Ultrastable complexes for in vivo use: A bifunctional chelator incorporating a cross-bridged macrocycle

Article abstract of DOI:10.1039/b406906d

The synthesis, copper(II) complexation and biotin conjugation of a bifunctional chelator incorporating a cross-bridged macrocycle are described.

Full text of DOI:10.1039/b406906d

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Ultrastable complexes for in vivo use: a bifunctional chelator
incorporating a cross-bridged macrocycle{
Elizabeth A. Lewis, Ross W. Boyle and Stephen J. Archibald*
Department of Chemistry, University of Hull, Cottingham Road, Kingston upon Hull, UK HU6 7RX.
E-mail: s.j.archibald@hull.ac.uk; Fax: 144(0)1482 466410; Tel: 144(0)1482 465488
Received (in Cambridge, UK) 10th May 2004, Accepted 8th July 2004
First published as an Advance Article on the web 20th August 2004
The synthesis, copper(II) complexation and biotin conjugation
of a bifunctional chelator incorporating a cross-bridged
macrocycle are described.
and the biologically relevant molecule, biotin, has also been
demonstrated.
A synthetic route has been developed that affords 10 in seven
steps (Scheme 1) in reasonable overall yield (ca. 20%). We have
previously described the efficient synthesis of the starting bisaminal,
3, in which a 4-nitrobenzyl group is appended at the 6-position of
the tetraazacyclotetradecane ring.9 Highly regioselective di-sub-
stitution at non-adjacent N-positions of 3 is achieved by stirring an
acetonitrile solution with an excess of benzyl bromide (ca. 10 equiv.)
for 8 d to give 4 in good yield (75%).} Treatment of 4 with a large
excess (ca. 50 equiv.) of sodium borohydride in methanol results in
double reductive ring expansion and formation of 5 (78%)., On
hydrogenolysis of 5, debenzylation and nitro reduction occur
efficiently in a single step to yield 6 (79%). The primary amine site is
selectively protected over the two secondary amine ones in 6 using
one equivalent of benzaldehyde in methanol. After stirring at RT
overnight 7 is isolated in excellent yield (93%). 7 can be considered a
useful precursor to a range of di-N-substituted cross-bridged
systems by choice of appropriate electrophile. In the current
synthetic route, addition of two equivalents of tert-butylbromoa-
cetate to 7 in the presence of anhydrous potassium carbonate in dry
acetonitrile leads to the formation of 8 (50%) on stirring at RT for
3 d. Treatment of 8 with an aqueous 6 M HCl solution heated to
100 uC for 3 d allows both imine and ester hydrolysis to be achieved
quantitatively, to afford 9.5HCl. For bioconjugation purposes, 9
can be converted to the isothiocyanate derivative 10 by reaction
with thiophosgene under biphasic conditions (aqueous solution,
pH 8/CHCl3, w95%).
A bifunctional chelator (BFC) is a chelating ligand with a func-
tional group that can be used for attachment to a bio-targeting
vector. There is much current interest in the use of BFCs for
medical imaging and therapy e.g. MRI contrast agents, radio-
immunotherapy.1,2 The ability of a BFC to bind and retain the
appropriate metal ion (e.g. Gd, 90Y, 64Cu) in vivo, allowing its
localisation at the target site (e.g. tumour or organ) and limiting its
potentially harmful release, is crucial. Consequently, there is an
ongoing requirement for the development of novel, high stability
chelators for bioconjugation purposes.
Recent research efforts to develop BFCs have focused on the use
of macrocyclic systems, which typically impart relatively high
stability to their metal complexes compared to acyclic ones. In
particular, systems based on tetraazacyclotetradecane-N,N’,N@,N--
