which signi?cantly increases the emission intensity. Xu
chelate. which limited the sensitivity of the ?uorescence states. ‘The strong ?uorescence of the lanthanide chelates is
measurement; and in a loss of spatial information due to the based on the fact that the ligand absorbs the excitation
separation of the ?uorophore from the binding moieties. energy, whereafter the energy is transferred from the triplet
25
Because of the separation of the ?uorescent tag from the level of the ligand to the resonance level of the lanthanide.”
speci?c binding molecule, this technique is incompatible Xu further states that. ‘The co?uorescence is based on an
with immuno?uorescence or similar measurements on intermolecular energy transfer that occurs from the chelate
single cells, or other particles by either ?ow cytometly or of the ion increasing ?uorescence, the energy donor. to the
microscopy. chelate of the ?uorescent ion, the energy acceptor, provided
30
Mukkala et al.. US. Pat. No. 5.216.134 (1993). described that the co?uorescence complex is in solution as a suspen
a number of poly-chelating ligands consisting of two sion or in solid form as aggregated particles and that the
N-donor heterocyclic rings joined at the 2-carbon positions solun'on contains a large excess of the chelate containing ion
increasing the ?uorescence.” The embodiment described by
by a bridging group suitable for the formation of a five-or
that inventor requires that the original chelate containing the
six-membered ring chelate ring with a lanthanide(l]I) ion. 35
Each of the N-donor heterocyclic rings also carried at the ?uorescent ion be dissociated from the biomolecule. fol
2'-carbon position a pendant substituent terminating in car lowed by the addition of the yttrium co?uorescence species
boxylate or phosphonate groups for additional chelation to and incubation with the synergistic compounds for 1 to 15
the lanthanide?ll) ion. In addition. a group carrying a minutes prior to measurement.
coupling functionality was substituted for any one of the Cell labeling with microcrystals of the phosphor yttrium
hydrogen atoms of the parent chelating structure. Table 1 of (111) oxysul?de doped with europium?ll) was reported by
said US. Pat. No. 5,216,134 described the ?uorescence Beverloo, H. B.; van Schadewijk, A.; van Gelderen-Boele.
properties of the Eu(l]I) and Tb(l1I) complexes of some of 5.; and Tanke, H. “Inorganic Phosphors as New Lumines
the above mentioned ligands, end speci?cally of various cent Labels for Immunocytochemistry and Time-Resolved
ring-substituted 6.6'-bis(N,N-bis(carboxymethyl) Microscopy”: Cytometry, 11, pages 784-792. 1990. Cells
45
aminomethyl)-2,2'-bipyridines. The excitation wavelengths speci?cally labeled with this phosphor could be detected in
for the Eu(l1I) chelates were in the 272-342 nm range in a time-gated mode even in the presence of the very bright,
borate bu?‘er at pH 8.5 buffer and in the 272-344 nm range red emitting DNA stain, ethidium bromide. A disadvantage
in ethanol The excitation wavelengths for the Tb([[I) che of these 50-500 nm phosphor particles was their tendency to
lates were in the 272-325 nm range in borate buffer at pH agglutinate. Coating the particles with a polycarboxylic acid,
8.5 and in the 272-325 nm range in ethanol. Monoclonal which can then be displaced by negatively charged ions such
antibodies to human larninine and keratin were labeled with as phosphate, citrate, or ionized ethylene-diamine tetracar
a IOO-fold molar excess of the isothiocyanate derivatives of boxylic acid, may serve to prevent aggluntination. However.
the Eu(llI) chelates (82) and (83). These antibodies were the negative charge of the coated phosphors causes nonspe
applied to ?xed cryostat sections which were subsequently 55 ci?c binding to slide-coating agents such as poly-l-lysine or
dehydrated with ethanol and viewed at 613 nm with a bovine serum albumin. In a subsequent paper, these authors
?uorescence microscope under excitation at 340 to 380 nm. reported discouraging results with these phosphors. Because
Kankare et al., US. Pat. No. $324,825 (1994), described of the short excitation Wavelengths (below 300 nm). com
a number of poly-chelating ligands capable of forming plete quartz optics were required (Beverloo et al., 1992,
luminescent complexes with appropriate metal ions. These “Preparation and Microscopic Visualization of Multicolored
Luminescent Irnmunophosphors”: Cytometry, 13, pages
polychelate ligands consisted of a 2,2':6'.2" terpyridine as
the parent structure, with substituents terminating in car 561-570). The authors concluded, “Nevertheless, there is a
boxylate groups at each of the 6 and 6“ positions. In need to develop soluble time-resolved dyes with long lumi
nescence life times with high quantum e?iciency which do
addition, a group containing a coupling functionality was
substituted for any one of the hydrogen atoms of the parent 65 not signi?cantly fade upon excitation”.
