Facile, Efficient Conjugation of a Trifunctional Lanthanide Chelate to a Peripheral Benzodiazepine Receptor Ligand

Article abstract of DOI:10.1021/ol017155b

matrix presented Receptor-mediated imaging and therapy of diseased tissue is rapidly gaining favor in the medical community. The synthesis and facile aqueous/ organic coupling of a peripheral-type benzodiazepine receptor ligand to a cyclen-based fluoropho

Full text of DOI:10.1021/ol017155b

ORGANIC
LETTERS
2002
Vol. 4, No. 7
1075-1078
Facile, Efficient Conjugation of a
Trifunctional Lanthanide Chelate to a
Peripheral Benzodiazepine Receptor
Ligand
H. Charles Manning, Timothy Goebel, John N. Marx, and Darryl J. Bornhop*
Department of Chemistry and Biochemistry, Texas Tech UniVersity,
Lubbock, Texas 79409
darryl.bornhop@ttu.edu
Received November 30, 2001
ABSTRACT
Receptor-mediated imaging and therapy of diseased tissue is rapidly gaining favor in the medical community. The synthesis and facile aqueous/
organic coupling of a peripheral-type benzodiazepine receptor ligand to a cyclen-based fluorophore is described herein. The contrast agent
QM-CTMC-PK11195, when chelated with lanthanides, produces bright luminescence and good MRI contrast and can potentially serve as an
imaging and demarcation agent for certain types of cancers.
Benzodiazepines continue to be among the most highly
prescribed central nervous system (CNS) drugs as a result
of their pharmacological properties.1 Overexpressed by
certain types of cancerous tissue, the peripheral-type ben-
zodiazepine receptor (PBR) is a class of binding sites for
benzodiazepines. It has been well documented that this
receptor also binds other types of small organic molecules
(e.g., isoquinolines,2 imidazopyridines,3 and indole4 deriva-
tives) with very high affinity.
process couples high transport capacity with ligand-specific
cell targeting, yielding a methodology to efficiently deliver
therapeutic and contrast agents to diseased tissue.7 To this
end, the ability to conjugate brightly luminescent fluoro-
phores to biologically up-regulated substrates is a crucial step
toward synthesizing contrast agents that clearly demarcate
diseased tissue.8 Recently, it has been shown that a PBR
ligand conjugate is able to retain the biological activity of
the native ligand.1 In an analogous way, by conjugating a
well-established tumor-specific ligand (PK-11195 analogue)
to one of our unique trifunctional lanthanide chelates, it is
believed that the resulting agent can be used for in vitro and
in vivo visual imaging of certain cancer bearing tissues.9
Receptor-mediated endocytosis can be employed as an
attractive means of affording cell-selective targeting.5,6 This
(1) Kozikowski, A. P. et al. J. Med. Chem. 1997, 40, 2435-2439.
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(8) Leopold, P. L. et al. Hum. Gene Ther. 2000, 11, 151-165.
(9) Manning, H. C.; Goebel, T. S.; Hopkins, J.; Thompson, R.; Bornhop,
D. J. Unpublished work.
10.1021/ol017155b CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/15/2002

Scheme 1
Development of this new cell-specific, visual contrast
can be efficiently synthesized and provide significant in vivo
contrast between normal and diseased (cancer) tissue,
facilitating early detection.20-22
agent is of paramount importance. Despite aggressive treat-
ment strategies including surgical resection, irradiation, and
chemotherapy, many cancer patients succumb to tumors
(within weeks to months),10,11 especially those of the intrac-
ranial region.12-15 Current brain tumor imaging methods
(detection of edema and the use of MRI)16 are severely
limited because they image the tumor indirectly and fail to
fully demarcate the boundaries of disease.17-19 Because
clinical outcome is so closely linked to surgical resection,
there is a critical need to develop new strategies for
intraoperative imaging of brain cancer. The ideal brain cancer
imaging modality would be tumor-specific, allowing infiltrat-
ing tumor margins to be well resolved, and provide real-
time intraoperative fluorescence that correlates with anatomic
imaging (MRI). We report methodology here to synthesize
such an imaging agent.
Recently we have reported a new trifunctional marker
that exhibits bright fluorescence (φ ∼0.25, Stokes shift
ca. 300 nm ), is chemically stable, and also has the neces-
sary functionality to be conjugated to many types of
biologics.23
Here, we report synthetic methodology used to prepare
and conjugate a PK11195 analogue to our lanthanide chelate
for use as a site-directed imaging agent. Our procedure is of
particular interest because, by using a well-documented
water-stable coupling agent (TSTU),24 we are able to
complete the conjugation under mixed aqueous/organic
conditions, unlike typical anhydrous peptide couplings. This
procedure will prove to be especially useful for the conjuga-
tion chemist whose substrate(s) are not fully soluble in the
absence of an aqueous solvent component.
The first step toward the synthesis of the conjugate was
to create the conjugable, trifunctional, macrocyclic ligand
1. This molecule possesses two phosphonic acid pendant
arms for strong chelating ability, an energy absorbing/
transmitting quinoline chromophore, and a carboxylic acid
Chart 1. Compound 1: Trifunctional Cyclen-Based
Lanthanide Chelate, QM-CTMC
(14) Enneking, W. F.; Conrad, E. U. Clinical Symposia; Ciba-Geiegy:
Summit, NJ, 1989; Vol. 41, pp 3-32.
(15) Rossi, M.; Zetter, B. R. Proc. Natl. Acad. Sci. U.S.A 1992, 89, 6197-
6201.
(16) Earnest, F. I.; Kelly, P. J.; Scheithauer, B. W. et al. Radiology 1988,
166, 823-827.
(17) DeAngelis, L. M. Brain Tumors. N. Engl. J. Med. 2001, 344(2),
114-123.
In previous work reported elsewhere, we have demon-
strated that our novel class of nontoxic lanthanide chelates
(18) Gordon, J. et al. Magn. Reson. Imaging. 1999, 17(10), 1495-
1502.
(19) Black, K. L.; Ciacci, J. R. West. J. Med. 1993, 158, 65-66.
(20) Houlne, M. P.; Agent, T. S.; Kiefer, G. E.; McMillan, K.; Bornhop,
D. J. Appl. Spectrosc. 1996, 50, 1221-1228.
(21) Hubbard, D. S.; Houlne, M. P.; Kiefer, G. E.; Janssen, H. F.; Hacker,
C.; Bornhop, D. J. Lasers Med. Sci. 1998, 13, 14-21.
(22) Houlne, M. P.; O’Briant, S. P.; Goebel, T.; Bornhop, D. J. Anal.
Chim. Acta 1999, 397, 267-278.
(23) Griffin, J. M.; Skwierawska, A. A.; Manning, H. C.; Bornhop, D.
J. Tetrahedron Lett. 2001, 42, 3823-3825.
(24) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Tetrahedron
Lett. 1989, 30, 1927-1930.
(10) Cotton, P. B. Pratical Gastrointestinal Endoscopy, 3rd ed.; Sci-
ence: Oxford, 1990.
(11) Melville, D. M.; Jass, J. R.; Morson, B.; Pollock, D. J.; Richman,
P. I.; Shepard, N. A.; Ritchie, J. K.; Love, S. B.; Lennard-Johnes, J. E.
Hum. Pathol. 1989, 20, 1008.
(12) Fadul, C.; Wood, J.; Thaler, H. et al. Neurology 1988, 38, 1374-
1379.
(13) Cancer Facts and Figures; American Cancer Society: Atlanta, 1997;
p 17.
1076
Org. Lett., Vol. 4, No. 7, 2002