tetraacetic acid (TETA) have shown potential as candidates for
labelling biomolecules for diagnostic imaging and targeted radio-
therapy, with clinical trials in progress.3 However, there is evidence
of transchelation from radiometal labelled TETA-bioconjugates
in vivo. For example, Anderson and co-workers have demonstrated
that 64Cu dissociates from the peptide conjugate TETA-D-Phe1-
octreotide in the liver and binds to superoxide dismutase in high
concentrations.4 Similarly, Meares and co-workers have reported
67Cu transfer from an antibody-TETA conjugate to ceruloplasmin
in lymphoma patients.5
The coordination chemistry of cross-bridged macrocycles has
been studied in detail by a number of groups.{6,10 In order to
confirm that we observed similar reactivity towards Cu(II) as
reported by Weisman and co-workers for CB-TE2A,6 we
performed a complexation reaction with ligand 9. Combination
of an equimolar amount of copper(II) nitrate trihydrate and 9.5HCl
in a 1:5 mixture of methanol and aqueous sodium hydroxide
(5 equiv.) affords the neutral species Cu(9) as a dark purple solid, in
good yield (69%) [single peak observed by ES1 corresponding to
Cu(9)Na1]. Radiolabelling of the BFC is often carried out after a
protein or antibody conjugate has been formed, necessitating the
use of mild complexation conditions.2 It is noteworthy therefore,
that Cu(9) is formed easily at RT in aqueous solution.
In order to demonstrate the high reactivity of 10 towards
biologically relevant molecules, the biotin ethylenediamine- con-
jugate has been formed.** A solution of 10 was stirred with biotin
ethylenediamine (ca. 0.9 equiv.) in an aqueous sodium carbonate
(ca. pH 10) solution for 4 h at RT. Negative ion electrospray mass
spectrometry (Fig. 1) clearly shows loss of the isothiocyanate-
functionalised macrocycle (10) and the formation of the biotin-
conjugate (11). A three step procedure whereby a patient is
administered first with biotinylated anti-tumor antibodies followed
by avidin or streptavidin and finally a biotinylated chelator-
conjugate has been used successfully to image target sites.11
Experiments to investigate the potential of 11 for use in pretargeted
diagnosis and therapy are underway.
An analogue of TETA in which two non-adjacent nitrogen
atoms are linked by an ethylene bridge has been developed by
Weisman and co-workers (so-called ‘cross-bridged’ macrocycle,
CB-TE2A, 2).6 It exhibits far greater complex stability and
overcomes these problems.{7 The 64Cu complex of CB-TE2A is
much less susceptible to transchelation in vivo than that of TETA:
64Cu-CB-TE2A resulted in substantially lower values of protein-
associated 64Cu than observed for 64Cu-TETA [13 ¡ 6% vs. 75 ¡
9% at 4 h].7b Importantly, these findings indicate that a BFC based
on CB-TE2A has the potential to be a powerful tool for medical
imaging and therapy.8 Herein, we present the synthesis and
characterisation of the first BFC incorporating a cross-bridged
macrocycle, 4,11-diacetic acid-6-(4-isothiocyanatobenzyl)-1,4,8,11-
tetraazabicyclo[6.6.2]hexadecane (NCSBz-CB-TE2A).§ The reac-
tivity of our functionalised cross-bridged systems towards Cu(II)
In summary, we report the efficient synthesis of a BFC that
incorporates a cross-bridged macrocycle and is an ideal candidate
for use in medical imaging and therapy. The ability of the system to
{ Electronic supplementary information (ESI) available: Further experi-
mental details and characterisation for compounds 9–11 and Cu(9); See
http://www.rsc.org/suppdata/cc/b4/b406906d/
2 2 1 2
C h e m . C o m m u n . , 2 0 0 4 , 2 2 1 2 – 2 2 1 3
T h i s j o u r n a l i s ß T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