chelan'ng structure. The europium?il) and terbiurn?lI) com A modi?ed ?ow cytometer has been employed to detect
plexes of 6,6"-Bis[N. N-bis(carboxymethyl)aminomethyl] the time-gated luminescence of biotinylated liposomes con
5,696,240
5 6
taining a europium?II) chelate (Condrau M. A. Schwen templated Schiif-base condensation of 2.6-diacetylpyridine
dener R. A.. Zimmermann M. Muser M H.. Graf U.. Nied with ethylenediarnine in the presence of the nitrate salts of
erer P., and Anliker M. ‘Time-Resolved Flow Cytometry for lanthanurn(I[l) and cerium(Il1). The conditions selected for
the Measurement of Lanthanide Chelate Fluorescence: II. the condensation were. however. reportedly unsuccessful for
Instrument Design and Experimental Results". Cytometry. the preparation of analogous complexes from the heavier
1994 16 pp 195-205). The biotin of the europium-labeled
members of the lanthanide series. Because of the high
liposomes was at?nity-linked to the streptavidin bound to
kinetic stability of these complexes and their resistance to
biotinylated CD8. which in turn was bound to human
dissociation in dilute aqueous solutions. the authors sug
lymphocytes. These authors stated. “However. the immuno
gested their potential use as aqueous NMIR shift reagents.
liposome method employed for the selective tagging of cell
10 The synthesis of kinetically stable macrocyclic complexes
surface antigens needs to be improved. particularly in terms
of the lanthanide(lII) ions by metal templated condensation
of the reduction of the nonspeci?c adsorbtion of the ?uo
techniques has recently been extended to include all ele
rescent complexes to the cell membrane."
ments within the lauthanide series. except radioactive
A series of luminescent metalloporphyrins have been promethium. glPm.
reported (Savitsky A. P.. Ponomarev G. V.. Lobanov O. I.
15 The following additional references describe known non
and Sadovsky A. “Room-Temperature Phosphorescence of
functionalized macrocyclic complexes. List A includes pub
Metalloporphyrins and its Application to Immunoassay”.
lications by at least one of the inventors. List B includes
Proceedings of Biochemical Diagnostic Instrumentation.
additional publications by others.
Progress in Biomedical Optics. Ed. R. F. Bonner, G. E.
Cohn. T. M. Lane. and A. V. Priezzhev. SPlE Proceedings LIST A
20
Series 2136. pages 285- 298. 1994). Several of these com L. De Cola. D. L. Smailes. L. M. Vallarino. X Convegno
plexes had excitation maxima at 381 um and thus could be Nazianale Di Fotochimica, 1985. p. 4, E?‘ect of Heteroli
excited by the same light source used for as europium gands on the Luminescence Properties of Cationic Macro
complexes. The emissions of the metalloporphyrins cyclic Complexes of Eu(]]I) and Tb(I[I).
occurred further in the red (646 to 707 nm) compared to L. De Cola. D. L. Smailes. L. M. Vallarino. Inarganica
25
those of europium(ll1) (614 to 620 nm). so that both species Chimica Acta, 110, 1985. L1-L2.Metal-templated Synthesis
could be simultaneously detected. In solution. these metal of Novel Macrocyclic Complexes of the Uranyl Ion.
loporphyrins were sensitive to oxygen and required the L. De Cola. D. L. Smailes. L. M. Vallarino. Inorganic
presence of an oxygen scavenger. The binding to the anti Chemistry 1986, 25. 1729. Hexaaza Macrocyclic Com
body was achieved via carboxylate groups attached to the 30 plexes of the Lanthanides.
periphery of the organic ligands.
G. Bombieri, F. Benetollo, A. Polo. L. De Cola. D. L.
The lanthanide?ll) macrocyclic complexes with pendant
Smailes. L. M. Vallarino. Inorganic Chemistry 1986. 25.
functionalities described in the present invention otl'er a 1127. Synthesis, Characterization. and Crystal Structure of a
unique combination of high luminescence intensity. chemi l-lexaaza Macrocyclic Complex of Lutetium(lII).
cal resistance to decomposition, and ability to link
35 N. Sabbatini. L. De Cola. L. M. Vallarino. G. Blasse. J.
covalently to biosubstrates. (R. C. Leif and L. M. Vallarino.
Phys. Chem. 1987. 91. 4681-4685. Radiative and Nonta
“Rare-Earth Chelates as Fluorescent Markers in Cell Sepa
diativc Transitions in the Eu(]]I) Hexaaza Macrocyclic
ration and Analysis”. ACS Symposium Series 464. Cell
Complex [Eu (C22 H26 N6)(CH3COO)](CH3COO)
Separation Science and Technology, D. S. Kampala and P.
Cl*2H20.
W. Todd Editors, American Chemical Society, Washington.
G. Bombieri, L. De Cola, A. Polo. D. L. Smailes. L M.
DC, pages 41-58, 1991 ;. L. M. Vallarino. P. M. Harlow and
Vallarino. Virginia Journal of Science, 1987. 38. 95. Con
R. C. Leif; “Lanthanide Macrocyclic Complexes. “Quantum
densation of 2.6-Diacetylpyridine and 1,2-Diaminobenzene
Dyes”: Optical Properties and Signi?cance”. Proceedings of
in the Presence of Lanthanide Ions.
Advances in Fluorescence Sensing Technology. I. R.