Scheme 2
residue for conjugation. When compound 1 is complexed
reagent TSTU. This was exceedingly important when we
discovered that 1 would not remain soluble throughout the
course of reaction in organic solvents alone. Using this
methodology, compound 8 was obtained pure in reasonably
high yield.
with Eu3+, the methylquinoline antenna absorbs light at 320
nm and the complex emits the characteristic lanthanide
emission pattern from 580 to 700 nm. This large Stokes shift
allows complete removal of the signal (fluorescence) from
the background noise (excitation).
Compound 8 is easily complexed at pH 6 with various
lanthanides. An indication of complex stability, when Eu3+
is chelated with compound 8, aqueous solutions are brightly
luminescent upon excitation with a low energy TLC plate
reader lamp (as one would expect from sensitized emission),
and these solutions remain luminescent for at least 1 month.
To demonstrate the typical luminescence of the conjugated-
chelated complex, Figure 1 shows a photograph of a 1 µM
With 1 synthesized and characterized, we were ready to
prepare the conjugable form of PK11195 (Scheme 1).
Initially guided by previously demonstrated methodology,25
first compound 2 was synthesized by coupling racemic
norephedrine with 2-chloro-benzoyl chloride in basic me-
thylene chloride. The compound was easily purified by
recrystallization in EtOH and obtained in high yield. Next,
a condensation with P2O5 in o-dichlorobenzene at high
temperature formed the isoquinoline ring, yielding compound
3. Compound 3 was then brominated under free-radical
conditions with 2 equiv of NBS, yielding the dibromo
compound 4. Next, 4 was hydrolyzed with the aid of AgNO3
to the aldehyde 5. Then the same reagent oxidized 5 to the
carboxylic acid 6. Finally, compound 6 is coupled with the
hexanediamine linker in dry DMF, yielding a conjugable
PK11195 analogue 7 (Scheme 2).
The final procedure in the synthesis of the target compound
8 was to conjugate the fluorophore 1 to the PBR ligand 7.
As seen in Scheme 2, this was accomplished under mixed
aqueous/organic conditions utilizing the water-stable coupling
Figure 1. Photograph of 1 µM solution of Eu-QM-CTMC-
PK11195.
(25) Goodman, M. M. et al. U.S. Patent 5,998,624, 1999.
Org. Lett., Vol. 4, No. 7, 2002
1077

Figure 2. Absorbance spectrum of Eu-QM-CTMC-PK11195.
Figure 3. Fluorescence spectrum of Eu-QM-CTMC-PK11195
(excitation 320 nm).
aqueous solution of Eu-QM-CTMC-PK11195 when il-
luminated by a TLC plate reader lamp. Note the stark contrast
in color between the pink fluorescence (complex) and blue
light reflected off the skin (tissue). Figures 2 and 3 show,
respectively, the absorbance (antenna) and fluorescence
(Eu3+) spectra for the complex.
Acknowledgment. This work was funded by the Whi-
taker Foundation. The authors recognize contributions from
Robert A. Flowers for technical discussions regarding the
preparation of this manuscript.
In summary, we have demonstrated that we can conjugate
a PBR ligand to our class of exogenous fluorophores utilizing
mixed aqueous/organic conditions, yielding a bioconjugate
that could facilitate brain cancer imaging. In general, this
procedure should also be applicable to the synthesis of other
bioconjugates, particularly those requiring an aqueous en-
vironment to facilitate solubility.
Supporting Information Available: Detailed experi-
mental procedures and multinuclear NMR shifts and CHN/
HRMS characterization is provided. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL017155B
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Org. Lett., Vol. 4, No. 7, 2002