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Scheme 1 Reagents and conditions: (i) 10 equiv. benzyl bromide, CH3CN, N2, RT, 8 d; (ii) 50 equiv. NaBH4, EtOH, N2, RT, 14 d, neutral alumina
chromatography; (iii) H2, 10% Pd/C, HOAc, RT, 3 d; (iv) 1 equiv. benzaldehyde, CH3OH, N2, RT, o/n; (v) 2 equiv. tert-butylbromoacetate, K2CO3,
CH3CN, N2, RT, 3 d, neutral alumina chromatography; (vi) 6 M HCl, 100 uC, 2 d; (vii) 1.1 equiv. Cl2CS, CHCl3/H2O, pH 8, RT, o/n.
spectroscopy arising from the relative positions of the ethylene bridge and
4-substituted phenyl group in the free ligand.
** An ethylenediamine spacer has been introduced in the biotin-conjugate
of 10 to minimise interference from the chelator on binding to avidin/
streptavidin; longer spacers can also be introduced.
1 (a) S. Lui and D. S. Edwards, Bioconjugate Chem., 2001, 12, 7; (b) The
Chemistry of Contrast Agents in Medicinal Magnetic Resonance Imaging,
´
ed. E. To´th and A. E. Merbach, Wiley, Chichester 2001.
2 (a) C. F. Meares, A. J. Chumura, M. S. Orton, T. M. Corneillie and
P. A. Whetstone, J. Mol. Recognit., 2003, 16, 255; (b) C. J. Anderson,
M. A. Green and Y. Fujibayashi in Handbook of Radiopharmaceuticals,
ed. M. J. Welch and C. S. Redvanly, Wiley, New York 2003, pp. 401–422.
´
3 Specific examples include: (a) R. Ruloff, E. To´th, R. Scopelliti,
R. Tripier, H. Handel and A. E. Merbach, Chem. Commun., 2002, 2630;
(b) C. J. Mathias, M. J. Welch, M. A. Green, H. Diril, C. F. Meares,
R. J. Gropler and S. R. Gergmann, J. Nucl. Med., 1991, 32, 475;
(c) G. L. DeNardo, D. L. Kukis, S. Shen, D. A. DeNardo, C. F. Meares
and S. J. DeNardo, Clin. Cancer Res., 1999, 5, 533.
Fig. 1 Biotin conjugate synthesis inset ESMS2 of (a) 10, m/z 510 (25, M 1
Na 2 2)2, 488 (100, M 2 1)2; (b) 11, m/z 796 (60, M 1 Na 2 2)2, 774 (100,
M 2 1)2 (for full MS see ESI).
4 L. A. Bass, M. Wang, M. J. Welch and C. J. Anderson, Bioconjugate
Chem., 2000, 11, 527.
5 G. R. Mirick, R. T. O’Donnell, S. J. DeNardo, S. Shen, C. F. Meares
and G. L. DeNardo, Nucl. Med. Biol., 1999, 26, 841.
bind Cu(II) ions has been demonstrated and the biologically
relevant biotin-conjugate formed.
We acknowledge the Wellcome Trust (E. A. L., grant
no. 069719) for funding. We also thank Dr Trevor Dransfield
(University of York) and the EPSRC National Service (Swansea)
for electrospray mass spectrometry.
6 E. H. Wong, G. R. Weisman, D. C. Hill, D. P. Reed, M. E. Rogers,
J. S. Condon, M. A. Fagan, J. C. Calabrese, K.-C. Lam, I. A. Guzei and
A. L. Rheingold, J. Am. Chem. Soc., 2000, 122, 10561.
7 (a) X. Sun, M. Wuest, G. R. Weisman, E. H. Wong, D. P. Reed,
C. A. Boswell, R. Motekaitis, A. E. Martell, M. J. Welch and
C. J. Anderson, J. Med. Chem., 2002, 45, 469; (b) C. A. Boswell, X. Sun,
W. Nui, G. R. Weisman, E. H. Wong, A. L. Rheingold and
C. J. Anderson, J. Med. Chem., 2004, 47, 1465.
Notes and references
{ Electronic supplementary information (ESI) available: Further experi-
mental details and characterisation for compounds 9–11 and Cu(9); See
http://www.rsc.org/suppdata/cc/b4/b406906d/
{ On binding to metal ions all four nitrogen lone pairs of the cross-bridged
ligand converge upon a cleft that encapsulates the cation with an enforced
cis-V6 ligand conformation; this conformational fixing leads to the
remarkable kinetic stability.10b
8 The biological applications of cross-bridged macrocycles have been
recognised in a number of patents including: (a) T. J. Hubin and
T. J. Meade, Novel Macrocyclic Magnetic Resonance Imaging Contrast
Agents, PCT Int. Appl., WO 02/06287 A2, 2002; (b) C. M. Perkins and
D. T. Reed, Metal Complexes for Use in Medical and Therapeutic
Applications, PCT Int. Appl., WO 02/26748 02, 2000.
9 E. A. Lewis, C. C. Allan, R. W. Boyle and S. J. Archibald, Tetrahedron
Lett., 2004, 45, 3059.
10 For example: (a) T. J. Hubin, J. M. McCormick, S. R. Collinson,
N. W. Alcock and D. H. Busch, J. Chem. Soc., Chem. Commun., 1998,
1675; (b) T. J. Hubin, Coord. Chem. Rev., 2003, 247, 27.
§ The high reactivity of the isothiocyanate functionality toward free amine
sites of biomolecules is well established making 10 the BFC of choice.12
} Highly regioselective bisquaternisation is
a result of macrocycle
conformation;6 two diastereoisomers are formed in 1:1 ratio as previously
described.9
11 E. A. Bayer and M. Wilchek in Immunoassay, ed. E. P. Diamandis and
T. K. Christopoulus, Academic Press, San Diego, CA, 1996, pp. 237–267.
12 G. T. Hermanson, Bioconjugate Techniques, Academic Press, London,
1996, p. 303.
, Ring inversion, whereby the ethylene bridge passes through the plane of
the 14-membered ring, is slow on the NMR timescale and so there are two
possible conformational isomers in solution observed by 1H and 13C NMR
C h e m . C o m m u n . , 2 0 0 4 , 2 2 1 2 – 2 2 1 3
2 2 1 3