Lakowicz and R. B. Thompson Editors, A. Katzir Progress F. Benetollo, G. Bombieri, W. T. Hawkins. A. Polo. L. M.
45 Vallarino. Division of Inorganic Chemistry 1987. 193.
in Biomedical Optics Series Editor. SPIE Proceedings Series
Synthesis. Structure. and Properties of Y(III) Macrocyclic
1885 376-385, 1993; R. C. Leif, L. M. Vallarino. and P. M.
Complexes.
Harlow; Production. Fixation, and Staining of Cells on
Slides for Maximum Photometric Sensitivity”. Proceedings A. Polo. J. R. Quagliano, L. M. Vallarino. Virginia Jour
of Biochemical Diagnostic Instrumentation, Progress in nal of Science, 1988. 39. 140, Lanthanide Complexes of a
50 Six-Nitrogen Analog of 18-C1'own-6.
Biomedical Optics. Ed. R. F. Bonner, G. E. Cohn, T. M.
Laue. and A. V. Priezzhev. SPIE Proceedings Series 2136, W. T. Hawkins 1]. A. Polo. L. M. Vallarino. Journal of
pp. 255—262, 1994). These luminescent species consists of American Chemical Society, 1987, 19, 36. Synthesis of
two parts, the functionalized macrocycle which binds the Six-Nitrogen-Donor Macrocyclic Complexes of Yttrium
lanthanide ion to the biomolecule and the enhancer which $5 (III)
absorbs the light and transfers the energy to the lanthanide. LIST B
Since the enhancer is a separate entity. both its chemical A. M. Arif. J. D. J. Backer-Dirks, C. J. Gray. F. A. Hart.
nature and its concentration may be tuned to provide higher M. B. Hursthouse. J. Chem. 800., 1987. 1665, Syntheses,
emission intensity. Thus the separation of the substrate X-Ray Structures, and Properties of Complexes of Macro
bound light-emitting europium center from the photo-trap. cyclic Hexamines with Lanthanide Nitrates.
the organic enhancer. permits the independent optimization K. K. Abid, D. E. Fenton. Inorganica Chimica Acta,
of both. 82(1984)223—226, The Synthesis of Macrocyclic Lan
The preparation of inert, kinetically stable. non thanide Complexes Derived from 2.5-Furandialdehyde and
functionalized macrocyclic complexes of a limited number alpha, omega-Alkanediamines.
of lanthanide(l]1) ions was ?rst reported in the technical 65 B. Bombieri, Inorganica Chimica Acta, 139(1987) 21-32.
literature by Backer-Dirks et al., J.C.S. Chem. Comm. (1979) New Trends in the Structural Chemistry of Actinide and
774. The synthesis described in this paper involved a metal Lanthanide Coordination Compounds.
5 .696, 240
7 8
W. Radecka-Paryzek. Inorganica Chimica Acta, 109 The derivatization of the chelates also contemplated the
(1985) 221-23. The Template Synthesis and Characteriza‘ presence of a “spacer" unit, which reportedly was linked to
tion of Hexaaza 18-Membered Macrocyclic Complexes of the chelate and could serve as a point of further derivatiza
Cerium(IlI). Praseodymium(lII) and Neodymium(IlI)
tion or attachment to the target molecules. The use of
Nitrates.
“spacer” units was recommended where the chelates were to
K. K Abid. D. E. Fenton. Inorganica Chimica Agra,
be used in conjunction with “biologically active target
95(1984) 119-125. Lanthanide Complexes of Some Mac
rocyclic Schitf Bases Derived from Pyridine-2,6 molecules", i.e. therapeutic drugs, enzymes. hormones. pep
dicarboxyaldehyde and alpha. omega-Primary Diamines. tides and other types of macromolecules which are typically
W. Radecka-Paryzek. Inorganica Chimica Acta, 45(1980) analyzed by immunoassay. Wieder’ s disclosure was.
Ll47-148. Template Synthesis and Characterization of however, relatively uninformative as to What type of spacer
lit-Member ed Hexaaza Macrocyclic Complex of
units were to be used, the criteria for their selection. and how
lanthanumfIlI) Perchlorate.
and under what conditions the chelate could be appropriately
V. K. Manchanda, C. A. Chang. Anal. Chem, 1987,
modi?ed to incorporate such spacer units.
813-818. Solvent Extraction Studies of Europium(l]I),
Ytterbium??). and Lutetium(I[l) with Ionizable Macrocy Notwithstanding the relative success reported by Wieder
clic Ligands and Thenoyltri?uoroacetone.
for his species-linked diarninetriacetic acid compounds as
W. Radecka-Paryzek, Inorganica Chimica Acta, 52(1981)
chelating agents for lanthanide ions. further improvement is
261-268. The Synthesis and Characterization of the Mac
still needed in the development of lanthanide-ligand com
rocyclic and Ring-opened Complexes Formed in the Reac
plexes for use in bioanalytical environments. and specifi
tion of the Lanthanides with 2,-6-diacetylpyridine and
Hydrazine. cally for immunoassay. In particular, there still exists a need
E. E. Fenton, P. A. Vigato, Chem. Soc. Rev., 1988, 17. for a lanthanide composition which can be readily adapted
69-90, Macrocyclic Schilf Base Complexes of Lanthanides (conjugated) to biologically active materials without sub
and Actinides. stantial alteration in its kinetic stability and desired physical
In the syntheses described in the Vallarino papers cited in 25 properties. The desired properties can include. for example.
List A. macrocyclic complexes were prepared from all
high ?uorescence excitation e?iciency and suitable lifetime
lanthanide(IlI) ions, except radioactive promethium. by
for complexes intended as ?uorescent probes, or high relax
metal templated condensation of 2,6-diacetylpyridine and
ivity toward the protons of water forcomplexes intended as
1.2-diaminoethane (ethylenediamine). The success of the
Vallarino technique in preparing macrocyclic complexes 30 in vivo magnetic resonance contrast agents.
from the heavier lanthanides was believed to be favorably
This invention has addressed the de?ciencies in the prior
in?uenced by the speci?c counterion present in the reaction
medium More speci?cally. when Vallarino carried out the art by providing a series of water-soluble hexa-aza
metal templated condensation in the presence of a lanthanide macrocyclic complexes. also hereinafter referred to as “mac
acetate. instead of the previously used nitrate or perchlorate 35 roq'clic complexes", incorporating a lanthanide, actinide or
salts. the formation of the macrocycles was extended to yttrium ion, wherein such complexes possess high kinetic
lutetium; furthm'more, greatly improved yields and purity of stability and pendant functional group(s) that can be readily
the resultant complex were observed. The use of a lan coupled/conjugated to a biologically active molecule such as
thanide acetate as the source of lanthanide ions was thus an antibody or antigen. or to a biologically compatible
preferred.
ionically uncharged macromolecule. such as a linear or
The macrocyclic complexes obtained by Vallarino et al. cross-linked polysaccharide. The functional group(s) can be
exhibited a kinetic stability and resistance to dissociation in incorporated into the macrocyclic structure either before or
dilute aqueous solutions that made them suitable for the
after the condensation reaction. Rn'thennore. the macrocy
staining of cytological specimens. Adaptation of these lan
clic complexes possess the properties desired for their
thanide macrocyclic complexes to the imaging science, as 45 intended use. For example. the complexes of europium?ll)
well as to ?uorescent immunoassay, requires their derivati
and terbium(III) possess a long-lived ?uorescence intensity
zation to permit their coupling to a biological substrate or to
that can be substantially increased by interaction with a
other reactive groups and/or entities. Such functionalization
suitable enhancer; whereas. the complexes of gadolinium
has not as yet been reported.
(1H) possess high relaxivity.
There have been other attempts at exploiting the unique
properties of lanthanides in both the industrial and the
biological environments. In each instance, the lanthanide SUMMARY OF THE INVENTION
(III) ion was associated with a chelating ligand. Some
The macrocyclic complexes of this invention. having a
processes involved, or were related to, the use of lanthanide
ligand complexes in a “time-gated” ?uorescence detection lanthanide, actinide, or yttrium ion as the metal center and
system. reportedly suitable in both solid and ?uid phase carrying peripheral coupling functionalities. are represented
?uorescence microscopy. Wieder et al. in US. Pat. No. by the general formulas I-lJI depicted below. In Formula I
4,352,751 described the chelation of lanthanide ions with a the substituent Q is attached directly to the macrocycle ring,
species-linked diaminetetraacetic acid, and the use of such and in Formula II the substituent Q is attached to the
chelates in conjunction with a “sensitizer". The sensitizer aromatic moiety. In Formula III. substituents A and B are
was used to increase the ?uorescence excitation e?iciency of attached respectively to each of the aliphatic side-chains,
these lanthanide chelates. The lanthanide chelates could be and substituents C and D are attached respectively to each of
further derivatized by incorporation of so-called “target the aromatic moieties (pyridine, furan, or thiophene) of the
molecules”; that is, of compounds which immunochemically macrocycle ring.
mimicked the analyte of interest and, thus. could compete
with the analyte of interest for an antibody speci?c to the Schematic formulas of symmetrically disubstituted mac
analyte. rocyclic complexes:
5,696,240
9 10
n is an integer selected from the group consisting of 2 and
I 3;
l' (C 1
"_\ Y is a negatively charged ion. including acetate,
carboxylate, sulfonate, halide, nitrate. perchlorate,
X thiocyanate, and picrate, with the proviso that such negative
ion does not limit the solubility of the resultant complex or
N x i " N
otherwise interfere with either the coupling procedure or the
out tIM-"II' 0 (Y1).
energy transfer leading to ?uorescence;
o" E ~‘~ N
I i
m is the ionic charge of the metal ion in the macrocyclic
_.._ R complex, and;
1. (CH). 1,, y is the ionic charge of the counterion in the macrocyclic
complex.
The macrocyclic complexes depicted above are prepared
and 20
by the lanthanide, actinide, or yttrium templated cyclic
II Schitf-base condensation of a 2.6-dicarbonylpyridine, or its
T 9 * furan or thiophene analog, with a 1.2-diaminoethane, with
either the dicarbonyl- or the diamino- precursor carrying the
(Ciel)
25 desired pendant coupling functionality. In the alternative, the
("x coupling functionality is added in a subsequent reaction.
'.__j R
X
The functionalized diamino- precursor is prepared by
[ N _ \IjMgH jI" ' N ] (Yr). 30 converting a substituted alpha-amino acid to the correspond
ing l.2-diaminoethane derivative. Where the amino acid has
other functional groups which are sensitive to reduction or
N "l N
deanimation, these groups are protected in the conventional
l ,'( R manner.
35
Once the functionalized 1,2-diaminoethane derivative has
(Cuff-4) been prepared, it is combined with a 2,6-dicarbonyl
pyridine. or its furan or thiophene analog, and with a salt of
i Q i J’
a lanthanide, actinide or yttrium ion. preferably the acetate
salt. The metal ion provides a geometric template for the
cyclic 2:2 condensation of the dicarbonyl compound with
the 1,2-diamino derivan've. The condensation is carded out
wherein in both formulas I and II:
preferably in an anhydrous alcohol, such as methanol or
R is a substituent selected from the group consisting of 45 ethanol. The reaction product, notably the lanthanide,
hydrogen, straight-chain alkyl, or branched-chain alkyl;
aryl-substituted alkyl, aryl, and alkyl-substituted aryl, actinide or yttrium macrocyclic complex, is recovered as a
solid residue from the reaction medium by vacuum evapo
with the proviso that such substituent does not limit the
solubility of the resultant complex or otherwise interfere ration. The solid residue is then recrystallized to yield a
with the cyclization of such complex during its synthesis; 50 crystalline product. the composition and structure of which
M is a metal ion selected from the group consisting of a correspond to the desired macrocyclic complex, as con
lanthanide having atomic number 57-71. an actinide having ?rmed by chemical and spectroscopic analysis.
atomic number 89-103 and yttriumflII) having atomic num
ber 39;
X is selected from the group consisting of nitrogen, sulfur 55 The functionalized dicarbonyl precursor can be prepared
and oxygen which forms a pad of a ring structure selected from the corresponding functionalized heterocycle by any
from the group consisting of pyridine, thiophene or furan, one of the appropriate methods lmown in the literature. If
respectively. at the positions marked X; necessary, the functional group is protected prior to the
Q is a substituent selected from the group consisting of transformation into the dicarbonyl derivative. The function
functionalized methyl. functionalized straight-chain alkyl, 60 alized dicarbonyl compound is then reacted with 1,2
functionalized branched-chain alkyl; functionalized aryl
diarninoethane in the presence of a metal salt as a template,
substituted alkyl. functionalized aryl. and functionalized
under the above indicated conditions, to produce the metal
alkyl-substituted aryl, with the proviso that groups of said
macrocycle functionalized at the heterocyclic moiety.
substituent provide coupling functionality between said sub
stituent and a bridging/linldng moiety to permit the deriva~ 65
tization thereof with a receptor molecule or an entity for Schematic formulas of unsymmetrically disubstituted
which there is a corresponding receptor molecule; macrocyclic complexes:
5,696,240
11 12
substituted 1.2-diiminoethane moieties, with each of the
diiminoethane moieties carrying one of the substituents A
and B.
In all three above mentioned procedures. either or both of
the pyridine moieties can be substituted by furan or
thiophene analogs. In an alternative procedure, any one of
the four substituents A, B. C, and D is added to the
pre-formed macrocyclic complex in subsequent reactions.
The reactants used in the templated synthesis of the
macrocyclic complexes of Formula III are obtained as
10
follows:
(Yr), The substituted 1,2-diaminoethane and substituted 2,6
dicarbonylpyridine, and its furan and thiophene analogs, are
obtained according to established literature procedures, as
outlined above for the complexes of Formulas I and H.
15
The dicarbonyl three-quarter-cycle is prepared by the
Schi?-base condensation, in the absence of a metal salt
template, of: (1) a substituted 1,2-diaminoethane and a
substituted 2,6-dicarbonylpyridine, in a 1:2 mole ratio; or (2)
a substituted 1,2-diaminoethane with a substituted 2,6
20 dicarbonylpyridine, in a 1:1 mole ratio. followed by the
Schilf-base condensation of the resulting compound with a
substituted 2,6-dicarbonylpyridine, in a 1:1 mole ratio and in
the absence of a metal salt template.
The diarnino three-quartcr-cycle is obtained by the Schi?
Wherein in Formula I[[:
25 base condensation, in the absence of a metal salt template,
R, M, X, Y. m, and y have the same meaning as in
of: (1) a substituted 1,2-diarninoethane with a substituted
Formulas I and 11. and 2,6-dicarbonylpyridine, in a 2:1 mole ratio; or (2) a substi
A, B, C, and D are selected from the groups consisting of tuted 1,2-diaminoethane with a substituted 2,6
hydrogen, straight-chain alkyl, or branched-chain alkyl; dicarbonylpyridine. in a 1:1 mole ratio, followed by the
aryl-substituted alkyl, aryl, or allcyl-substituted aryl; reactive 30 Schiff-base condensation of the resulting compound with a
functionality, functionalized alkyl, functionalized aryl substituted 1,2-diaminoethane, in a 1:1 mole ratio and in the
substituted alkyl, functionalized aryl. or functionalized absence of a metal salt template.
alkyl-substituted aryl. In the above mentioned syntheses of the dicarbonyl
three-quarter-cycle and diarnino tree-quarter-cycles, the sub
At least one of the substituents A,B,C, and D is selected
stituted diamine and substituted pyridine precursors carry
from the group consisting of a reactive functionality, func
three of the four of the A, B, C, and D substituents in the
tionalized alkyl, functionalized aryl-substituted alkyl, func
manner required to produce, after metal-templated
tionalized aryl, or functionalized alkyl-substituted aryl, with
cyclization, the macrocyclic complexes depicted by Formula
the proviso that groups of said substituent provide coupling
IH; furthermore, one or both of the pyridine moieties can be
functionality between said substituent and a bridging/linking substituted by furan or thiophene analogs.
moiety to permit the derivatization thereof with a receptor
The macrocyclic complexes have various uses depending
molecule or an entity for which there is a corresponding upon the unique properties of each central metal ion, upon
receptor molecule. the other constituents of the complex, and upon the inter
One or more of the three remaining substituents AB, C, actions between the lanthanide ion and any other constitu
and D. selected from the group consisting of reactive ents of the complex.
functionality, functionalized alkyl, functionalized aryl 45 The pendant functional groups are further reacted
substituted alkyl, functionalized aryl, or functionalized (conjugated) with biologically active molecules, i.e.
alkyl-substituted aryl, can incorporate a moiety capable of antibodies, antigens, binding proteins. polysaccharides,
providing additional features, such as increased solubility, nucleotides, polynucleotides and/or analytes of interest. This
greater stability, enhanced luminescence, or a combination conjugate is then used in analytical procedures, eg ?uo
thereof. with the proviso that said substituent or substituents 50 rescent immunoassay; in the identi?cation of biological
do not hinder the coupling of the macrocycle to the biosub products, i.e. cells, proteins and low molecular weight
substances; in histological staining; in ?uorescence in situ
strate.
hybridization of nucleic acids and other ?uorescence label
The macrocyclic complexes of Formula lII depicted
ing of nucleic acids; in nuclear magnetic resonance or
above are prepared by the lanthanide, actinide, or yttrium
SS nuclear imaging; and in immunotherapy. More specifically,
templated cyclic Schi?-base condensation of:
the conjugates of this invention are used as an injectable
(1) an A-substituted 1,2-diaminoethane, a B-substituted
solution for administration to a recipient for in vivo obsm
1,2-diaminoethane, a C-substituted 2,6-dicarbonylpyridine,
vation and/or labelling of a target tissue in accordance with
and a D-substituted 2,6-dicarbonylpyridine; standard diagnostic imaging protocols; or, alternatively, in
(2) an A-substituted 1.2-diaminoethane and a dicarbonyl treatment of a disease associated with speci?c antigens. i.e.
compound (referred to as “dicarbonyl three-quarter~cycle") neoplasia and autoimmune disorders.
consisting of a B-substituted 1,2-diiminoethane moiety and
two substituted pyridine moieties, with each of the pyridine DETAILED DESCRIPTION OF THE
INVENTION INCLUDING PREFERRED
moieties carrying one of the substituents C and D; and
EMBODIMENTS
(3) a C-substituted 2,6-dicarbonylpyridine and a diarnino 65
compound (referred to as “diarnino three-qum'ter-cycle”) The macrocyclic complexes of this invention me unique
consisting of one D-substituted pyridine moiety and two in several signi?cant respects. The combination of proper
5,696,240
13 14
ties which sets them apart from other complexes of the In one of the preferred embodiments of this invention, the
lanthanide, actinide or yttrium ions includes: kinetic inert functionalized 1,2-diaminoethane is obtained from a substi
ness (chemical stabilization) in dilute aqueous solution; high tuted amino acid by the following general reaction scheme.
?uorescence excitation e?iciency of the europium?ll) and Initially, the substituted amino acid is converted to the
terbium?ll) derivatives in the presence of suitable enhanc corresponding methyl ester hydrochloride by reaction with
ers; high relaxivity of the gadolinium?ll) complexes; ability anhydrous methanol and hydrogen chloride gas. This ester
to react with, i.e. covalently bond to, biomolecules, i.e. intermediate is further reacted with gaseous ammonia in
antigens, antibodies. binding proteins etc., either directly or anhydrous methanol to produce the corresponding amide
through an intermediate bridging moiety; and relative ease hydrochloride. The amide hydrochloride is then changed to
and predictability of synthesis. The conjugation of these the haze amide by further reaction with ammonia in anhy
complexes to a desired substrate through their pendant drous tetrahydrofuran. If necessary, the pendant functional
functional groups is virtually limitless. Furthermore, such group to be used as a coupling link is protected by the use
conjugation is effected without adversely altering the ?uo of one of the many protective groups known in the literature.
rescent behavior, relaxivity, or kinetic stability of the com This protected, tree amide is then subjected to reduction
plexes in comparison to the non-functionalized analogs. with either boron hydrideltetrahydrofm'an or lithium alumi
15
The complexes of this invention are prepared from start num hydride/tetrahydrofuran, followed by acid hydrolysis to
produce a functionalized diarnine hydrochloride. This
ing materials which are either commercially available, or
which can themselves be synthesized from commercially diamine hydrochloride is converted to the free diamine by
available starting materials in accordance with established reaction with a base, e.g. sodium methoxide or potassium
synthetic procedures or adaptations thereof. As noted above, hydroxide, in anhydrous methanol at 0° C. Substituted
one of the primary objectives of this invention is to provide amino acids which satisfy the foregoing criteria include, for
example: tyrosine, aminophenylalanine, lysine and its
a synthetic pathway for the preparation of these complexes.
namely, the macrocyclic lanthanide, actinide or yttrium analogs, cystine. cysteine, and serine.
complexes, by the metal templated Schi?-base condensation In another embodiment of this invention, a functionalized
dicarbonyl precursor is prepared from 3-hydroxypyridine by
of: (1) a Q-functionalized LZ-diamine with a 2,6 25
dicarbonylpyridine, or its furan or thiophene analog; (2) a the following scheme. The 3-hydroxypyridine is reacted
Q-functionalized dicarbonyl-pyridine, furan or thiophene with formaldehyde (37% in water) in the presence of a
with 1,2-diaminoethane, (3) a substituted 1,2-diaminoethane strong base (sodium hydroxide, NaOH) at 90° C., to give
and a dicarbonyl three-quarter-cycle containing two aro 3-hydroxy-2.6-bis(hydroxymethyl)pyridine. This is then
matic heterocyclic moieties, (4) a 2,6-dicarbonyl-substimted oxidized with manganese(IV) oxide. in the presence of
aromatic heterocycle and a diamino three-quarter-cycle con potassium carbonate (K2CO3). Acidi?cation of the ?nal
taining a single aromatic moiety. and (5) two substituted product gives the hydrochloride of the 3-hydroxy-2,6
diformylpyridine.
1.2-diamino-ethanes and two subsituted 2,6-dicarbonyl
substituted aromatic heterocycles; with each of the precur The following structural formulas are representative of
sors in syntheses (3), (4), and (5) carrying one of the some of the materials which are suitable in the preparation
35
substituents A, B. C, and D in the manner required for the of these complexes.
formation of a macrocyclic complex depicted by Formula A. Dicarbonyl Reactants
111 In each of the above mentioned syntheses, the aromatic
moieties may consist of pyridine, furan, or thiophene sys Q C
terns. I I
(CHIIL I-1) (CuIm -l)
In the macrocyclic complexes of Formulas I and II, the
functionalized portion of the diarnine or dicarbonyl precur __‘ I- i’ -_‘ i
sor which does not participate in the Schi?-base condensa ‘r’ R R ‘w’ R
tion reaction, are selected to afford compatibility with sub x x
sequent coupling to proteinaceous or poly- saccharide 45 o o o o
materials. In the macrocyclic complexes of Formula 111, at
least one of the four substituents A, B, C, and D is selected D
to a?ord compatibility with subsequent coupling to proteina (CDIIH MI)
ceous or poly-saccharide materials; whereas, one or more of
the three remaining substituents can be selected to provide
additional desired features, such as increased solubility, R ~—’ a
x
greater stability, enhanced luminescence, or a combination
thereof, with the proviso that such substituents do not hinder o 0
the coupling of the macrocycle to the biosubstrate.
The synthesis of these macrocyclic complexes follows S5 wherein
established techniques for the lanthanide templated conden n is an integer selected from the group consisting of 2 and
sation of 2,6-diacetylpyridine or 2.6-diformylpyridine and 3;
1,2-diaminoethane. However. the ability to accomplish such X is selected from the group consisting of nitrogen, sulfur
synthesis with a functionalized diamine or dicarbonyl pre and oxygen which forms a part of a ring structure selected
cursor is unique to this process and permits the further from the group consisting of pyridine, thiophene or furan,
coupling of the macrocycle to a biolog'cally compatible respectively, at the positions marked X;
synthetic macromolecule, or to what can be collectively R is a substituent selected from the group consisting of
classi?ed as a “biomolecule", i.e. cell surface marker, hydrogen, methyl, straight-chain or branched alkyl, aryl
polypeptide or genetic polynucleotide sequence. This cou substituted alkyl; aryl, and alkyl-substituted aryl,
pling is e?‘ected through the unreacted coupling functional 65 with the proviso that such substituent does not limit the
ity of the macrocycle. or through a bridging moiety attached solubility of the resultant complex or otherwise interfere
to such coupling functionality. with the cyclization of such complex during its synthesis;
5,696,240
15 16
Q is a substituent selected from the group consisting of Wherein Q is a substituent selected from the group
reactive functionality, functionalized straight-chain alkyl. consisting of reactive functionality. functionalized methyl,
functionalized branched-chain alkyl; functionalized aryl functionalized straight-chain alkyl. functionalized branched
alkyl. functionalized aryl. and functionalized alkyl-aryl with chain alkyl; functionalized aryl-alkyl, functionalized aryl.
the proviso that groups of said substituent provide coupling
and functionalized alkyl-aryl. with the proviso that groups of
functionality between said substituent and a bridginglinking
said substiment provide coupling functionality between said
moiety to permit the derivatization thereof with a receptor
substituent and a bridginglinldng moiety to permit the
molecule or an entity for which there is a corresponding
receptor molecule; and derivatization thereof with a receptor molecule or an entity
C and D are selected from the groups consisting of for which there is a corresponding receptor molecule; and
hydrogen. straight-chain alkyl. or branched-chain alkyl;
aryl-substituted alkyl. aryl. or alkyl-substituted aryl; reactive A and B are selected from the groups consisting of
functionality. functionalized aryl-substituted alkyl. function hydrogen. straight-chain alkyl. or branched-chain alkyl;
alized aryl. or functionalized alkyl-substituted aryl. When C aryl-substituted alkyl. aryl. or alkyl-substituted aryl; reactive
or D. or both. are selected from the group consisting of functionality. functionalized alkyl. functionalized aryl
reactive functionality. functionalized aryl-substituted alkyl.
substituted alkyl. functionalized aryl. or functionalized
functionalized aryl. or functionalized alkyl-substimted aryl.
alkyl-substituted aryl. When A or B. or both. are selected
either the C or D substituent can incorporate a functionality
from the group consisting of reactive functionality, func
suitable for coupling to a biosubslrate, whereas the other
substituent can incorporate a functionality that provides 20 tionalized aryl-substituted alkyl. functionalized aryl. or
additional features such as increased solubility. greater functionalized alkyl-substituted aryl. either the A or B sub
stability. enhanced luminescence. or a combination thereof. stituent can incorporate a functionality suitable for coupling
with the proviso that groups of said substituent do not hinder to a biosubstrate, whereas the other substituent can incor
the coupling of the mauocycle to the biosubstrate. porate a functionality that provides additional features such
The ring structures depicted above speci?cally include the 25 as increased solubility. greater stability. enhanced
pyridine. furan and thiophene rings.
luminescence, or a combination thereof. with the proviso
B. Metal Ion Salts
that groups of said substituent do not hinder the coupling of
the macrocycle to the biosubstrate.
(MW). 30
For the macrocyclic complexes having the structures of
wherein M is a metal ion selected from the group con Formulas I and II. the metal templated Schiff-base cycliza
sisting of a lanthanide having atomic number 57-71. an
tion involves only one functionalized precursor. eithm' the
actinide having atomic number 89-103 and ytlrium?Il)
dicarbonyl compound or the diarnine. The functionalized
having atomic number 39;
35 macrocyclic compounds derived from precursors having Q
Y is a negatively charged ion, including acetate.
attached to the diamine result in compounds having the
carboxylate. sulfonate. halide. nitrate. perchlorate.
thiocyanate, and picrate. with the proviso that such negative structure of Formula I. The functionalized macrocyclic
compounds derived from precursors having Q attached to
ion does not limit the solubility of the resultant complex or
the heterocyclic moiety result in compounds having the
otherwise interfere with either the coupling procedure or the
energy transfer leading to ?uorescence; structure of Formula II.
m is the ionic charge of the metal ion in the macrocyclic
complex; and. For the macrocyclic complexes having the structure of
y is the ionic charge of the counterion in the macrocyclic Formula III, the substituent (A. B. C. or D) that carries the
complex.
coupling functionality is attached either to one of the aro
45
C. Diamine Reactants matic moieties of the dicarbonyl precursors or to one of the
aliphatic chains of the diamino precursors; the remaining
NH N11; NH; NIH; three substituents are attached to the other precursors in the
Q2 A
manner required to produce the macrocycle of Formula 1]].
NH; NH;
An example species of Formula I in which Q is a
(4-aminophenyl)methyl group attached to the diamine pre
cursor is represented by the following formula:
5,696,240
17 18
Hsc 0N
N I N
E \ + /
HZN HzC - - / M'I" \ - - CH; NH; (‘11'’),
QKLNj/L
N N
l. H3 C) 4,
Another example species of Formula I in which Q is a The macrocyclic complexes that have been prepared by
(4-hydroxyphenyl)methyl group attach?d t0 the dl'?mille these procedures have been isolated as crystalline solids
precursor is represented by the following formula:
H3C\”J\/Oj\"/ cm
N
N N
E \ + / 3
HO HZC - - MP" - - CH; OH (10-),
/ \
N | N
| N
“3 C) CH3
l. i ,
An example species of Formula II in which Q is a hydroxy with correct chemical analysis and spectroscopic properties.
group attached to the heterocyclic ring of the dicarbonyl Most compounds were isolated as solvates.
precursor is represented by the following formula: 40
An example species of Formula 111. in which A is a
4-hydroxyphenylmethyl group attached to one of the ali
phatic side-chains and B, C, and D are hydrogen atoms, is
45 represented by the following formula:
50
55